Effects of allergen immunotherapy on follicular regulatory T cells

Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al. [35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al. [48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express. While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com. The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients. Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum. As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express. While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com. The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients. Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum. As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express. While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com. The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients. Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum. As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express. While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com. The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients. Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum. As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express. While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com. The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients. Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum. As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express. While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com. The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients. Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum. As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express. While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com. The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients. Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.Purpose of reviewEmerging evidence indicating that the dysfunction of T follicular regulatory (TFR) cells contributes to excessive immunoglobulin E (IgE) production and the development of allergic diseases. Conversely, allergen immunotherapy (AIT) modulates TFR cells abundance and function to promote immune tolerance. This review focus on the role of TFR cells in allergic diseases and AIT, with the objective of providing novel insights into the mechanisms underlying immune tolerance of AIT and proposing the potential targeting of TFR cells in the context of allergic diseases.Numerous studies have consistently demonstrated that TFR cells play a pivotal role in the inhibition of class switch recombination to IgE in both humans and specific murine models. This suppression is attributed to the actions of neuritin and IL-10 secreted by TFR cells, which exert direct and indirect effects on B cells. In patients with allergic rhinitis, reduced frequencies of circulating or tonsillar TFR cells have been reported, along with impaired functionality in suppressing IgE production. AIT, whether administered subcutaneously or sublingually, reinstates the frequency and functionality of TFR cells in allergic rhinitis patients, accompanied by changes of the chromatin accessibility of TFR cells. The increase in TFR cell frequency following AIT is associated with the amelioration of clinical symptoms.TFR cells exert an inhibitory effect on IgE production and demonstrate a correlation with the clinical efficacy of AIT in patients with allergic rhinitis, suggesting TFR cells hold promise as a therapeutic target for allergic diseases and potential biomarker for AIT.Papers of particular interest, published within the annual period of review, have been highlighted as:Allergen immunotherapy (AIT) is a highly effective treatment for allergic rhinitis and asthma, which involves the repeated administration of high-dose allergens to patients, either subcutaneously or sublingually [1-3]. As the only disease-modifying therapy, AIT has been reported to provide long-term clinical benefits and tolerance, even after discontinuation following a course of over three years [1-3]. However, a significant percentage, ranging from 20 to 50%, of patients exhibit a poor response to this treatment, reflecting our limited understanding of the mechanisms of AIT [4-6]. Over the past few decades, significant progress has been made in unraveling the mechanisms involved in AIT. The establishment of long-term clinical tolerance to allergens involves a complex network of interactions, including the depletion of pathogenic T cell responses, modulation of basophil and mast cell functions, induction of allergen-specific regulatory T and B cells, and the production of IgG4 and IgA 'protective' antibodies [7-9]. Despite these advancements, the mechanisms underpinning AIT are still far from completely revealed. Recently, the identification of novel subsets of T helper (TH) cells, such as follicular helper T (TFH) and follicular regulatory T (TFR) cells, has expanded our understanding of the intricate immune networks involved in allergic diseases [10,11]. Consequently, investigations into the roles of TFH and TFR cells in the context of AIT have gained momentum.As a specialized subset of CD4+ T cells, TFH cells are primarily localized in the B-cell follicle and play a critical role in antibody affinity maturation and the formation of humoral memory [12,13]. Emerging evidence has highlighted the crucial role of TFH cells in regulating IgE class switching and the production of allergen-specific IgE in both mice and humans, through secreting interleukin (IL)-4 and IL-13 [14,15]. Conversely, TFR cells, characterized by co-expression of the regulatory T (TREG) cells transcription factor forkhead box P3 (Foxp3) and the germinal center defining transcription factor B cell lymphoma 6 (Bcl-6), act as germinal center resident T cells that counterbalance the effects of TFH cells [16]. TFR cells regulate various aspects of the germinal center reaction, including B cell activation, differentiation, isotype switching, and emergence of self-reactivity, thereby maintaining immune homeostasis in secondary lymphoid organs [16]. TFR cells majorly inhibit the IgE production by regulating the activation of TFH and B cells, although they have also been reported to promote IgE response in some allergic conditions [17-21]. Additionally, it has been demonstrated that AIT can induce TFR cells and restore the balance between TFR cells and type 2 TFH (TFH2) cells in allergic patients [14,17,22]. The induction of TFR cells closely associated with the efficacy of AIT [17], highlighting the importance of TFR cells in the development and maintenance of immune tolerance during AIT.This review aims to provide an overview of the recent evidence regarding the function of TFR cells in allergic diseases and the impact of AIT on TFR cells, offer further insights into the mechanisms of immune tolerance in AIT, and provide a perspective on the potential targeting of TFR cells in the management of allergic diseases. no caption availableTFR cells were initially identified in 2011 as a distinct subtype of TREG cells that are located within B-cell follicles of secondary lymphoid organs, by three independent groups [23-25]. TFR cells exhibit a unique phenotype characterized by co-expressing markers associated with both TREG cells and TFH cells, including the transcription factor Bcl-6, the chemokine receptor C-X-C motif receptor (CXCR5), inducible co-stimulator (ICOS), co-inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and transcription factors Foxp3 and B lymphocyte induced maturation protein 1 (Blimp1) [23-25]. The development of TFR cells involves a multifactorial process similar to, but distinct from, that of TFH cells [16,26]. Like TFH cells, the development of TFR cells require T-cell receptor (TCR) stimulation and depend on the signaling including CD28, signaling lymphocytic activation molecule (SLAM)-associated protein, and ICOS to induce and maintain Bcl-6 expression [27,28]. IL-21 is essential for TFH cell differentiation, but it restricts the differentiation and proliferation of TFR cells [29,30]. IL-2 has been proposed as a negative regulator for the differentiation of TFH and TFR cells [31,32]; however, a recent study demonstrated that IL-2 promotes the tonsillar TFH to TFR transition in vitro[33]. It is likely that IL-2 exerts dose-dependent effects on the differentiation of TFH and TFR cells [33,31].TFR cells were initially believed to derive from thymic TREG cells based on various adoptive transfer experiments in mice [24,25]. Indeed, TCR sequencing revealed that TFR cells exhibited a TCR repertoire that more closely resembles to TREG cells than TFH cells in immunized mice [34]. Nevertheless, Aloulou et al.[35] reported that in addition to developing from thymic derived TREG cells, murine TFR cells, although in a minority, can also arise from naive T cells and peripheral TREG cells in response to the immunodominant peptide (MOG35-55) and Incomplete Freund's Adjuvant. Similarly, the dominant maturation of TFR cells follows a bifurcated trajectory from precursor TREG cells, with one pointing to circulating TFR cells while the other to the most mature germinal center TFR cells, as observed through transcriptional changes of TFH, TREG, and TFR cells isolated from the blood, lymph nodes, and tonsils in human [36]. More recently, using in-silico and in-vitro models, Coz et al.[33] found that although the majority of TFR cells derive from TREG cells, some TFR cells derive from TFH cells in tonsils. Consistently, Jacobsen et al.[37] demonstrated that an end-stage TFH population is able to express Foxp3 and become TFR cell in OVA-immunized mice. The results indicate the heterogeneity of TFR cells, which could potentially be attributed to the diverse repertoires of TCR they express.While the initial definition of TFR cells in both mice and humans described them as CXCR5+PD-1+ICOS+Bcl-6+CD25+Foxp3+ T cells, recent research has highlighted their heterogeneity [10,23-25]. Indeed, the origin and anatomical location of TFR cells may correspond to different developmental stages with distinct phenotypes [38]. For instance, although CD25 is highly expressed by TREG cells, CD25- TFR cells were observed in lymphoid organs of both mice and humans, appearing to preferentially localize within germinal center [39-41]. Using highly multiplexed quantitative imaging, Sayin et al.