Extended Data Fig. 2: Effects of CCR4 deficiency on atherosclerosis and CCL17-induced migration via CCR4.
(a) Experimental scheme of Apoe−/− or Apoe−/−Ccr4−/− mice fed a Western-diet (WD) for 12 weeks; (b, c) Flow cytometric quantification of CD45+CD3+CD4+CD25+FoxP3+ Tregs, axillary (b) and inguinal LNs (c) of Apoe−/− (b, n = 13; c, n = 16) or Apoe−/−Ccr4−/− (b, n = 18; c, n = 20) mice after 12 weeks of WD; (d-f) Quantification of the percentage of lesional Mac2+ macrophages (d), SMA+ SMCs (e) and collagen content (f) in Apoe−/− (d, e n = 20; f, n = 19) or Apoe−/−Ccr4−/− (d, n = 19; e, n = 20; f = 16) mice fed a WD for 12 weeks; (g) Analysis of Annexin-V (AnnV) expression on Tregs (CD45+CD3+CD4+CD25+FoxP3+) from isolated LNs (para-aortic, axillary and inguinal combined) of Apoe−/− (n = 8) or Apoe−/−Ccl17e/e (n = 6); (h) Transwell migration assay with CD4+ T cells (isolated from Apoe−/− mice) towards recombinant mouse CCL17 (100 ng/ml) or CCL22 (50 ng/ml) in the presence or absence of the CCR4 inhibitor C021 dihydrochloride (0.5 µM), number of replicates in parentheses over number of independent experiments per bar from left to right: n = (40)8, (40)8, (27)6, (27)6, (25)5, (25)5; (i) Transwell migration assay with CD4+ T cells (isolated from human blood PBMCs) towards recombinant human CCL17 (100 ng/ml) or CCL22 (50 ng/ml), in the presence or absence of the CCR4 inhibitor C021 dihydrochloride (0.5 µM), number of replicates in parentheses over number of independent experiments per bar from left to right: n = (37)7, (33)7, (31)5, (28)5, (16)4, (16)4; (h,i) All migrated cells were quantified by flow cytometry, chemotactic index calculated as the ratio of chemokine-stimulated and unstimulated migration is depicted; (a-i) Data represent mean ± SEM. Two-sided P values as indicated and analyzed by unpaired Student’s t-test, Mann-Whitney U (b-h), or nested ANOVA with Holm-Å Ãdák’s post hoc test (h, i).
