SARS-CoV-2 specific CD4+ T cell longevity correlates with Th17-like phenotype

•Th17-like CD4+ T cells showed a longer half-life than other CD4+ T cell subsets•Anti-RBD-IgG titers were associated with Th2- and Tfh-like CD4 T cells•CD45RA+CD8+ T cells were correlated with disease severity during the early phase Determinants of memory T cell longevity following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain unknown. In addition, phenotypes associated with memory T cell longevity, antibody titers, and disease severity are incompletely understood. Here, we longitudinally analyzed SARS-CoV-2-specific T cell and antibody responses of a unique cohort with similar numbers of mild, moderate, and severe coronavirus disease 2019 cases. The half-lives of CD4+ and CD8+ T cells were longer than those of antibody titers and showed no clear correlation with disease severity. When CD4+ T cells were divided into Th1-, Th2-, Th17-, and Tfh-like subsets, the Th17-like subset showed a longer half-life than other subsets, indicating that Th17-like cells are most closely correlated with T cell longevity. In contrast, Th2- and Tfh-like T cells were more closely correlated with antibody titers than other subsets. These results suggest that distinct CD4+ T cell subsets are associated with longevity and antibody responses. Determinants of memory T cell longevity following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain unknown. In addition, phenotypes associated with memory T cell longevity, antibody titers, and disease severity are incompletely understood. Here, we longitudinally analyzed SARS-CoV-2-specific T cell and antibody responses of a unique cohort with similar numbers of mild, moderate, and severe coronavirus disease 2019 cases. The half-lives of CD4+ and CD8+ T cells were longer than those of antibody titers and showed no clear correlation with disease severity. When CD4+ T cells were divided into Th1-, Th2-, Th17-, and Tfh-like subsets, the Th17-like subset showed a longer half-life than other subsets, indicating that Th17-like cells are most closely correlated with T cell longevity. In contrast, Th2- and Tfh-like T cells were more closely correlated with antibody titers than other subsets. These results suggest that distinct CD4+ T cell subsets are associated with longevity and antibody responses. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is an ongoing pandemic that has killed more than 5 million people worldwide as of December 2021. SARS-CoV-2 infection triggers virus-specific cellular and humoral immune responses. While the coordination of both arms of adaptive immunity is deemed critical for resolving COVID-19 (Bert et al., 2020Le Bert N. Tan A.T. Kunasegaran K. Tham C.Y.L. Hafezi M. Chia A. Chng M.H.Y. Lin M. Tan N. Linster M. et al.SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.Nature. 2020; 584: 457-462https://doi.org/10.1038/s41586-020-2550-zCrossref PubMed Scopus (956) Google Scholar; Moderbacher et al., 2020Rydyznski Moderbacher C. Ramirez S.I. Dan J.M. Grifoni A. Hastie K.M. Weiskopf D. Belanger S. Abbott R.K. Kim C. Choi J. et al.Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.Cell. 2020; 183: 996-1012.e19https://doi.org/10.1016/j.cell.2020.09.038Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar; Sekine et al., 2020Sekine T. Perez-Potti A. Rivera-Ballesteros O. Strålin K. Gorin J.-B. Olsson A. Llewellyn-Lacey S. Kamal H. Bogdanovic G. Muschiol S. et al.Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19.Cell. 2020; 183: 158-168.e14https://doi.org/10.1016/j.cell.2020.08.017Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar; Sette and Crotty, 2021Sette A. Crotty S. Adaptive immunity to SARS-CoV-2 and COVID-19.Cell. 2021; 184: 861-880https://doi.org/10.1016/j.cell.2021.01.007Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar), accumulating evidence suggests that the kinetics and magnitude of SARS-CoV-2-specific T cells and antibody responses do not necessarily correlate with each other. For example, SARS-CoV-2-specific antibody titers usually correlate with disease severity in COVID-19 patients, i.e., severe COVID-19 patients possess higher antibody titers than mild or moderate COVID-19 cases (Chen et al., 2020Chen X. Pan Z. Yue S. Yu F. Zhang J. Yang Y. Li R. Liu B. Yang X. Gao L. et al.Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19.Signal Transduct. Target. Ther. 2020; 5: 180https://doi.org/10.1038/s41392-020-00301-9Crossref PubMed Scopus (135) Google Scholar; Garcia-Beltran et al., 2021Garcia-Beltran W.F. Lam E.C. Astudillo M.G. Yang D. Miller T.E. Feldman J. Hauser B.M. Caradonna T.M. Clayton K.L. Nitido A.D. et al.COVID-19-neutralizing antibodies predict disease severity and survival.Cell. 2021; 184: 476-488.e11https://doi.org/10.1016/j.cell.2020.12.015Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar; Piccoli et al., 2020Piccoli L. Park Y.-J. Tortorici M.A. Czudnochowski N. Walls A.C. Beltramello M. Silacci-Fregni C. Pinto D. Rosen L.E. 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Kunasegaran K. Zhuang Y. Tham C.Y.L. Chia A. Smith G.J.D. et al.Early induction of functional SARS-CoV-2 specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients.Cell Rep. 2021; 34: 108728https://doi.org/10.1016/j.celrep.2021.108728Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). In addition, serum antibody titers are poorly predictive of the presence of SARS-CoV-2-specific CD4+ T cells (Dan et al., 2021Dan J.M. Mateus J. Kato Y. Hastie K.M. Yu E.D. Faliti C.E. Grifoni A. Ramirez S.I. Haupt S. Frazier A. et al.Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.Science. 2021; 371: eabf4063https://doi.org/10.1126/science.abf4063Crossref PubMed Scopus (1176) Google Scholar). It is currently unknown whether the relationship between disease severity, T cell responses, and antibody titers changes over time. Longitudinal analysis of SARS-CoV-2-specific immune responses at multiple time points in each case is essential for understanding the factors that influence T cell longevity as well as the basis of disparity between the T cell and antibody response. Identifying determinants for T cell longevity is urgently needed due to rapidly emerging variants of concern (VOCs) that can evade antibody neutralization (Cele et al., 2021Cele S. Jackson L. Khan K. Khoury D.S. Moyo-Gwete T. Tegally H. Scheepers C. Amoako D. Karim F. Bernstein M. et al.SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires ACE2 for infection.medRxiv. 2021; (Preprint at)https://doi.org/10.1101/2021.12.08.21267417Crossref Scopus (0) Google Scholar; Karim and Karim, 2021Karim S.S.A. Karim Q.A. Omicron SARS-CoV-2 variant: a new chapter in the COVID-19 pandemic.Lancet. 2021; 398: 2126-2128https://doi.org/10.1016/s0140-6736(21)02758-6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Nemet et al., 2021Nemet I. Kliker L. Lustig Y. Zuckerman N.S. Erster O. Cohen C. Kreiss Y. Alroy-Preis S. Regev-Yochay G. Mendelson E. et al.Third BNT162b2 vaccination neutralization of SARS-CoV-2 Omicron infection.medRxiv. 2021; (Preprint at)https://doi.org/10.1101/2021.12.13.21267670Crossref Scopus (0) Google Scholar). T cells, particularly CD4+ T cells, are versatile and able to differentiate into distinct subsets, such as Th1, Th2, Th17, and so on, each of which expresses a unique chemokine receptor (DuPage and Bluestone, 2016DuPage M. Bluestone J.A. Harnessing the plasticity of CD4+ T cells to treat immune-mediated disease.Nat. Rev. Immunol. 2016; 16: 149-163https://doi.org/10.1038/nri.2015.18Crossref PubMed Scopus (282) Google Scholar). Information on T cell subsets in COVID-19 is still limited. Tfh cells are required for most Immunoglobulin G (IgG) responses and potently neutralizing antibodies and distinguished by CXCR5 expression. Puzzlingly, reduced disease severity was correlated with circulating CXCR5+ SARS-CoV-2-specific CD4+ T cell frequencies but not with antibody titers (Long et al., 2020Long Q.