Fig. 4: C19S-specific CD4⁺ T cells exhibit distinct transcriptional states marked by peripheral activation and clonal expansion.

a, Representative flow cytometry plot (left) showing co-staining with InsB_12-20:I-A^g7 and InsB_12-20(C19S):I-A^g7 tetramers, revealing two distinct, register-specific populations. The bar graph (right) summarizes the frequencies of the tetramer-binding populations (n = 12). b, t-distributed stochastic neighbor embedding (t-SNE) plot showing T cell clusters merged from InsB_12-20-specific, InsB_12-20(C19S)-specific and Tet^⁻ CD4⁺ T cells (n = 4,787). c, Feature plots showing the expression of functional genes classifying CD4⁺ T cell clusters. d, t-SNE plots (upper) showing cluster distribution for InsB_12-20:I-A^g7 (n = 719) and InsB_12-20(C19S):I-A^g7 (n = 1,256) tetramer-binding CD4⁺ T cells. The bar graph (lower) shows the relative frequency of each cluster. e, Heat map showing the expression of indicated T cell activation and cycling genes among all clusters. f, GSEA showing enrichment of cluster 5 for memory T cell signatures from dataset GSE9650. g, GSEA showing enrichment of cluster 6 for effector T cell signatures from dataset GSE9650. h, GSEA showing enrichment of both clusters 5 and 6 for intra-islet, terminally activated CD4⁺ T cells depicted in dataset GSE262101. i, Pie charts showing clonal overlap of TCRs identified in tetramer-negative, InsB_12-20:I-A^g7 and InsB_12-20(C19S):I-A^g7 tetramer-binding CD4⁺ T cells. Clonal overlap was defined as TCRs with identical amino acid sequences in the full CDR3 regions of both α and β chains. j, Representative flow cytometry plots (left) showing indicated activation markers on InsB_12-20:I-A^g7 and InsB_12-20(C19S):I-A^g7 tetramer-binding CD4⁺ T cells in SLOs of 8-week-old female NOD mice. The dot plots (right; mean ± s.e.m.) summarize the frequencies of CD44^hiCD62L⁻ (n = 14), CD44^hiCD11a^hi (n = 22) and CD44^hiCXCR6⁺ (n = 23) cells in the tetramer-binding T cells. In a and j, n is the number of mice; each dot is one mouse. In b and d, n is the number of cells; each dot is one cell. For statistical analysis, two-tailed paired t-test was performed for a. One-way ANOVA with Dunnett’s multiple comparisons test was performed for j. The data represent two (b–i) and 6-10 (a, j) independent experiments.

Source data