Extended Data Fig. 1: Intra-Islet CD8⁺ T cells upregulate markers of exhaustion but are a heterogeneous population.

Phenotypic quantification of exhaustion markers in the NOD model of diabetes. (a-f) Spectral flow cytometry for CD8⁺ T cell functional markers was completed over a time course of 6-14-week-old female WT NOD mice. Representative flow plots are derived from total intra-islet CD8⁺ T cells (gated on lymphocytes, single cells, Live, Thy1.2⁺, CD8b⁺) of 12-week-old female NOD. Data were accumulated from a total of 5 experiments, each experiment had mice of several ages, with n = 10 mice per timepoint, n = 50 total mice. Each point on the graph is representative of a single mouse. (a) Representative flow plotting demonstrating gating strategy to obtain CD8⁺ T cells. (b) High dimensional analysis at 12 weeks of age was preformed using Cytobank³⁸ viSNE map analysis (Methods). viSNE maps are shown portraying the 11 markers are used to create FlowSOM clustering analysis. (c) Representative flow plots of intra-islet CD8⁺ T cell PD1 and LAG3 (6 vs 12 weeks p = .0355), TIGIT (6 vs 12, 14 weeks p = .0005, 0.0011), TIM3, CTLA4, and ICOS expression are shown islets and expression of IRs are quantified from the ndLN, pLN, and islets. (d) Co-expression of multiple IRs and the transcription factor TOX are represented in Simplified Presentation of Incredibly Complex Evaluations (SPICE)⁶³ plots showing bulk CD8⁺ T cells from 6- and 12-week-old islet samples. (e) Representative flow plots and quantification of bulk intra-islet CD8⁺ T cell expression of TCF1 and TOX populations. (TCF1–TOX + 6 vs 12, 14 weeks, p = 0.0029, 0.0021) (f) % TOX⁺ correlation to PD1 (p < .0001), TIGIT (p < .0001), and LAG3 (p < .0001). Pearson’s correlation coefficients and r² values were calculated. (c and e) A two-sided nonparametric Mann-Whitney was preformed. Graphs portray the median. P = * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Unlabeled indicates not statistically significant.

Source data