Fig 4. Assessment of IL-10-producing T cells, TNF-α expression in the lesions and cytokine levels in B6;129S-Ldlr ^tm1Her Apob ^tm2Sgy /J mice fed a high-fat diet after immunization with constructs AHHC, RHHC, and RPHC.

(A) Photomicrographs showing dual-IHC staining for IL-10 (red) and CD4 (green) (scale bar: 100 μm and 12.5μm for magnified ones). (B) Scatter plot showing mean of IL-10-positive area co-localized with CD4⁺ area (%) (N = 6). (C) Photomicrographs showing IHC staining for TNF-α (green) of lesions (scale bar: 100 μm and 12.5μm for magnified ones). (D) Scatter plot showing mean of anti- TNF-α-stained area in the lesion versus total lesion area (N = 6). (E-H) Cytokine levels measured in plasma. (I-L) Cytokine levels measured in the supernatant of splenocytes stimulated with ConA. (M) Representative analysis of CD4⁺IL-4⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometer. (N) Percentages of IL-4⁺ expressing CD4⁺ spleen cells. Data are expressed as mean of 3 analyses ± SEM. Differences between groups are shown. (O) Representative analysis of IL-17A expression by CD4⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometry. (P) Levels of IL-17A expression in spleen cells. Data are expressed as mean of 3 analyses ± SEM. Differences between groups are shown. (Q) Representative analysis of CD4⁺IL-2⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometer. (R) Levels of IL-2⁺ expressing CD4⁺ in spleen cells. Data are expressed as mean of 3 analyses ± SEM. Differences between groups are shown.