Extended Data Fig. 4: A subset of intra islet CD8⁺ T cells upregulate markers of exhaustion, as well as effector cell markers.

(a-d) Spectral flow cytometry for CD8⁺ T cell functional markers was completed and representative flow plots and graphs appear as described in Extended Data Fig. 1, with the added sub gate of PD1⁺ and PD1^–. (a) representative flow plot of intra-islet CD8⁺ T cells PD1 expression. (b) Representative flow plots and quantification of LAG3 and TIGIT expression on PD1⁺ and PD1^– intra-islet CD8⁺ T cells. (LAG3⁺TIGIT⁺: PD1⁺ vs PD1^– 6, 8, 10-14 weeks p = .0005, 0.000002, <.000001, 6 vs 12, 14 weeks PD1⁺ p = .0039, 0.0065. LAG3^–TIGIT^–: PD1⁺ vs PD1^– 6, 8-14 weeks p = .000174, <.000001, 6 vs 10, 12, 14 weeks PD1⁺ p = .028, 0.006, 0.005). (c) Representative flow plots of TCF1 and TOX staining on PD1⁺ and PD1^– intra-islet CD8⁺ T cells. (d) quantification of (c) (TCF1⁺TOX^–: PD1⁺ vs PD1^– p = .000011 at all time points, 6 vs 10, 12, 14 weeks PD1⁺ p = .0027, 0.0019, 0.0064. TCF1⁺TOX⁺: PD1⁺ vs PD1^– p = <.000001 at all timepoints. TCF1^–TOX⁺: PD1⁺ vs PD1^– 6, 8-14 weeks p = .000262, <.000001, 6 vs 8, 10, 12, 14 weeks p = .0355, 0.0355, 0.0147, 0.0014). (e-f) Total intra-islet CD8⁺ T cells from 12-week-old female WT NOD mice were analyzed by spectral flow cytometry including ndLN and pLN controls (n = 10, 2 independent experiments). (e) Representative flow plots (islets) and quantification of CD73 and CD39 expression (CD73⁺: islets vs ndLN, pLN p = <.0001, 0.06. CD39⁺CD73⁺: islets vs ndLN, pLN, p = <.0001, 0.0142. CD39⁺: islets vs ndLN, pLN, p = <.0001, 0.0315). (f) Representative flow plots (islets) and quantification of Tbet and Eomes expression (Tbet⁺: islets vs ndLN, pLN, p = .0056, 0.0003. Eomes⁺: islets vs ndLN, pLN, p = .0005, <.0001). (g-i) intra-islet CD8⁺ T cells were isolated and from 12-week-old female WT NODs and analyzed by flow cytometry for metabolic markers or cytokines (n = 10, 2 independent experiments, techniques described in methods) (g) intra-islet CD8⁺ T cells are stained for TMRM (islets vs ndLN, pLN p = <.0001, 0.0106), (h) MitoSOX (islets vs. ndLN and pLN, p < .0001) and CellROX (islets vs ndLN p = .0019), and for (i) cytokine production. (a-i) Each data point corresponds to a single mouse. A two-sided nonparametric Mann-Whitney was preformed, where P = * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Unlabeled indicates not statistically significant. Graphs portray the median.

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