Fig. 5: Epitope availability and inflammatory cues regulate peripheral activation, tissue infiltration and diabetogenic potential of C19S-specific CD4⁺ T cells.

a, IL-2 production by 9B9 and S5 T cells in response to InsB_9-23, InsB_9-23(C19S) and InsB_9-23(Y16A) peptides. b, Bar graph (mean ± s.e.m.) summarizing the frequencies of InsB_12-20(C19S):I-A^g7 tetramer-binding CD4⁺ T cells in SLOs of 8-week-old female NOD (n = 5) and NOD.B16A (n = 6) mice. c, Representative flow cytometry plot (left) and bar graph (right; mean ± s.e.m.) showing the percentage of CD44^hiCD62L⁻ T cells within InsB_12-20(C19S):I-A^g7 tetramer-binding CD4⁺ T cells in SLOs of 8-week-old female NOD (n = 5) and NOD.B16A (n = 6) mice. d–f, Representative flow cytometry plots (left) and bar graphs (right; mean ± s.e.m.) showing the expression of indicated activation markers on InsB_12-20(C19S):I-A^g7 tetramer-binding CD4⁺ T cells in SLOs of 8-week-old female NOD (n = 9) and NOD.Tnfrsf1a/1b^⁻/⁻ (n = 8) mice. g, Feature plots illustrating the expression of CD44, CD11a and CXCR3 on InsB_12-20:I-A^g7 versus InsB_12-20(C19S):I-A^g7 tetramer-binding CD4⁺ T cells from SLOs of diabetic NOD mice. Shown are equal numbers of tetramer-binding T cells for either epitope merged from eight mice. h, Bar graph (mean ± s.e.m.) showing the frequencies of CD44^hiCD62L^⁻ cells within tetramer-negative (n = 21), InsB_12-20(C19S):I-A^g7 (n = 8), CP1d:I-A^g7 (n = 7), 6.9HIP:I-A^g7 (n = 8) or 2.5HIP:I-A^g7 (n = 6) tetramer-binding CD4⁺ T cells in SLOs from 8-week-old female NOD mice. i, Representative flow cytometry plot (left) showing co-staining of intra-islet CD4⁺ T cells with the InsB_12-20(C19S):I-A^g7 (n = 7) tetramer, along with the 6.9HIP:I-A^g7 (n = 4) or 2.5HIP:I-A^g7 (n = 3) tetramer. The bar graph (right; mean ± s.e.m.) summarizes the frequencies of indicated tetramer-binding cells among intra-islet CD4⁺ T cells. j, Percentage of activated CD44^hiCD62L⁻ and CD44^hiCD11a^hi T cells among indicated intra-islet tetramer-negative (n = 3), InsB_12-20(C19S):I-A^g7 (n = 3), or 2.5HIP:I-A^g7 (n = 3) tetramer-binding CD4⁺ T cells. In b, c–f, h–j, n is the number of mice; each dot is one mouse. For statistical analysis, two-tailed Mann-Whitney tests were performed for b–f. One-way ANOVA with Dunnett’s multiple comparisons test was performed for h. One-way ANOVA with Tukey’s multiple comparisons tests were performed for i and j. The data in all panels represent at least three independent experiments.

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