Phenotypic Analysis of Disease-Relevant T Cells in Dermatitis Herpetiformis.

Gluten-specific CD4+ T cells are central players in the pathogenesis of celiac disease (CeD), an inflammatory disease driven by exposure to dietary gluten proteins. Patients with CeD are treated with a lifelong gluten-free diet. When gluten is reintroduced to the diet of patients in remission, there is a wave of activated (CD38+) gluten-specific CD4+ T cells in the blood that peaks on days 6‒8 after the first gluten exposure. This increase in the frequency of activated gluten-specific CD4+ effector-memory T cells (TEM) in the blood can be detected by IFN-γ enzyme-linked immunospot assay (Anderson et al., 2000Anderson R.P. Degano P. Godkin A.J. Jewell D.P. Hill A.V. In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope.Nat Med. 2000; 6: 337-342Crossref PubMed Scopus (491) Google Scholar; Tye-Din et al., 2010Tye-Din J.A. Stewart J.A. Dromey J.A. Beissbarth T. van Heel D.A. Tatham A. et al.Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease.Sci Transl Med. 2010; 2: 41ra51Crossref PubMed Scopus (363) Google Scholar) or by HLA-DQ:gluten tetramers (Ráki et al., 2007Ráki M. Fallang L.E. Brottveit M. Bergseng E. Quarsten H. Lundin K.E. et al.Tetramer visualization of gut-homing gluten-specific T cells in the peripheral blood of celiac disease patients.Proc Natl Acad Sci USA. 2007; 104: 2831-2836Crossref PubMed Scopus (122) Google Scholar; Sarna et al., 2018bSarna V.K. Skodje G.I. Reims H.M. Risnes L.F. Dahal-Koirala S. Sollid L.M. et al.HLA-DQ:gluten tetramer test in blood gives better detection of coeliac patients than biopsy after 14-day gluten challenge.Gut. 2018; 67: 1606-1613Crossref PubMed Scopus (49) Google Scholar; Zühlke et al., 2019Zühlke S. Risnes L.F. Dahal-Koirala S. Christophersen A. Sollid L.M. Lundin K.E. CD38 expression on gluten-specific T cells is a robust marker of gluten re-exposure in coeliac disease.United European Gastroenterol J. 2019; 7: 1337-1344Crossref PubMed Scopus (20) Google Scholar). The gluten-specific CD4+ T cells in the blood that increase in frequency also alter their phenotype to that of gluten-specific CD4+ T cells in the gut mucosa of untreated CeD (Christophersen et al., 2021Christophersen A. Zühlke S. Lund E.G. Snir O. Dahal-Koirala S. Risnes L.F. et al.Pathogenic T cells in celiac disease change phenotype on gluten challenge: implications for T-cell-directed therapies.Adv Sci (Weinh). 2021; 8e2102778PubMed Google Scholar, Christophersen et al., 2019Christophersen A. Lund E.G. Snir O. Solà E. Kanduri C. Dahal-Koirala S. et al.Distinct phenotype of CD4+ T cells driving celiac disease identified in multiple autoimmune conditions.Nat Med. 2019; 25: 734-737Crossref PubMed Scopus (87) Google Scholar). Parallel with the increase of gluten-specific CD4+ T cells in the blood, there is an increase in the frequency of gut-homing and activated CD8+ and γδ T cells (Christophersen et al., 2021Christophersen A. Zühlke S. Lund E.G. Snir O. Dahal-Koirala S. Risnes L.F. et al.Pathogenic T cells in celiac disease change phenotype on gluten challenge: implications for T-cell-directed therapies.Adv Sci (Weinh). 2021; 8e2102778PubMed Google Scholar; Han et al., 2013Han A. Newell E.W. Glanville J. Fernandez-Becker N. Khosla C. Chien Y.H. et al.Dietary gluten triggers concomitant activation of CD4+ and CD8+ αβ T cells and γδ T cells in celiac disease.Proc Natl Acad Sci USA. 2013; 110: 13073-13078Crossref PubMed Scopus (151) Google Scholar; Risnes et al., 2021Risnes L.F. Eggesbø L.M. Zühlke S. Dahal-Koirala S. Neumann R.S. Lundin K.E.A. et al.Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease.Mucosal Immunol. 