Circulating gluten‐specific, but not CMV‐specific, CD39+ regulatory T cells have an oligoclonal TCR repertoire

Figures

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  Figure 1. TCR repertoire of gluten‐peptide‐specific CD39+ T cells. (a) PBMCs were isolated from n = 3 coeliac disease patients (#0174, #0512 and #0251) 6 days following oral gluten challenge, stimulated with gluten peptide DQ2.5‐glia‐α1/α2 for 44 ‐h and the antigen‐specific CD4+CD25+OX40+CD39+ Tregs sorted. (b) The TCR clonotype repertoires of the CD4+CD25+OX40+CD39+ Treg populations are shown along with the number of clonotypes analysed for each patient. Coloured segments indicate clonotypes (including variants of these clonotypes that differed by a single residue in the CDR3 region) detected in more than one individual (this study and Qiao et al. 10), and clonotype sequences are in Table 1. (c) TCR clonotype repertoires of unstimulated total CD39+ Tregs sorted from coeliac disease patients #0174 and #0512 prior to gluten challenge, and CD4+ cells not specific for gluten peptides from coeliac patient #0251 that were sorted from an OX40 assay as CD25−OX40− cells post‐gluten challenge (n = number of clonotypes analysed for each patient). Clonotype sequences are in Supplementary tables 2–4; data are from a single experiment for each patient.

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  Figure 2. TCR repertoire of CMV‐peptide‐specific CD39+ and CD39− CD4+ T cells. For n = 3 healthy donors, PBMCs were stimulated with 10 μg mL−1 antigen for 44 h and the antigen‐specific CD4+CD25+OX40+ cells sorted into CD39− cells and CD39+ Tregs, as shown in the FACS plots. TCR repertoire was assessed by 5′RACE and Sanger sequencing, and the percentage usage of each TCR Vβ family is shown for (a) donor 1 CMV‐P1‐specific cells, (b) donor 2 CMV‐P1‐specific cells and (c) donor 3 CMV‐P4‐specific cells. The number of TCR clonotype sequences analysed is represented by n for each subset. There was insufficient CMV‐P4‐specific CD39− cells sorted from donor 3 to enable analysis. Clonotype sequences are in Supplementary tables 5–9; data are from a single experiment for each donor.

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  Figure 3. Effect of antigen concentration on proportions of CD39+ Tregs in recall responses. (a) For CMV‐P1 and CMV‐P4 responses, the proportion of CD39+ Tregs and CD39− Tconv cells are shown for a log dilution of antigen concentrations from 10 to 1 μg mL−1. Data represent 3–4 independent experiments for each donor, median ± interquartile range. Black arrows indicate the CMV‐P1 concentrations used to sort higher and lower avidity responses for donor 1. (b) Percentage usage of TCR Vβ families is shown for CD39+ cells and CD39− cells responding to 2.5 and 10 μg mL−1 of CMV‐P1 from donor 1. The number of TCR clonotype sequences analysed is shown (Supplementary tables 5, 6, 10 and 11); data are from a single experiment.