C-C chemokine receptor 4 (CCR4) deficiency accelerates the development of early atherosclerotic lesions characterized by an inflammatory plaque phenotype.

A, Representative photomicrographs of Oil Red O staining and quantitative analysis of atherosclerotic lesion area at 5 different levels and the average area in the aortic sinus of 18-week-old apolipoprotein E-deficient (Apoe−/−) mice (n=24) or CCR4-deficient mice on an Apoe−/− background (Ccr4−/−Apoe−/−; n=23). B, Representative photomicrographs of Oil Red O staining and quantitative analysis of atherosclerotic lesion area in the aorta of 18-week-old Apoe−/− (n=15) or Ccr4−/−Apoe−/− mice (n=15). C-E, Representative sections and quantitative analyses of MOMA-2+ macrophages (C), CD4+ T cells (D), and collagen (E) in the aortic sinus. Arrowheads indicate the CD4+ T cells. n=10 per group. F, mRNA expression of pro- or anti-inflammatory cytokines and helper T cell-associated transcription factors in aorta. The expression levels of the target genes were normalized so that the mean values in Apoe−/− mice were set to 1. n=8 to 10 per group. Eighteen-week-old Apoe−/− or Ccr4−/−Apoe−/− mice were used for all experiments. Black bars represent 50, 200, or 500 μm as described. Data points represent individual animals. Horizontal bars represent means. Error bars indicate s.d. *P<0.05, **P<0.01; Mann-Whitney U-test: A and F Il1b; 2-tailed Student’s t-test: C, D, E, and F Il6, Tbx21, and Rorc.

C-C chemokine receptor 4 (CCR4) deficiency augments effector T cell immune responses in peripheral lymphoid tissues.

A and B, Representative flow cytometric analysis of CD4+ forkhead box P3 (Foxp3)+ regulatory T cells (Tregs) (A) and CD4+CD44highCD62Llow effector memory T cells (B) in the spleen of 8-week-old apolipoprotein E-deficient (Apoe−/−) mice or CCR4-deficient mice on an Apoe−/− background (Ccr4−/−Apoe−/−). The graphs represent the total numbers and proportions of CD4+Foxp3+ Tregs (A) and CD4+CD44highCD62Llow effector memory T cells (B) in the peripheral lymph nodes (LNs) and spleen of 8- or 18-week-old Apoe−/− or Ccr4−/−Apoe−/− mice. n=9 to 10 per group. C and D, The graphs represent the proportions of Ki-67 positive cells among CD4+Foxp3+ Tregs (C) and CD4+Foxp3− non-Tregs (D) in the peripheral LNs and spleen of 8-week-old Apoe−/− or Ccr4−/−Apoe−/− mice, as assessed by flow cytometry. n=13 per group. E, Expression levels of activation-associated molecules cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and CD103 were analyzed by gating on CD4+Foxp3+ Tregs in the peripheral LNs of 8- or 18-week-old Apoe−/− or Ccr4−/− Apoe−/− mice. n=9 to 10 per group. F, mRNA expression of Treg-associated markers in splenic Tregs from 8-week-old Apoe−/− or Ccr4−/−Apoe−/− mice. n=8 per group. G, Expression levels of activation-associated molecules CTLA-4 and CD103 were analyzed by gating on CD4+Foxp3− non-Tregs in the peripheral LNs of 8- or 18-week-old Apoe−/− or Ccr4−/−Apoe−/− mice. n=9 to 10 per group. H, mRNA expression of activation-associated molecules in splenic non-Tregs from 8-week-old Apoe−/− or Ccr4−/−Apoe−/− mice. n=8 per group. The expression levels of the target genes were normalized so that the mean values in Apoe−/− mice were set to 1 (F, H). Data points represent individual animals. Horizontal bars represent means. Error bars indicate s.d. *P<0.05, **P<0.01; Mann-Whitney U-test: A second (8w) from the left, B second (8w) and third (8w) from the left, C left, and H Cd44 and Cd103; 2-tailed Student’s t-test: A first, second (18w), and third from the left, B first, second (18w), third (18w), and fourth from the left, C right, D, E, G, and H Ctla4. MFI indicates mean fluorescence intensity.