[39] identified a population of TFR cells with low PD-1 expression residing at T-B and germinal center-mantle borders in human mesenteric lymph nodes. On the basis of CD38 expression, Coz et al. [33] characterized two distinct populations of TFR cells within the germinal center with unique distribution and function [33]. CD38+ TFR cells reside within the germinal center and acquire suppressive function on inhibiting the proliferation of T responder cells while maintaining their capacity to help B cells, whereas CD38- TFR cells were identified as highly efficient suppressors primarily localized in the follicular mantles [33]. In addition, a subset of T follicular cells lacking Foxp3 expression but displaying high levels of CD25, CTLA-4, and IL-10 in children tonsils as well as human mesenteric lymph nodes has been found to exert suppressive effects on T cell proliferation and B cell class switching to IgE in vitro[42]. Hence, caution should be exercised to define TFR cells given their heterogeneity.TFR cells play a crucial role in inhibiting TFH cell-induced germinal center responses by modulating the activation and proliferation of TFH cells and B cells [23-25,43]. CTLA-4 and IL-10 have been implicated in the suppressive activity of TFR cells. Deletion of CTLA-4 in TFR cells enhances germinal center responses, as evidenced by increased TFH cells, germinal center B cells, and IgG levels [44]. Additionally, Canete et al. [42] demonstrated that IL-10 derived from follicular T cells repressed B cell class switching to IgE in vitro[42]. However, Xie et al. [20] show that IL-10-producing TFR cells promoted germinal center responses and IgE production in a mouse model of food allergy [20]. This discrepancy may be attributed to differences in the specific models used to induce allergic responses. It is worth noting that some TFR cell function is still preserved in patients lacking CTLA-4 and in mice with IL-10 knockout, suggesting the existence of additional mechanisms, such as secreting neuritin, by which TFR cells exert their actions.The investigation of TFR cells in allergic diseases was first conducted by Clement et al. [19] who developed a TFR cell-deleter mouse by simultaneous deleting FOXP3 and CXCR5 genes. Deleting TFR cells before germinal center initiation, but not afterwards, led to increased production of HDM-specific IgE and aggravated inflammation in the lung in a HDM-induced allergic airway model [19]. TFR cell deletion did not alter the numbers of total TFH cells, germinal center B cells, IgG1+ GC B cells, and IgE+ germinal center B cells but significantly increased the numbers of IgE+ plasma cells, suggesting that TFR cells may control allergen-specific IgE production by suppressing plasma cell differentiation in mice [19]. The suppressive role of TFR cells in IgE responses was confirmed by using Bcl6fl/flFOXP3cre mice [18]. Increased total and antigen-specific IgE production and allergic tissue inflammation were observed in the absence of TFR cells [18]. Recently, Gowthaman et al.[15] identified a population of TFH cells termed as TFH13 cells, which produce both IL-4 and IL-13 and are responsible for the generation of high-affinity but not low-affinity IgE in HDM-sensitized mice. In in-vitro cell coculture system, Clement et al. [19] revealed that TFR cells suppress IL-13-expressing TFH cells and reduce the numbers of IgE+ and IgG1+ B cells, suggesting that TFR cells may restrict the affinity maturation of IgE. Indeed, Gonzalez-Figueroa et al.[45] found that TFR cell-deficient mice exhibited enhanced affinity of antigen-specific germinal center B cells, despite a decrease in the overall number of these cells, in an OVA-immunized model. By generating mice that selectively deficient in neuritin, a neuropeptide, in Foxp3+ T cells, Gonzalez-Figueroa et al.[45] revealed that neuritin, secreted by TFR cells, acts directly on B cells to suppress plasma cell differentiation and limit IgE production by restricting IgE class switch recombination. In contrast to most published studies, Xie et al.[20] demonstrated that TFR cells actively promote antigen-specific IgE production by secreting IL-10 in a mouse model of peanut-induced food allergy. In this model, TFR cell deficiency (Bcl6fl/flFOXP3cre) results in reduced antigen-specific IgE titers and anaphylaxis responses, and an increase in nonspecific IgE titers, suggesting a facilitatory effect of TFR on antigen-specific IgE responses [20]. The observed dual role of TFR cells, both inhibitory and facilitatory, in antigen-specific IgE production in mouse models may be attributed to differences in the specific models used to induce allergic responses [21]. Whether the different functions of TFR cells are related to the heterogeneity of TFR cells is an interesting topic to study [46]. In addition, the clinical relevance of these findings remains to be elucidated.A great obstacle in understanding how TFR cells regulate IgE in humans is the challenge in obtaining human samples of secondary lymphoid tissues. Canete et al.