-X. Liu B.-Z. Deng H.-J. Wu G.-C. Deng K. Chen Y.-K. Liao P. Qiu J.-F. Lin Y. Cai X.-F. et al.Antibody responses to SARS-CoV-2 in patients with COVID-19.Nat. Med. 2020; 26: 845-848https://doi.org/10.1038/s41591-020-0897-1Crossref PubMed Scopus (1684) Google Scholar; Moderbacher et al., 2020Rydyznski Moderbacher C. Ramirez S.I. Dan J.M. Grifoni A. Hastie K.M. Weiskopf D. Belanger S. Abbott R.K. Kim C. Choi J. et al.Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.Cell. 2020; 183: 996-1012.e19https://doi.org/10.1016/j.cell.2020.09.038Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar; Robbiani et al., 2020Robbiani D.F. Gaebler C. Muecksch F. Lorenzi J.C.C. Wang Z. Cho A. Agudelo M. Barnes C.O. Gazumyan A. Finkin S. et al.Convergent antibody responses to SARS-CoV-2 in convalescent individuals.Nature. 2020; 584: 437-442https://doi.org/10.1038/s41586-020-2456-9Crossref PubMed Scopus (955) Google Scholar). Thus far, the association between circulating Tfh (cTfh) and antibody titers remains inconclusive. Th1 and Th17 are often characterized by CXCR3 (Ward et al., 1998Ward S.G. Bacon K. Westwick J. Chemokines and T Lymphocytes more than an attraction.Immunity. 1998; 9: 1-11https://doi.org/10.1016/s1074-7613(00)80583-xAbstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) and CCR6 (Singh et al., 2008Singh S.P. Zhang H.H. Foley J.F. Hedrick M.N. Farber J.M. Human T cells that are able to produce IL-17 express the chemokine receptor CCR6.J. Immunol. 2008; 180: 214-221https://doi.org/10.4049/jimmunol.180.1.214Crossref PubMed Scopus (287) Google Scholar), respectively, and CXCR3+ and CCR6+ SARS-CoV-2-specific CD4+ T cells were both detected during acute COVID-19 (Juno et al., 2020Juno J.A. Tan H.-X. Lee W.S. Reynaldi A. Kelly H.G. Wragg K. Esterbauer R. Kent H.E. Batten C.J. Mordant F.L. et al.Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19.Nat. Med. 2020; 26: 1428-1434https://doi.org/10.1038/s41591-020-0995-0Crossref PubMed Scopus (219) Google Scholar; Moderbacher et al., 2020Rydyznski Moderbacher C. Ramirez S.I. Dan J.M. Grifoni A. Hastie K.M. Weiskopf D. Belanger S. Abbott R.K. Kim C. Choi J. et al.Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.Cell. 2020; 183: 996-1012.e19https://doi.org/10.1016/j.cell.2020.09.038Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). However, their durability and role in pathogenesis have not been adequately addressed. SARS-CoV-2-specific CD8+ T cells are also detectable in acute COVID-19 cases, albeit less consistently than CD4+ T cells, and their presence was also correlated with a better outcome of COVID-19 (Bert et al., 2021Le Bert N. Clapham H.E. Tan A.T. Chia W.N. Tham C.Y.L. Lim J.M. Kunasegaran K. Tan L.W.L. Dutertre C.-A. Shankar N. et al.Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection.J. Exp. Med. 2021; 218 (e20202617)https://doi.org/10.1084/jem.20202617Crossref PubMed Google Scholar; Moderbacher et al., 2020Rydyznski Moderbacher C. Ramirez S.I. Dan J.M. Grifoni A. Hastie K.M. Weiskopf D. Belanger S. Abbott R.K. Kim C. Choi J. et al.Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.Cell. 2020; 183: 996-1012.e19https://doi.org/10.1016/j.cell.2020.09.038Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar; Sekine et al., 2020Sekine T. Perez-Potti A. Rivera-Ballesteros O. Strålin K. Gorin J.-B. Olsson A. Llewellyn-Lacey S. Kamal H. Bogdanovic G. Muschiol S. et al.Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19.Cell. 2020; 183: 158-168.e14https://doi.org/10.1016/j.cell.2020.08.017Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar; Tan et al., 2021Tan A.T. Linster M. Tan C.W. Le Bert N. Chia W.N. Kunasegaran K. Zhuang Y. Tham C.Y.L. Chia A. Smith G.J.D. et al.Early induction of functional SARS-CoV-2 specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients.Cell Rep. 2021; 34: 108728https://doi.org/10.1016/j.celrep.2021.108728Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Whether any particular CD8+ T cell subset contributes to disease severity remains unknown. In this study, we analyzed the frequency and phenotypes of SARS-CoV-2-specific CD4+ and CD8+ T cell responses in peripheral blood mononuclear cells (PBMCs) of a unique cohort that consists of similar numbers of mild, moderate, and severe cases. Most subjects contributed blood samples four times for up to 15 months after disease onset, allowing us to determine the half-life of T cell responses and the correlation between T cell responses and antibody titers without being affected by subject-to-subject variations. The study identifies distinct CD4+ T cell subsets uniquely associated with T cell longevity, antibody titers. Thirty-eight individuals with PCR-confirmed COVID-19 were enrolled in this study. None of them were vaccinated against COVID-19. The disease severity was evaluated according to the NIH severity of illness categories (available: https://www.covid19treatmentguidelines.nih.gov/) (2021). The cohort consists of 15 cases with mild, 10 with moderate, and 13 with severe COVID-19. The demographics and clinical characteristics of participants are summarized in Figure 1A . Subjects with mild COVID-19 were younger than moderate and severe cases, but statistical significance was only observed between mild and severe cases (mild vs. moderate: p = 0.1916, mild vs. severe: p = 0.0311). All subjects provided samples at least twice, and most subjects (28 of 38 subjects) contributed four times, over 14 months post symptom onset. Accordingly, we divided the convalescent period into 4 phases as follows: T1 (<2.5 months), T2 (2.5–5 months), T3 (5–8 months), and T4 (>8 months), and the number of samples from mild, moderate, and severe cases in each phase was indicated above the schema (Figure 1B). SARS-CoV-2-specific T cell responses were longitudinally analyzed by activation-induced marker assay using overlapping peptide pools spanning the spike (S) and nucleocapsid (N) proteins as described in the STAR METHODS and Figure S1. Immunoglobulin G titers against spike receptor-binding domain (RBD) and N protein were also measured by enzyme-linked immunosorbent assay (ELISA). S-specific and N-specific CD4+ and CD8+ T cell responses were induced in virtually all subjects and similarly maintained for approximately 1 year (Figure 2A and S2A). When the T cell responses were compared among three groups based on disease severity, no clear difference was observed in each time point, although the frequency of antigen-specific T cells tended to be lower in the mild cases. In contrast to T cell responses, anti-RBD IgG titers were significantly lower in mild cases, especially when compared with those in severe cases (Figure 2A). Anti-N IgG titers also tended to be lower in the mild cases, but no significant difference was observed among the three (mild, moderate, and severe) groups (Figure S2A). As reported elsewhere (Dan et al., 2021Dan J.M. Mateus J. Kato Y. Hastie K.M. Yu E.D. Faliti C.E. Grifoni A. Ramirez S.I. Haupt S. Frazier A. et al.Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.Science. 2021; 371: eabf4063https://doi.org/10.1126/science.abf4063Crossref PubMed Scopus (1176) Google Scholar; Rodda et al., 2021Rodda L.B. Netland J. Shehata L. Pruner K.B. Morawski P.A. Thouvenel C.D. Takehara K.K. Eggenberger J. Hemann E.A. Waterman H.R. et al.Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.Cell. 2021; 184: 169-183.e17https://doi.org/10.1016/j.cell.2020.11.029Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar; Wheatley et al., 2021Wheatley A.K. Juno J.A. Wang J.J. Selva K.J. Reynaldi A. Tan H.