C-C chemokine receptor 4 (CCR4) deficiency promotes proinflammatory CD4+ T cell immune responses in peripheral lymphoid tissues.

A and B, The graphs represent the frequencies of interferon (IFN)-γ+, interleukin (IL)-4+, IL-10+, and IL-17+ CD4+ T cells in the peripheral lymph nodes (LNs) (A) and spleen (B) of 8-week-old apolipoprotein E-deficient (Apoe−/−) mice or CCR4-deficient mice on an Apoe−/− background (Ccr4−/−Apoe−/−). n=10 to 11 per group. C and D, Purified splenic CD4+ T cells from 8-week-old Apoe−/− or Ccr4−/−Apoe−/− mice were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in vitro. The levels of various cytokines and chemokines in pooled cell supernatants from 8 mice in each group were determined semiquantitatively by a cytokine array kit (C). Data are representative of two independent experiments. Cytokine concentrations in the cell supernatants were measured by ELISA (D). n=8 per group. Data points represent individual animals. Horizontal bars represent means. Error bars indicate s.d. *P<0.05, **P<0.01; Mann-Whitney U-test: A, B first from the left, and D second from the left; 2-tailed Student’s t-test: B fourth from the left and D first, third, and fourth from the left.

C-C chemokine receptor 4 (CCR4) deficiency promotes T helper type 1 (Th1) cell responses in para-aortic lymph nodes (LNs) and atherosclerotic aorta.

A and B, Representative flow cytometric analysis of CD4+ forkhead box P3 (Foxp3)+ regulatory T cells (Tregs) (A) and CD4+CD44highCD62Llow effector memory T cells (B) in para-aortic LNs. The graphs represent the total numbers and proportions of CD4+Foxp3+ Tregs (A) and CD4+CD44highCD62Llow effector memory T cells (B) in para-aortic LNs. n=9 to 10 per group. C and D, mRNA expression of Treg-associated markers in Tregs (C) and mRNA expression of activation or helper T cell-associated molecules in non-Tregs (D) in para-aortic LNs. The expression levels of the target genes were normalized so that the mean values in apolipoprotein E-deficient (Apoe−/−) mice were set to 1. Tregs or non-Tregs purified from pooled para-aortic LNs of 9 to 10 mice were analyzed as a sample. n=4 per group. E, The graphs represent the frequencies of interferon (IFN)-γ+, interleukin (IL)-4+, IL-10+, and IL-17+ CD4+ T cells in para-aortic LNs. n=12 per group. F-H, Representative flow cytometric analysis of T-box expressed in T cells (T-bet) (F), GATA3 (G), and retinoic acid-related orphan receptor gamma t (RORγt) (H) expression in aortic CD3+CD4+CD45+ T cells. The graphs represent the frequencies of T-bet+ (F), GATA3+ (G), and RORγt+ (H) cells among aortic CD3+CD4+CD45+ T cells. n=9 per group. I, Representative flow cytometric analysis of Foxp3 expression in aortic CD3+CD4+CD45+ T cells. The graph represents the frequency of Foxp3+ Tregs among aortic CD3+CD4+CD45+ T cells. n=9 per group. J, The graph represents the ratio of CD4+T-bet+ Th1 cells to CD4+Foxp3+ Tregs (Th1 cell/Treg ratio). n=9 per group. Pooled aortic lymphoid cells from 2 mice were analyzed as a sample. Eighteen-week-old Apoe−/− or CCR4-deficient mice on an Apoe−/− background (Ccr4−/−Apoe−/−) were used for all experiments. Data points represent individual animals (A, B, and E) or individual pooled samples (C, D, F-J). Horizontal bars represent means. Error bars indicate s.d. *P<0.05, **P<0.01; Mann-Whitney U-test: D Cd103 and J; 2-tailed Student’s t-test: A, B, D Tbx21, E, and F.

C-C chemokine receptor 4 (CCR4) expression on regulatory T cells (Tregs) regulates T helper type 1 cell responses and mediates Treg migration to the aorta.