[42] described a subset of human tonsillar follicular T cells expressing high levels of IL-10 and CD25, referred to as CD25+ TF cells. These CD25+ TF cells share similarities with mouse TFR cells but do not express Foxp3, yet they are capable of inhibiting class switching to IgE in vitro[42]. Interestingly, in allergic patients, serum IgE levels were found to be inversely correlated with the number of CD25 TF cells [42]. By examining tonsillar TFR cells in individuals with or without concomitant allergic rhinitis, Yao et al.[17] discovered a significant decrease in tonsillar Foxp3+ TFR cells in germinal centers in allergic rhinitis patients compared with individuals without allergic rhinitis. A tight correlation between the frequencies of TFR cells in tonsils and those in paired blood samples was noted [17]. Furthermore, they observed a notable reduction in circulating TFR cells among allergic rhinitis patients, whereas the percentages of total TREG cells and CD45RAlowCXCR5- TREG cells were comparable between the two groups [17]. The selective reduction of TFR cells underscores the specific role of TFR cells in preventing allergic rhinitis development. Moreover, circulating TFR cells from allergic rhinitis patients exhibited a specific impairment in suppressing IgE production while maintaining their ability to regulate other types of immunoglobulins [17]. The mechanisms by which TFR cells regulate IgE induction were elucidated by Gonzalez-Figueroa et al.[45]. They demonstrated that neuritin, principally derived from human TFR cells, acts on B cells and triggers IRS1 and 4E-BP1 phosphorylation, thereby preventing B cell differentiation into plasma cells and inhibiting class switch recombination to IgE in humans [45]. Despite the valuable insights gained from these studies, the precise mechanisms employed by TFR cells to suppress IgE responses remain to be fully elucidated.AIT is an effective treatment for allergic rhinitis and asthma, with the goal of restoring immune tolerance. Several recent studies have shown that successful AIT is associated with the induction of TFR cells in patients with allergic rhinitis or asthma [11,17,22,47,48]. Schulten et al. [11] demonstrated that subcutaneous AIT significantly reduced the frequencies of circulating CD45RO+CXCR5+ TFH cells while increased the frequencies of CXCR5low TFH cells that exhibit a TFR cell phenotype with high FOXP3 expression, in individuals allergic to timothy grass. In in vitro, T-cell receptor stimulation suppressed CXCR5 expression on TFH cells and facilitated their differentiation into TFR cells [11]. In contrast, IL-2 partially attenuated the induction of TFR cells, and resulted in a decrease of approximately half of TFR cells compared to T-cell receptor stimulation alone [11]. In a phase III, multicenter, randomized, double-blind, placebo-controlled trial, Sharif et al.[22] reported that a 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide subcutaneous immunotherapy robustly increased the frequencies of circulating Foxp3+ and CTLA-4+ TFR cells, whereas decreased the frequencies of IL-4+, IL-21+, and IL-4+IL-21+ CXCR5+PD-1+ circulating TFH cells in patients with grass pollen-induced allergic rhinitis, suggesting an immune switch from TFH2 to TFR cell during AIT. More importantly, Yao et al.[17] discovered that subcutaneous AIT not only increased the abundance of circulating TFR cells but also enhanced their immunosuppressive effects on IgE production in allergic rhinitis patients allergic to HDM (Fig. 1). The mechanisms underlying the change of TFR cell function and abundance are not very clear. Recently, by employing ATAC-seq analysis of circulating TFR cells from grass pollen-allergic patients with and without AIT, Sharif et al.[48] revealed that subcutaneous AIT enhanced chromatin accessibility associated with macromolecular catabolism in connection with superior cell survival and development, while sublingual AIT enhanced chromatin accessibility associated with negative regulation of cell differentiation and proliferation and diverse molecular functions in TFR cells (Fig. 1). These findings suggest that AIT may modulate TFR cells by epigenetic mechanisms.Allergen immunotherapy alters the abundance and function of follicular regulatory T cells in patients with allergic rhinitis. TFH2 cells from patients with AR display a robust capacity to induce IgE production, whereas AR-derived TFR cells show a defective function in suppressing IgE production. AIT reduces the number of TFH2 cells and improves abundance and function of TFR cells, accompanied by chromatin landscape alteration in TFR cells. AIT may increase IL-10 and neuritin production by TFR cells to inhibit IgE production. AR, allergic rhinitis; AIT, allergen immunotherapy; B, B cells; IL, interleukin; PB, plasmablasts; TFH2, type 2 follicular helper T; TFR, follicular regulatory T. Created with BioRender.com.The induction of allergen-specific IgG4 and IgA has been recognized as an important event associated with the establishment of immune tolerance during AIT [49,50]. These antibodies possess allergen neutralizing capacity and compete with IgE for allergen binding, thereby inhibiting the formation of allergen-IgE complexes and subsequent activation of mast cells and basophils [49]. Yao et al.