-X. Lee W.S. Wragg K.M. Kelly H.G. Esterbauer R. et al.Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19.Nat. Commun. 2021; 12: 1162https://doi.org/10.1038/s41467-021-21444-5Crossref PubMed Scopus (143) Google Scholar), antibody titers appeared to decay more rapidly than T cell responses. We then analyzed the half-life of SARS-CoV-2-specific T cell responses and antibody titers. So far, in many studies, the half-life was mostly calculated based on raw T cell frequencies (Dan et al., 2021Dan J.M. Mateus J. Kato Y. Hastie K.M. Yu E.D. Faliti C.E. Grifoni A. Ramirez S.I. Haupt S. Frazier A. et al.Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.Science. 2021; 371: eabf4063https://doi.org/10.1126/science.abf4063Crossref PubMed Scopus (1176) Google Scholar; Jung et al., 2021Jung J.H. Rha M.-S. Sa M. Choi H.K. Jeon J.H. Seok H. Park D.W. Park S.-H. Jeong H.W. Choi W.S. Shin E.C. SARS-CoV-2-specific T cell memory is sustained in COVID-19 convalescent patients for 10 months with successful development of stem cell-like memory T cells.Nat. Commun. 2021; 12: 4043https://doi.org/10.1038/s41467-021-24377-1Crossref PubMed Scopus (68) Google Scholar), which can be profoundly influenced by subject-to-subject variations. To estimate the half-life more precisely, we determined fold changes of T cell frequencies and antibody titers relative to the value of initial sampling for each subject. As shown in Figure 2B, the half-life of S-specific CD4+ T cells and CD8+ T cells were approximately 657.4 and 346.4 days, respectively, indicating that CD4+ T cell responses were more stable than CD8+ T cell responses. A similar tendency was observed with N-specific CD4+ and CD8+ T cells (Figure S2B), although the half-life of N-specific CD8+ T cells may not be accurate due to a significant sample-to-sample variation. When samples were stratified by disease severity, we did not see clear correlation between T cell longevity and disease severity. As expected, the half-lives of antibodies (123.5 days for anti-RBD IgG, 128.9 days for anti-N IgG) were shorter than those of T cells. To further characterize SARS-CoV-2-specific T cell responses, we analyzed SARS-CoV-2-specific CD4+ T cells for the expression of various chemokine receptors, such as CXCR5, CXCR3, and CCR6, which are often used to determine T cell subsets (Kasatskaya et al., 2020Kasatskaya S.A. Ladell K. Egorov E.S. Miners K.L. Davydov A.N. Metsger M. Staroverov D.B. Matveyshina E.K. Shagina I.A. Mamedov I.Z. et al.Functionally specialized human CD4+ T-cell subsets express physicochemically distinct TCRs.Elife. 2020; 9: e57063https://doi.org/10.7554/elife.57063Crossref PubMed Google Scholar; Long et al., 2021Long Q.-X. Jia Y.-J. Wang X. Deng H.-J. Cao X.-X. Yuan J. Fang L. Cheng X.-R. Luo C. He A.-R. et al.Immune memory in convalescent patients with asymptomatic or mild COVID-19.Cell Discov. 2021; 7: 18https://doi.org/10.1038/s41421-021-00250-9Crossref PubMed Scopus (16) Google Scholar; Morita et al., 2011Morita R. Schmitt N. Bentebibel S.-E. Ranganathan R. Bourdery L. Zurawski G. Foucat E. Dullaers M. Oh S. Sabzghabaei N. et al.Human blood CXCR5+CD4+ T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion.Immunity. 