2021; 14: 842-851Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Although dermatitis herpetiformis (DH) is considered an extraintestinal manifestation of CeD, little is known about the gluten-induced T-cell responses in patients with DH. Recently, Kalliokoski et al., 2020Kalliokoski S. Mansikka E. de Kauwe A. Huhtala H. Saavalainen P. Kurppa K. et al.Gliadin-induced ex vivo T-cell response in dermatitis herpetiformis: A predictor of clinical relapse on gluten challenge?.J Invest Dermatol. 2020; 140: 1867-1869.e2Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar performed IFN-γ enzyme-linked immunospot assay on PBMCs from patients with DH and CeD on day 6 after a 3-day gluten challenge as part of an oral challenge for a period of up to 1 year. Only 47% of the patients with DH displayed reactivity to deamidated gluten, leading to the conclusion that early IFN-γ response to selected gluten peptides does not predict clinical relapse in patients with DH on long-term gluten provocation. The study was approved by the Finnish Regional Ethics Committee of Tampere University Hospital (Tampere, Finland), and all patients gave written informed consent. Using cryopreserved PBMCs that were available from 7 of the 19 patients from the original challenge study (Mansikka et al., 2019Mansikka E. Hervonen K. Kaukinen K. Ilus T. Oksanen P. Lindfors K. et al.Gluten challenge induces skin and small bowel relapse in long-term gluten-free diet-treated dermatitis herpetiformis.J Invest Dermatol. 2019; 139: 2108-2114Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), in this study, we aimed to identify the phenotypic markers of disease-relevant T cells in patients with DH by performing multiparametric flow cytometric analysis of CD4+, CD8+, and γδ T cells. We sought to identify gluten-reactive T cells by use of HLA-DQ2.5:gluten tetramers (termed HLA-tetramers in the remaining part of this paper) representing a mixture of the immunodominant DQ2.5-glia-α1a, DQ2.5-glia-α2, DQ2.5-glia-ω1, DQ2.5-glia-ω2, and DQ2.5-hor3 epitopes (Tye-Din et al., 2010Tye-Din J.A. Stewart J.A. Dromey J.A. Beissbarth T. van Heel D.A. Tatham A. et al.Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease.Sci Transl Med. 2010; 2: 41ra51Crossref PubMed Scopus (363) Google Scholar). We stained the T cells for CD45RA and CD62L as well for gut-homing marker integrin β7, cutaneous lymphocyte antigen (CLA), and CCR4 (for further details, see Supplementary Materials and Methods). We detected CD4+ TEM (CD45RA‒CD62L‒) cells that bound HLA tetramers in five of the seven patients investigated, but only two patients displayed a clear increase in the frequency of such cells on day 6 (Figure 1a and b). The number of cells retrieved from the cryopreserved PBMC samples ranged from 0.35 to 3.9 million cells (median = 1.1) with a viability of 78‒97% (median = 93%). Ideally, more cells should have been analyzed. Although the tetramer enrichment method is highly sensitive and distinguishes patients with CeD from HLA-matched controls, precise cell number estimates require higher numbers (>10 million) of PBMCs, especially for the baseline samples because the method detects low-frequency cells (typically one cell per million CD4+ T cells) (Sarna et al., 2018aSarna V.K. Lundin K.E.A. Mørkrid L. Qiao S.W. Sollid L.M. Christophersen A. HLA-DQ-gluten tetramer blood test accurately identifies patients with and without celiac disease in absence of gluten consumption.Gastroenterology. 