Fig. 5: scRNA-seq analysis reveals distinct signatures in CD4⁺ T cells specific for epitopes presented in the thymus versus pancreatic islets.

a Tetramer-binding CD4⁺ T cells for indicated epitopes in SLOs of 6–8-week-old female NOD mice. The FACS plots depict double-positive tetramer-binding populations (gated on CD45⁺B220⁻CD8⁻CD11b⁻I-A^g7−Thy1.2⁺CD4⁺) in cells with magnetic enrichment. The dot plot shows the numbers of tetramer-binding T cells for each epitope (mean ± s.e.m) quantified from 4 to 8 individual mice in four independent experiments (n = 4: 2.5HIP, n = 4: 6.9HIP, n = 4: CP1d, n = 8: G20, n = 7: E21, n = 4: CP2, n = 4: DBP, n = 4: ZnT8). b tSNE plots (upper) showing cellular heterogeneity of indicated T cell populations. The pie charts (lower) depict the composition of different transcriptional clusters in the three T cell populations. The data are merged from two independent scRNA-seq analyses of tetramer-binding CD4⁺ T cells sourced from SLOs of 6–8-week-old female NOD mice. T cells for the thymic and islet epitope set were sorted from 24 and 10 mice included in two independent experiments, respectively. c Violin plots depicting function-related genes that were differentially expressed among CD4⁺ T cell clusters (Benjamini–Hochberg correction for multiple tests). For each gene, the Log2 expression level range is 0–5. d Signature genes that were upregulated in the two early effector clusters compared to the naïve T cell cluster. All the genes described in this Figure meet the statistical cut-off (adjusted P < 0.05) using the Benjamini–Hochberg correction for multiple tests. e GSEA analysis of the early effector and naïve T cell clusters identified in this study using a previous dataset depicting intermediate priming of a G20-specific TCR transgenic T cell (8F10) sourced from inguinal lymph nodes of WT NOD or NOD.B16A mice. The weighted Kolmogorov–Smirnov statistic was performed. The data show that genes upregulated in the early effector cluster were significantly enriched for genes upregulated in 8F10 T cells from the antigen-replete NOD condition, whereas the naïve T cell cluster resembled 8F10 T cells sourced from the antigen-delete NOD.B16A condition. f Signature genes representing the T_H1-like cluster as compared to the naïve cluster, based on nonparametric Wilcoxon rank sum test with Benjamini–Hochberg correction for multiple testing.