[14,17] reported that 12 months of subcutaneous AIT resulted in a reduction of allergen-specific IgE and the induction of blocking IgG4 antibodies, which correlated with clinical improvement. Notably, a distinct cluster of antibody-expressing cells with abundant IGHG4 and IGHE expression was observed in patients with nasal polyps, suggesting a potential transition from IgE to IgG4 [51,52]. IL-10 has been reported to promote IgG4 but inhibit IgE production in humans [42,53]. In addition, 12 months of subcutaneous AIT significantly increased the numbers of IL-10+ TFR cells [47]. It raises a possibility that IL-10-secreting TFR cells are germane to the IgG4 class switching in AIT [47]. Indeed, a significantly increased frequencies of IL-10-producing circulating TFR cells was observed in patients with IgG4-related disease, and their frequencies positively correlated with levels of IgG4 in serum [54]. Using Bcl6fl/flFOXP3cre mice, Zhang et al.[55] found that TFR cell deletion did not affect intestinal peanut-specific IgA production, indicating that IgA induction occurs through a pathway distinct from IgG4 production and independent of TFR cells. However, given the distinct roles of TFR cells observed in food allergy compared to allergic airway diseases, further investigation is needed to explore the role of TFR cells in IgA production using other allergic disease models, such as the HDM-induced allergic airway model.The identification of biomarkers that can monitor or predict the efficacy of AIT is highly beneficial for clinicians to select appropriate patients who are likely to benefit from treatment. Yao et al.[17] found that the elevated frequencies of TFR cells positively correlated with the improvement of combined symptom and medication score after 12 months subcutaneously AIT in patients with allergic rhinitis allergic to HDM. Interestingly, the ratio of TFR/TFH2 cells exhibited a stronger correlation with clinical benefits than TFR cells alone [17], suggesting a crucial involvement of rebalance of functionally antagonistic TFR and TFH2 cells in AIT efficacy. Despite the mechanisms underlying the restoration of TFH and TFR cell balance during AIT remain poorly understood, emerging evidence from recent investigations offers valuable insights into this process. Botta et al. [31] reported the accumulation of TFR cells later than TFH cells following immunization and infection. Jacobsen et al.[37] observed that a population of TFH cells upregulates Foxp3 expression at the late stage of germinal center, suggesting a slower generation kinetic of TFR cells. Therefore, extending the duration of allergen administration during AIT may prolong the accumulation of TFR cells and potentially enhance clinical benefits [31].A growing body of evidence suggests that TFR cells have a prominent role in regulating IgE production. In addition, defective TFR cells, as shown by lessened abundance and impaired functions, have been observed in allergic patients, which may contribute to the pathogenesis of these diseases. An important mechanism underlying the induction of allergen tolerance during AIT is the restoration of compromised TFR cells in allergic patients.Despite the progress made in understanding the involvement of TFR cells in allergic diseases and AIT, several important questions remain unanswered. Although certain effector molecules such as neuritin and IL-10 have been identified, they cannot fully explain the regulatory function of TFR cells. Thus, it is crucial to explore other effector pathways utilized by TFR cells. In addition, whether these identified pathways are involved in AIT-mediated restoration of TFR cells is unknown. Given the heterogeneity of TFR cells, it is important to investigate the impact of AIT on different subsets of TFR cells, including their abundance, TCR repertoires, chromatin landscapes, and functions. TFH2 cells promote the production of high-affinity IgE, which mediates anaphylactic reactions. Nevertheless, the role of AIT in regulating the affinity of IgE warrants further investigation. In conclusion, TFR cells and their associated effector molecules hold promise as therapeutic targets to enhance the clinical benefits of AIT. Continued research in this area is essential for advancing our understanding of TFR cell biology and optimizing the effectiveness of AIT.All authors participated in drafting and writing the manuscript and approved the manuscript.