2018; 154 (e886): 886Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The majority of the HLA-tetramer‒positive TEM cells expressed integrin β7, suggesting that these are gut-homing cells. Curiously, 31% of the HLA-tetramer‒positive TEM cells in one of the patients (DH4) expressed the skin-homing marker CLA (Figure 1a and c). The CLA+ HLA-tetramer‒positive T cells of this one patient also expressed high levels of CCR4. Whether additional patients with DH also have skin-homing gluten-specific CD4+ T cells in addition to gut-homing gluten-specific CD4+ T cells cannot be concluded from this single observation. More studies are warranted. Despite variation in the increase of numbers of HLA-tetramer‒positive TEM cells, in all the five patients, we observed a uniform increase in CD38 expression in HLA-tetramer‒positive TEM cells after gluten challenge (Figure 1d). We further showed that all the HLA-tetramer‒positive TEM cells clustered distinctly from the HLA-tetramer‒negative cells (Figure 1e). In line with previous observations of patients with CeD (Christophersen et al., 2021Christophersen A. Zühlke S. Lund E.G. Snir O. Dahal-Koirala S. Risnes L.F. et al.Pathogenic T cells in celiac disease change phenotype on gluten challenge: implications for T-cell-directed therapies.Adv Sci (Weinh). 2021; 8e2102778PubMed Google Scholar, Christophersen et al., 2019Christophersen A. Lund E.G. Snir O. Solà E. Kanduri C. Dahal-Koirala S. et al.Distinct phenotype of CD4+ T cells driving celiac disease identified in multiple autoimmune conditions.Nat Med. 2019; 25: 734-737Crossref PubMed Scopus (87) Google Scholar), the HLA-tetramer‒positive TEM cells on gluten challenge upregulated CD38 and PD-1 and downregulated CD127 (Figure 1f). To identify which epitope the gluten-specific T cells in patients with DH were reactive to, we sorted CD4+ HLA-tetramer‒positive TEM cells to generate T-cell clones and successfully established 10 T-cell clones from four patients. All the 10 T-cell clones displayed proliferative response against deamidated gluten and to at least one of the four immunodominant epitopes of wheat gluten that were represented in the HLA-tetramer cocktail used during sorting (Figure 1g). We also verified the HLA-tetramer binding of these T-cell clones by restaining them with individual HLA-tetramers used for analysis and sorting (Figure 1h). The results indicate that epitope specificities of gluten-specific CD4+ T cells of patients with DH are similar to those of regular patients with CeD. Clinical parameters and T-cell measurements of the study subjects are summarized in Supplementary Table S1. Furthermore, we investigated the response of CD8+ T cells and γδ T cells. For this purpose, we used the depleted PBMCs after enrichment for HLA-tetramer‒binding cells. Similar to the flow panel used for CD4+ T cells, we stained for CLA and CCR4 to investigate for potential skin homing. After gluten challenge, we observed an increase of CD103+CD38+ CD8+ T cells in six of seven patients (P = 0.02) and an increase of CD103+CD38+ γδ T cells in four of seven patients (Figure 2a and b). Moreover, the increase in the frequency of CD8+ T cells as fold change ranged from 2.2 to 104. Interestingly, patient DH5 had the lowest frequency of CD103+CD38+ CD8+ T cells on day 6, and this was the only patient who continued for the entire 12-months challenge period. CLA was expressed by a substantial proportion of total CD8+ (9‒76%, median = 32%) and γδ T cells (13‒63%, median = 36%). Also among CD103+CD38+ CD8+ T cells, some expressed CLA (range = 0‒51%, median = 13%) (Figure 2c and d). None of the CD103+CD38+ γδ T cells expressed the CLA skin-homing marker. Whether any of the activated CD8+ T cells found in the blood on day 6 after gluten challenge home to skin and exert effector functions there remains to be proven. Although further studies with a higher number of patients and cells are warranted, our study suggests that patients with DH have gluten-induced T-cell responses with similar characteristics to regular patients with CeD. Furthermore, CD38 expression in gluten-specific integrin β7+ CD4+ T cells and CD103+CD38+ CD8+ T cells are promising markers to predict clinical relapse on a short gluten challenge in patients with DH. The data are not publicly available owing to Finnish legislation concerning patient-related data. Louise F. Risnes: http://orcid.org/0000-0002-2580-1678 Markéta Chlubnová: http://orcid.org/0000-0002-9612-3106 Elio Magistrelli: http://orcid.org/0000-0001-7434-5499 Esko Kemppainen: http://orcid.org/0000-0002-5491-5391 Kaisa Hervonen: http://orcid.org/0000-0002-6759-8399 Eriika Mansikka: http://orcid.org/0000-0002-3358-3922 Katri Lindfors: http://orcid.org/0000-0001-7417-5151 Teea Salmi: http://orcid.org/0000-0001-7459-4938 Shiva Dahal-Koirala: http://orcid.org/0000-0002-0165-5098 Ludvig M. Sollid: http://orcid.org/0000-0001-8860-704X The authors state no conflict of interest. We thank Bjørg Simonsen for the production of HLA-DQ2.5:gluten tetramer reagents. The work was supported by grants from Stiftelsen KG Jebsen (project SKGJ-MED-017), the University of Oslo World-leading research program on human immunology (WL-IMMUNOLOGY), the South-Eastern Norway Regional Health Authority (project 2018068), the Academy of Finland (9X051), the Sigrid Juselius Foundation (9AA070) and the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital (9AB068). Conceptualization: LFR, LMS, KL, TS; Formal Analysis: LFR, MC, SDK, LMS; Funding Acquisition: LMS; Investigation: LFR, MC, EMag, SDK; Methodology: LFR, MC, EMag, SDK; Resources: LMS, TS; Supervision: LFR, SDK, LMS; Visualization: LFR, MC, SDK; Writing – Original Draft Preparation: LFR, SDK, LMS; Writing – Review and Editing: LFR, MC, EMag, EK, KH, EMan, KL, TS, SDK, LMS Monomeric HLA-DQ2.5:gluten molecules were multimerized on R-phycoerythrin (PE)-conjugated streptavidin (SA-PE S866, Invitrogen, Waltham, MA). Before tetramer staining, thawed and filtered PBMC samples were incubated at 37 °C for 10 minutes in a buffer containing 50 nM dasatinib (Sigma-Aldrich, St. Louis, MO). The samples were then directly stained with a mixture of five PE-conjugated HLA-DQ2.5:gluten tetramers representing the epitopes DQ2.5-glia-α1a, DQ2.5-glia-α2, DQ2.5-glia-ω1, DQ2.5-glia-ω2, and DQ2.5-hor3 (10 μg/ml of each tetramer) for 45 minutes at room temperature. Tetramer-binding cells were further enriched using anti-PE magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). After washing, we added 100 μg/ml human IgG (Sigma-Aldrich) for Fc blocking, Live/dead (Violet) marker, and 0.25 μg anti-PE (clone PE001) unconjugated antibody to the enriched samples for 10 minutes on ice before antibody staining. The samples were stained with a surface antibody mixture for 20 minutes on ice. For tetramer restaining of T-cell clones (TCCs), we stained with three different mixtures of HLA-DQ2.5:gluten tetramers consisting of one PE-conjugated and one allophycocyanin (APC)-conjugated tetramer to determine the epitope specificity of each TCC (indicated in Figure 1g). We designed the following 13-color flow panel for HLA-tetramer‒stained samples used for cell sorting: CCR4-AlexaFluor488 (clone 205410, R&D System, Minneapolis, MN), CD62L-PerCP-Cy5.5 (clone DREG-56, BioLegend, San Diego, CA), CD3-BV510 (clone OKT3, BioLegend), CLA-BV605 (clone HECA-452, BD Bioscience, San Jose, CA), Integrin β7-BV650 (clone FIB504, BD Bioscience), CD127-BV711 (clone A019D5, BioLegend), PD-1-PE-CF594 (clone EH12.1, BD Bioscience), CD39-PE-Cy7 (clone A1, BioLegend), CD38-APC (clone HB-7, BioLegend), CD45RA-AlexaFluor700 (clone HI100, BioLegend), and CD4-APC-Cy7 (clone SK3, BioLegend). We included a dump channel consisting of CD11c-V450 (clone B-Ly6, BD Bioscience), CD14-Pacific Blue (clone M5E2, BD Bioscience), CD19-Pacific Blue (clone HIB19, BioLegend), and CD56-Pacific Blue (clone MEM-188, BioLegend). The depleted PBMC samples after tetramer enrichment were stained with a 12-color flow panel with the following antibodies: CCR4-Alexa Fluor 488, CD8-PerCP (clone SK1, BioLegend), CD3-BV510, CLA-BV605, Integrin β7-BV650 (clone FIB504, BD Bioscience), CD27-BV711 (clone M-T271, BioLegend), γδTCR-PE (clone 5A6.E9, Invitrogen), CD103-PE-Cy7 (clone B-Ly7, eBioscience, San Diego, CA), CD38-APC (clone HB-7, BioLegend), CD45RA-Alexa Fluor 700, and CD39-APC-Fire750 (clone A1, BioLegend). In this study, we included a dump channel consisting of CD62L-PacificBlue (clone DREG-56, BioLegend), CD11c-V450, and CD14-Pacific Blue. The flow analyses were performed on Aria III cell sorter (BD Biosciences) for the tetramer-stained samples and BD Fortessa for the analyses of CD8+ and γδ T cells, at the Flow Cytometry Core Facility at Oslo University Hospital (Oslo, Norway). Data analysis was done with FlowJo software (FlowJo LLC, Ashland, OR). The number of HLA-DQ2.5:gluten tetramer‒positive effector-memory T cells was normalized to the number of million CD4+ T cells. Total PBMC was counted before enrichment, and the frequency of CD4+ T cells was analyzed in a pre-enriched sample. TCCs were generated by limiting dilution and antigen-free expansion. HLA-tetramer binding CD4+ T cells were sorted in a tube containing feeder mixture, which contains irradiated PBMCs (1 million/ml, 60 Gy) of three healthy donors in 10% human serum/RPMI medium with penicillin/streptomycin, phytohemagglutinin (1 μg/ml), IL-2 (20 IU/ml), and IL-15 (1 ng/ml). This T cell/feeder mixture suspension was cultured on Terasaki plates for 10 days. Subsequently, growing TCCs were transferred to 48-well plates containing 500 μl of feeder mixture and cultured for additional 10 days. Proliferative response of TCCs to transglutaminase 2‒deamidated CT-gluten and five immunodominant epitopes, namely DQ2.5-glia-α1a-epitope peptide (QLQPFPQPELPY, underlined 9-mer core sequence) (GenScript Biotech, Piscataway, NJ), the DQ2.5-glia-α2-epitope (PQPELPYPQPQL) (Research Genetics, Huntsville, AL), the DQ2.5-glia-ω1 epitope (PQQPFPQPEQPFP), the DQ2.5-glia-ω2 epitope (FPQPEQPFPWQP), and the DQ2.5-hor3a epitope (PEQPIPEQPQPYPQQP) (all three from GenScript Biotech), was measured in triplicates using thymidine incorporation assay. On the first day, antigen-presenting cells (Epstein-Barr virus‒immortalized B-cell line from HLA-DQ2.5 homozygous patients with Crohn's disease) irradiated at 75 Gy were incubated with gluten (10 μg) and epitopes (10 μM) on 96-well plate overnight at 37 °C. On the second day, TCCs were added to the plate and again incubated overnight at 37 °C. On the third day, 3H-thymidine (Hartman Analytics, Braunschweig, Germany) (1 μCi/well [0.037 MBq/well]) was added to each well. After 16 hours, the radioactivity of 3H-thymidine taken up by proliferating cells was measured by liquid stimulation counting (Wallac MicroBeta TriLux 1450, PerkinElmer, Waltham, MA) as counts per minute. The experiments were performed twice, and a representative experiment is shown.Supplementary Table S1Clinical Parameters and T-Cell Measurements of Study SubjectsPatients with DH1234567GFD before challenge (mo)824204022522Relapse (mo)43361214Skin rash at relapseyesyesyesyesnoyesnoSkin IgA at BLnegnegnegnegnegposnegSkin IgA at relapsenegposposposnegposnegSerum TG2 at BL (≥3.0 AU/ml)negnegnegnegnegnegnegSerum TG2 at relapse>100>1003.1negnegneg54.2Serum TG3 at BL (≥30 AU/ml)302333<2.3440Serum TG3 at relapse>189>189>1896372489Vh:CrD ratio at BL4.52.13.72.72.22.82.2Vh:CrD ratio at relapse10.40.80.70.81.31Events of HLA-tetramer‒positive TEM cells (BL)4809n.d.n.d.n.d.Events of HLA-tetramer‒positive TEM cells (D6)171105121620n.d.n.d.Fold change CD103+CD38+ CD8+ T cells (D6/BL)4.8104.381.12.2—395.8Fold change CD103+CD38+ γδ T cells (D6/BL)5.65.12.61—94.51.9Abbreviations: AU. Arbitrary unit; BL, baseline; CrD, crypt depth; D6, day 6; n.d., not determined; neg, negative; pos, positive; TEM, effector-memory T cell; TG2, transglutaminase 2; Vh, villus height. Open table in a new tab Abbreviations: AU. Arbitrary unit; BL, baseline; CrD, crypt depth; D6, day 6; n.d., not determined; neg, negative; pos, positive; TEM, effector-memory T cell; TG2, transglutaminase 2; Vh, villus height.