Extended Data Fig. 6: Impact of anti-PD1 blockade on plaque inflammation and plasma chemokines/cytokines.
IfngYFP/YFPApoe−/− mice were fed a high-cholesterol diet for 10 weeks before administration of bi-weekly i.p. injections of a murine blocking anti-PD1 antibody or isotype IgG control (n = 12/group) for 6 weeks. (a) Quantification of CD3+ cells in adventitia of aortic-root sections. Adventitia designated as 100 μm from smooth muscle layer of aortic valve. (b) Isotype IgG staining for CD3 immunohistochemical analysis of aortic root cross-sections. (c) Quantification of T cells (TCRβ+CD45+) per whole-aorta digest analyzed by flow cytometry (*p = 0.010). (d-h) In the short-term anti-PD1 cohort, IfngYFP/YFPApoe−/− mice were fed a high-cholesterol diet for 21 weeks before administration of bi-weekly i.p. injections of anti-PD1 antibody or isotype IgG control (n = 11-12/group). Mice received a total of six injections and were terminated after a total of 24 weeks of diet. (e) Average plaque area determined by Oil Red O staining of aortic subvalvular cross-sections. (f-g) Quantification and representative image of immunohistochemical analysis of CD3+ cells in aortic root plaques (***p = 0.0002). (h) Representative immunohistochemical staining images and confocal images of CD3 and CD19 staining of advanced adventitial foci. (i) Measurements of MC38 tumor diameter in HCD-fed IfngYFP/YFPApoe−/− mice with (n = 5) and without (n = 7) PD1 blockade. Data presented as mean values +/- SD. (j-k) Representative images of immunohistochemical staining of neutrophils (Ly6G) and macrophages (CD68) in IfngYFP/YFPApoe−/− mice treated with PD1 blockade or isotype IgG control for 6 weeks (n = 12/group). (l-p) Concentration of plasma cytokines CCL4 (*p = 0.023), IL-6, TNF-α, IL-4 (***p = 0.0009), and CCL2 (**p = 0.0013) in IfngYFP/YFPApoe−/− mice treated with anti-PD1 or isotype IgG control for 6 weeks. (a, c, f, m-p). Bars denote median, analyzed with two-sided Mann-Whitney U test. (e, l) Bars denote mean, analyzed with two-sided unpaired t test.
