Supplemental Table I Male / Mean ± SEM n Mean ± SEM n Body weight, g 29.2±0.4 17 29.7±0.5 17 Total cholesterol, mg/dl 534.0±30.8 17 561.6±26.1 17 HDL-cholesterol, mg/dl 9.6±0.8 17 10.1±0.7 17 Triglycerides, mg/dl 76.8±5.9 17 93.4±5.7 17 Systolic blood pressure, mmhg 118.5±2.2 10 118.6±2.1 10 Diastolic blood pressure, mmhg 70.5±3.4 10 68.0±3.8 10 Female / Mean ± SEM n Mean ± SEM n Body weight, g 23.0±0.3 19 23.0±0.3 19 Total cholesterol, mg/dl 407.3±16.7 15 395.1±16.1 14 HDL-cholesterol, mg/dl 5.5±0.4 15 6.9±0.3 14 Triglycerides, mg/dl 57.5±3.8 15 58.6±6.2 14 Systolic blood pressure, mmhg 111.7±1.7 9 116.8±2.1 10 Diastolic blood pressure, mmhg 72.77±2.0 9 71.19±2.5 10 Supplemental Table I. Analysis of body weight, plasma lipid profile, and blood pressure at 16 weeks. HDL, high-density lipoprotein.
Supplemental Figure I / Supplemental Figure I. Overexpression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) does not affect the proportions of CD11c + dendritic cells (DCs), natural killer (NK) cells, and NKT cells in spleen. n=4 to 5 per group. Error bars indicate s.e.m.
Supplemental Figure II 15.7 15.6 53.3 49.7 Counts Spleen Blood / / Ly6C Supplemental Figure II. Overexpression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) does not affect the proportion of Ly6C high monocytes in blood and spleen. Representative results of Ly6C expression in blood and splenic monocytes assessed by flow cytometry. The graph represents the percentage of Ly6C high monocytes within the blood and splenic CD11b + Ly6G - monocytes. n=4 to 5 per group. Error bars indicate s.e.m.
Supplemental Figure III / A B Supplemental Figure III. A, Overexpression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) does not affect the proportions of regulatory B cells (Bregs) or B2 cells such as CD19 + B220 + IgM high CD5 - marginal zone B cells or CD19 + B220 + IgM low CD5 - follicular B cells in spleen. The graph represents the percentage of Bregs or B2 cells within splenocytes. n=5 per group. B, Overexpression of CTLA-4 increases the proportion of CD19 + B220 low IgM high CD5 - B1b cells, but does not affect the proportion of CD19 + B220 low IgM high CD5 + B1a cells in the peritoneal cavity. The graph represents the percentage of B1 cells within peritoneal cells. n=4 to 5 per group. Error bars indicate s.e.m. *P<0.05; **P<0.01.
Supplemental Figure IV A 11.4 49.5 / 8.5 48.3 B / CD8 35.8 38.3 CD4 C / 3.6 4.6 93.7 1.4 CTLA-4 89.4 2.3 1.4 0.0 D Foxp3 Supplemental Figure IV. Overexpression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) does not affect CD4 + or CD8 + T cell development, whereas it prevents the development of CD4 + Foxp3 + regulatory T cells (Tregs) in the thymus. Lymphoid cells from thymus of 4-week-old apolipoprotein E-deficient ( ) or / mice were prepared. A, Representative results of CD4 and CD8 expression in TCR-β + T cells assessed by flow cytometry. B, The graphs represent the numbers and percentage of CD4 single positive (CD4 + ), CD4/CD8 double positive (CD4 + CD8 + ), and CD8 single positive (CD8 + ) T cells within TCRβ + thymocytes. C, Representative results of Foxp3 and CTLA-4 expression within the TCRβ + CD4 + CD8 - population. D, The graphs represent total numbers of TCR-β + CD4 + CD8 - Foxp3 + Tregs and percentage of CD4 + Foxp3 + Tregs within the TCR-β + CD4 + CD8 - population. n=5 to 6 per group. Data are representative of two independent experiments. Error bars indicate s.e.m. *P<0.05.
Supplemental Figure V PI 5.0 2.9 Annexin V / Supplemental Figure V. Overexpression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) suppresses the early stages of apoptosis in CD4 + T cells. Representative results of Annexin V and propidium iodide (PI) staining in splenic CD4 + T cells assessed by flow cytometry. The graph represents the percentage of Annexin V + PI - early apoptotic cells within the CD4 + population. n=5 per group. Error bars indicate s.e.m. **P<0.01.
Supplemental Figure VI A / B / 30.1 29.8 IFN-γ CD8 / Supplemental Figure VI. Overexpression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has a minimal effect on the activation of CD8 + T cells. A, The graphs represent the percentage of CD44 high, CD62L low, CD69 high cells within the CD8 + population in lymph nodes (LNs) and spleen. n=7 per group. Data are representative of two independent experiments. Error bars indicate s.e.m. B, Lymphoid cells from spleen were stimulated with phorbol 12-myristate 13-acetate and ionomycin in vitro. Intracellular cytokine staining was performed. Representative results and frequency of interferon (IFN)-γ + CD8 + T cells in the spleen of each group. n=5 per group. Error bars indicate s.e.m.
