Fig. 8

CCR5⁺CD4⁺Foxp3⁺ Tregs ameliorate JE via IL-10 production without affecting the accumulation of myeloid-derived leukocytes, CD4⁺ Th1, or Th17. a, b Detection of CCR5⁺CD4⁺Foxp3^GFP Tregs in the spleen and brain. Adoptively transferred CCR5⁺CD4⁺Foxp3^GFP and CCR5⁻CD4⁺Foxp3^GFP Tregs were detected by flow cytometry in the spleen (a) and brain (b) at 5 days following JEV (3.0 × 10⁷ pfu) infection. Values in representative dot-plots denote the average percentages of CCR5⁺CD4⁺Foxp3^GFP Tregs in CD4⁺ T cells. c, d The frequency and number of Ly-6C^hi monocytes and Ly-6G^hi granulocytes in the CNS of CCR5⁺ or CCR5⁻ Treg-injected CCR5-ablated mice. The frequency (c) and number (d) of Ly-6C^hi monocytes and Ly-6G^hi granulocytes in the CNS of CCR5⁺ (WT) or CCR5⁻ (KO) Treg-injected CCR5-ablated mice were determined by flow cytometric analysis at 5 dpi using vigorous heart perfusion. Values in representative dot-plots denote the average percentage of the indicated cell population after gating on CD11b⁺ cells. e–h Accumulated number of CD4⁺ Th1, Th17, and Tregs in the CNS of CCR5⁺ (WT) or CCR5⁻ (KO) Treg-injected CCR5-ablated mice. The absolute number of CD4⁺ Th1, Th17, and Tregs in the CNS and spleen of Treg-injected Ccr5^−/− recipients was determined at 5 dpi. e CD4⁺Foxp3⁺ Tregs in brain. f CD4⁺ Th1 and Th17 in the brain. g CD4⁺Foxp3⁺ Tregs in the spleen. h CD4⁺ Th1 and Th17 in the spleen. i IL-10 expression in adoptively transferred CCR5⁺CD4⁺Foxp3^GFP and CCR5⁻CD4⁺Foxp3^GFP Tregs. The expression of IL-10 in CNS-infiltrated CCR5⁺CD4⁺Foxp3^GFP and CCR5⁻CD4⁺Foxp3^GFP Tregs was evaluated by intracellular IL-10 staining at 5 dpi. Data are averages ± SD of values derived from at least three independent experiments (n = 3–4). *p < 0.05; **p < 0.01; p < 0.001 compared with the levels of the indicated groups