2011; 34: 108-121https://doi.org/10.1016/j.immuni.2010.12.012Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). Here, we defined CXCR3+/CXCR5-/CCR6-, CXCR3-/CXCR5-/CCR6-, CXCR3-/CXCR5-/CCR6+, and CXCR5+ CD4+ T cells as Th1-like, Th2-like, Th17-like, and Tfh-like cells, respectively (Figure S1). As shown in Figure 3A , S-specific Th2- and Th17-like cells were more frequent than Tfh- and Th1-like cells at every time point, suggesting that T cell subset hierarchy is maintained over an extended period. The hierarchy is also maintained when subjects are divided into three groups according to disease severity (Figure S3). We then calculated the half-life of each T cell subset as described above. As shown in Figure 3B, the half-life of Th1-, Th2-, Th17-, and Tfh-like cells were 478.5, 427.7, 1796.2, and 690.0 days, respectively, indicating that Th17-like cells are most closely associated with T cell longevity. Next, the correlation between SARS-CoV-2-specific T cell frequencies and antibody titers was examined at multiple time points. As shown in Figure 4A , S-specific CD4+ T cell frequencies only modestly correlated with anti-RBD IgG titers. Interestingly, the correlation tended to be stronger at T2 and T3 (i.e., between 2.5 and 8 months after onset) with statistical significance than at T1 (<2.5 months) or T4 (>8 months). We then examined the correlation between individual T cell subsets and anti-RBD IgG titers. As shown in Figure 4B, anti-RBD IgG titers were correlated most closely with the frequencies of Th2-like cells and least closely with those of Th17-like cells. At T4, none of the T cell subsets showed a significant correlation with anti-RBD IgG. N-specific CD4+ T cell frequencies showed virtually no correlation with anti-RBD IgG titers (Figure S4), raising the possibility that CD4+ T cells and B cells need to recognize the cognate antigen for antibody production. However, none of the N-specific T cell subsets correlated with anti-N IgG titers (Figure S5). We also determined CD4+ T cell frequencies specific for RBD using a set of overlapping peptides covering RBD. Because S-specific CD4+ T cell responses correlated weakly with anti-RBD IgG titers at T1 and T4 (Figure 4), we examined the extent to which RBD-specific CD4+ T cell responses correlate with anti-RBD IgG titers at T1 (n = 27) and T4 (n = 33) (two samples at T1 could not be analyzed due to the limited sample availability). Since previous reports indicate mutations in RBD confer resistance to cellular immunity (Motozono et al., 2021Motozono C. Toyoda M. Zahradnik J. Saito A. Nasser H. Tan T.S. Ngare I. Kimura I. Uriu K. Kosugi Y. et al.SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity.Cell Host Microbe. 2021; 29: 1124-1136.e11https://doi.org/10.1016/j.chom.2021.06.006Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), we also determined the frequencies of CD4+ T cells against RBD of wild type (WT), beta (containing K0417N, E0484K, N0501Y), and epsilon (containing L452R) and correlated the frequencies with anti-RBD IgG titers. As shown in Figure 5A , frequencies of CD4+ T cells responding to WT, beta, and epsilon RBD were similar. Interestingly, anti-RBD IgG titers correlated more strongly with the frequency of CD4+ T cells responding to WT-RBD than those to the entire S, beta-RBD, and epsilon RBD (Figure 5B). While previous reports suggested that SARS-CoV-2-specific T cell responses were inversely correlated with disease severity during disease progression (Mathew et al., 2020Mathew D. Giles J.R. Baxter A.E. Oldridge D.A. Greenplate A.R. Wu J.E. Alanio C. Kuri-Cervantes L. Pampena M.B. D’Andrea K. et al.Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications.Science. 2020; 369: eabc8511https://doi.org/10.1126/science.abc8511Crossref PubMed Google Scholar; Moderbacher et al., 2020Rydyznski Moderbacher C. Ramirez S.I. Dan J.M. Grifoni A. Hastie K.M. Weiskopf D. Belanger S. Abbott R.K. Kim C. Choi J. et al.Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.Cell. 2020; 183: 996-1012.e19https://doi.org/10.1016/j.cell.2020.09.038Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar), we did not observe significant differences in the frequencies of SARS-CoV-2-specific CD4+ and CD8+ T cells at the early memory phase in our cohort (Figures 2A and S2A). We then examined the association between disease severity and individual T cell subsets. CD4+ T cells were divided into Th1-, Th2- Th17-, and Tfh-like cells, while CD8+ T cells were divided into CD45RA+ and CD45RA- subsets. As shown in Figure 6, the frequencies of S-specific CD45RA+ CD8+ T cells were significantly higher in severe cases than in mild cases. There was no correlation between S-specific CD45RA+ CD8+ T cells and subjects’ age. CD45RA+ CD8+ T cells represent not only effector T cells and terminally differentiated effector memory T cells (CD45RA+ CCR7-) but also stem cell-like memory (Tscm) T cells (CD45RA+ CCR7+) (Jung et al., 2021Jung J.H. Rha M.-S. Sa M. Choi H.K. Jeon J.H. Seok H. Park D.W. Park S.-H. Jeong H.W. Choi W.S. Shin E.C. SARS-CoV-2-specific T cell memory is sustained in COVID-19 convalescent patients for 10 months with successful development of stem cell-like memory T cells.Nat. Commun. 2021; 12: 4043https://doi.org/10.1038/s41467-021-24377-1Crossref PubMed Scopus (68) Google Scholar). To distinguish these alternatives, we analyzed the frequencies of SARS-CoV-2 S-specific CD45RA+ CD8+ T cells in PBMCs collected later than 8 months after the symptom onset (T4). As shown in Figure S6A, there was no significant difference between mild and severe cases at T4, indicating that the higher frequency of CD45RA+ CD8+ T cells in severe cases at T1 was most likely attributed to the induction of short-lived T cells rather than Tscm. Collectively, these results indicate that effector or terminally differentiated effector memory CD8+ T cells may contribute to the pathogenesis of COVID-19 in the convalescent phase. Little information was available about factors that affect memory T cell longevity. In addition, the relationship between T cell responses and antibody titers after SARS-CoV-2 infection remains incompletely understood. Here, we analyzed T cell and antibody responses of a unique cohort in which similar numbers of mild (n = 15), moderate (n = 10), and severe (n = 13) COVID-19 cases were enrolled. In most cases, both plasma and peripheral blood samples were available at multiple time points, spanning approximately 1 year after COVID-19 infection. The sample availability at multiple time points enabled us to calculate the half-life of T cell and antibody responses without being affected by differences between individuals. The results revealed differential longevity among memory CD4+ T cell subsets. The study also identifies distinct CD4+ T cell subsets that are associated with anti-RBD IgG titers. Interestingly, anti-RBD IgG titers were more closely correlated with CD4+ T cell responses to RBD than to the entire S region during the memory phase. Immune memory induced by primary infection or vaccination provides the foundation of protective immunity from subsequent infection. While sterilizing immunity can only be achieved by pre-existing neutralizing antibodies, T cells are thought to prevent the onset of symptomatic or severe disease. Detailed information on T cell durability is urgently needed because VOCs that can evade antibody neutralization are rapidly emerging. It has been difficult to compare the durability between SARS-CoV-2-specific antibodies and T cells, because longitudinal data with at least three time points per subject are rarely available and immune responses to SARS-CoV-2 infections are notably heterogeneous (Bert et al., 2020Le Bert N. Tan A.T. Kunasegaran K. Tham C.Y.L. Hafezi M. Chia A. Chng M.H.Y. Lin M. Tan N. Linster M. et al.SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.Nature. 2020; 584: 457-462https://doi.org/10.1038/s41586-020-2550-zCrossref PubMed Scopus (956) Google Scholar; Grifoni et al., 2020Grifoni A. Weiskopf D. Ramirez S.I. Mateus J. Dan J.M. Moderbacher C.R. Rawlings S.A. 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Muschiol S. et al.Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19.Cell. 2020; 183: 158-168.e14https://doi.org/10.1016/j.cell.2020.08.017Abstract Ful