Supplemental Figure VII / Supplemental Figure VII. The effects of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) overexpression on interleukin (IL)-10, IL-12p35, and IL-12p40 expression in splenic CD11c + DCs. Expression of several cytokines in splenic DCs was quantified by quantitative real-time reverse transcription PCR and normalized to GAPDH. Fold change relative to each group is shown. n=5 per group. Error bars indicate s.e.m.
Supplemental Figure VIII / Supplemental Figure VIII. Overexpression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) does not change regulatory T cell (Treg)-associated molecule expression in CD4 + Foxp3 + Tregs. Lymphoid cells from lymph nodes (LNs) and spleen of apolipoprotein E-deficient ( ) or CTLA-4- Tg/ mice were prepared. The expression levels of Treg-associated markers were analyzed by flow cytometry gating on CD4 + Foxp3 + Tregs in LNs and spleen. The data are shown as mean fluorescence intensity (MFI). n=6 to 7 per group. Error bars indicate s.e.m. GITR, glucocorticoid-induced tumor necrosis factor receptor family-related gene/protein.
Supplemental Figure IX A / 0.45 0.25 IL-10 B Foxp3 LN / LN / 1.9 1.6 LAP Foxp3 / Spleen / Spleen 2.7 2.6 LAP Foxp3 / Supplemental Figure IX. CTLA-4 transgenic CD4 + CD25 - T cells are neither T regulatory type 1 cells nor T helper type 3 cells. A, Lymphoid cells from spleen were stimulated with phorbol 12-myristate 13-acetate and ionomycin in vitro. Intracellular cytokine staining was performed. The graph represents the frequency of IL-10 + cells within CD4 + Foxp3 - non-tregs. n=5 per group. B, Representative results of LAP expression in lymph node (LN) and splenic CD4 + Foxp3 - non-tregs. The graphs represent the percentage of LAP + cells within CD4 + Foxp3 - non-tregs in LNs and spleen. n=5 per group. Error bars indicate s.e.m. **P<0.01.
Supplemental Figure X A / B / C / / Counts CTLA-4
Supplemental Figure X. The effects of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) overexpression on inflammatory responses in atherosclerotic aortas. A, Total RNA was extracted from aortas of 10-week-old apolipoprotein E-deficient ( ) or / mice. Expression of interferon (IFN)-γ, CD4, CD80, CD86, CTLA-4, and regulatory T cell (Treg)-associated molecules (Foxp3 and interleukin (IL)-10) in atherosclerotic aortas was quantified by quantitative real-time reverse transcription PCR and normalized to GAPDH. Fold change relative to each group is shown. n=5 to 6 per group. B, Representative staining and quantitative analysis of Foxp3 + cells in the aortic sinus plaques. The white bar represents 200 μm. White arrowheads indicate the Foxp3 + cells. n=12 per group. C, Cell suspensions from collagenasedigested aortas of 16-week-old or / mice were stained with antibodies against CD4, CD3, CD45, and CTLA-4. Representative results of intracellular CTLA-4 expression in aortic CD3 + CD4 + CD45 + T cells assessed by flow cytometry. The data are shown as mean fluorescence intensity (MFI). n=3 to 6 per group. Error bars indicate s.e.m. *P<0.05; **P<0.01.
Supplemental Figure XI (A, B) Cell-extrinsic suppression CD80/CD86 CD28 APC MHC classⅡ Antigen TCR Treg CTLA-4 Activated Teff Naïve T cell Teff (C) Cell-intrinsic suppression CTLA-4 Proinflammatory cytokine Activated Teff Atherosclerotic plaque Supplemental Figure XI. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) regulates proatherogenic immune responses in cell-intrinsic and -extrinsic manners. There are several possible mechanisms for CTLA-4-mediated suppression of atherosclerosis. A, B, Cell-extrinsic mechanisms of CTLA-4; CTLA-4 negatively regulates proatherogenic immune responses by ligating CD80 and CD86 on dendritic cells (DCs) and inhibiting CD28-CD80/CD86 interactions. CTLA-4 on both regulatory T cells (Tregs) (A) and activated effector T cells (Teffs) (B) may downregulate the CD80 and CD86 expression on DCs and reduce the ability of DCs to activate other T cells, which contributes to reduction of atherosclerosis. C, Cell-intrinsic mechanism of CTLA-4; CTLA-4 on activated Teffs receives negative signals by binding to CD80 and CD86 on DCs, leading to suppression of their function and atherosclerosis development. APC, antigen-presenting cell; MHC, major histocompatibility complex; TCR, T cell receptor.