Extended Data Fig. 3: Functional characteristics of PD1-expressing aortic T cells.
IfngYFP/YFPApoe−/− mice were fed a high-cholesterol diet (12-16 weeks per experiment) and Ki67 expression and T-cell cytokine co-produciton was analyzed. (a-b) Digested whole-aortas were pooled (n = 2/pool; 8 pools total) and analyzed for proliferation along with splenocytes (n = 16). (a) Representative flow cytometry plots of Ki67-staining in naïve (CD44−) and PD1 subsets of aortic CD4 and CD8 aortic T cells. (b) Quantification of high Ki67 expression within PD1 subsets of splenic CD4 (top-down, ****p = 2.3E-5, 3E-6, 4.8E-5) and CD8 T cells (****p = 4E-8, 2E-6, **p = 0.0074). (c-e) Digested and pooled whole-aortas (n = 3-4/pool, 3 pools total) and spleens (n = 11) were stimulated ex vivo with PMA/ionomycin with Brefeldin A for 4 hr followed by flow cytometry. (c) Flow cytometry plot of enrichment of IFN-γ-APC expression in stimulated PD1+ aortic CD4 and CD8 T cells. (d) Frequency of co-production of IFN-γ (measured here with anti-IFN-γ-APC antibody) and IL-2 or IFN-γ and TNF-α by stimulated aortic CD4 and CD8 PD1 subsets. (e) Analysis of cytokine production and co-production of IFN-γ (measured here with anti-IFN-γ-APC antibody), TNF-α, and IL-2 with stimulated splenic PD1 CD4 and CD8 T cell subsets. (f-g) Frequency of Tox expression within aortic CD4 PD1 subsets and CD8 PD1 subsets (**p = 0.0046). (b) Bars denote median, analyzed with two-sided Kruskal Wallis test. (h-i) Quantification of PSGL1 and Ly6C on aortic IFN-γ+ CD4 T cell PD1-subsets. (j-k) Quantification of KLRG1 and IL7Rα on aortic IFN-γ+ CD8 T cell PD1-subsets. Representative flow cytometry plots of aorta-draining lymph node and aorta. (f-g) Bars denote mean, analyzed with two-sided one-way ANOVA test.