A, The suppressive function of Tregs was assessed by evaluating the proliferation of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled conventional T cells (Tconv) cocultured with Tregs from apolipoprotein E-deficient (Apoe−/−) mice or CCR4-deficient mice on an Apoe−/− background (Ccr4−/−Apoe−/−). Data are presented as the results of triplicate wells and are representative of 2 independent experiments. Data are expressed as the mean±s.d. B and C, CD80 and CD86 expression in live splenic dendritic cells (DCs) after 2 days of coculture with Tconv from Apoe−/− mice, or a mixture of Tregs from Apoe−/− or Ccr4−/−Apoe−/− mice and Tconv from Apoe−/− mice in the presence of an anti-CD3 antibody. Data points represent the results of quintuplicate wells. Data are representative of 2 independent experiments. B and D, Tconv from Apoe−/− mice and DCs were cocultured with or without Tregs from Apoe−/− or Ccr4−/−Apoe−/− mice in the presence of an anti-CD3 antibody. Interferon (IFN)-γ concentrations in cell supernatants were measured by ELISA. Data points represent the results of sextuplicate wells. E, Eighteen-week-old Apoe−/− mice fed a high-cholesterol diet for 10 weeks received transfer of Tregs from Apoe−/−Kaede-Tg or Ccr4−/−Apoe−/−Kaede-Tg mice, and the accumulation of Kaede+ Tregs in the peripheral lymphoid tissues and aorta was analyzed by flow cytometry 20 hours later. F-I, Representative flow cytometric analysis and the proportions of Kaede+ Tregs among CD4+ T cells in the peripheral lymph nodes (LNs) (F), spleen (G), para-aortic LNs (H), and aorta (I) of Apoe−/− mice that received Apoe−/−Kaede+ Tregs or Ccr4−/−Apoe−/−Kaede+ Tregs. n=9 to 10 per group (F-H). Pooled aortic lymphoid cells from 5 mice in each group were used for analysis. The results are presented as the mean ±s.d. of 3 independent experiments. Treg migration was normalized so that the number of migrated Apoe−/−Kaede+ Tregs was set to 1 (I). Data points represent individual animals (F-H) or individual pooled samples (I). Horizontal bars represent means. Error bars indicate s.d. *P<0.05, **P<0.01; 1-way ANOVA followed by Tukey’s multiple comparisons test: C and D; 2-tailed Student’s t-test: A and I. MFI indicates mean fluorescence intensity.

C-C chemokine receptor 4 (CCR4) expression on regulatory T cells (Tregs) is critical for limiting aortic inflammation and the development of early atherosclerosis.

A, Tregs purified from the peripheral lymph nodes (LNs) and spleen of apolipoprotein E-deficient (Apoe−/−) mice or CCR4-deficient mice on an Apoe−/− background (Ccr4−/−Apoe−/−) were intravenously transferred into 12-week-old Apoe−/− mice fed a standard chow diet, and atherosclerotic lesions were analyzed at 16 weeks of age. As a control without cell transfer, 12-week-old Ccr4−/−Apoe−/− mice were intravenously injected with saline and atherosclerotic lesions were analyzed at 16 weeks of age. B, Representative photomicrographs of Oil Red O staining and quantitative analysis of atherosclerotic lesion area at 5 different levels and maximal lesions in the aortic sinus of Apoe−/− mice injected with saline (n=19), Apoe−/− Tregs (n=17), or Ccr4−/−Apoe−/− Tregs (n=20). C-E, Representative sections and quantitative analyses of MOMA-2+ macrophages (C), CD4+ T cells (D), and collagen (E) in the aortic sinus of Apoe−/− mice injected with saline, Apoe−/− Tregs, or Ccr4−/−Apoe−/− Tregs. Arrowheads indicate the CD4+ T cells. n=10 per group. Black bars represent 50, 200, or 500 μm as described. Data points represent individual animals. Horizontal bars represent means. Error bars indicate s.d. *P<0.05, **P<0.01; 1-way ANOVA followed by Tukey’s multiple comparisons test.