Fig. 4: Aortic PD-1+ T cells express CD69 and show signs of recent TCR signaling. | Nature Cardiovascular Research

Fig. 4: Aortic PD-1+ T cells express CD69 and show signs of recent TCR signaling.

From: Progenitor exhausted PD-1+ T cells are cellular targets of immune checkpoint inhibition in atherosclerosis

Fig. 4

a–c, TCR signaling reporter mice (Nur77wt/GFPApoe−/−) were generated and fed an HCD for 12 weeks, and, at euthanization, T cell phenotype was analyzed (n = 9). a, Flow cytometry plots of Nur77–GFP expression on aortic T cells. b, MFI of Slamf6 (**P = 0.004), PD-1 (**P = 0.0019) and Tim3 (***P = 0.0002) of Nur77− and Nur77+ (recent TCR activation) aortic CD4. c, MFI of Slamf6 (****P = 9.2 × 10−5), PD-1 and Tim3 (*P = 0.01) of Nur77− and Nur77+ aortic CD8 T cells. d, Flow cytometry plots of CD69 and CD103 expression on aortic CD8 T cells of IfngYFP/YFPApoe−/− mice fed an HCD for 14 weeks (n = 9). e,f, Quantification of percent CD69+ T cells (CD4 ****P = 8.5 × 10−5, *P = 0.049; CD8 ****P = 5.6 × 10−9, ***P = 0.0004) and CD69+CD103+ T cells (CD4 *P = 0.012, CD8 *P = 0.011) within PD-1 subsets in the aorta. g,h, Flow cytometry analyzing CD69 and Nur77–GFP expression within PD-1 subsets of aortic CD4 T cells from IfngYFP/YFPNur7wt/GFPApoe−/− mice fed an HCD for 10 weeks (n = 6). Quantification of non-activated CD4 TRM-like cells (CD69+Nur77–GFP−) and recently activated CD4 T cells (CD69−Nur77–GFP+ and CD69+Nur77–GFP+). i–j, Flow cytometry analyzing CD69 and Nur77–GFP expression within PD-1 subsets of aortic CD8 T cells. k–m, In a separate cohort, IfngYFP/YFPApoe−/− mice were fed an HCD for 10 weeks before receiving intraperitoneal injections of anti-IL-2 antibodies or control IgG (n = 9 per group) for 2 weeks. k,l, Flow cytometric analysis of aortic memory T cells to assess composition of PD-1 subsets and subsets of cells expressing Slamf6 and/or Tim3. m, Flow cytometric analysis of IFNγ production by aortic CD44+CD4 and CD44+CD8 T cells. b,l, Bars denote median, analyzed with two-sided Mann–Whitney U-test. c,m, Bars denote mean, analyzed with two-sided unpaired t-test. e,f,h,j, Bars denote median, analyzed with two-sided Kruskal–Wallis test. h,j, P values compared to PD-1− subset (CD4 PD-1int **P = 0.0088, CD4 PD-1high *P = 0.0016, CD8 PD-1int *P = 0.031, CD8 PD-1high ***P = 0.0005). act, activated; Ctrl, control.

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