Fig. 6

Early skewed IL-17⁺CD4⁺ Th17 response of CCR5-ablated mice during JE progression. a, b Frequency and number of CD4⁺Foxp3⁺ Tregs, IFN-γ⁺CD4⁺ Th1, and IL-17⁺CD4⁺ Th17 cells in the spleen of CCR5-ablated mice. c, d Frequency and number of CD4⁺Foxp3⁺ Tregs, IFN-γ⁺CD4⁺ Th1, and IL-17⁺CD4⁺ Th17 cells in the brain of CCR5-ablated mice. The frequency and absolute number of CD4⁺Foxp3⁺ Tregs (a, c), IFN-γ⁺CD4⁺ Th1, and IL-17⁺CD4⁺ Th17 cells (b, d) in the spleen (a, b) and brain (c, d) of Ccr5^+/+ and Ccr5^−/− mice were determined by flow cytometric analysis at 3 and 5 days following JEV (3.0 × 10⁷ pfu) infection. CD4⁺Foxp3⁺ Tregs were detected with intracellular Foxp3 and surface CD4 staining, and the frequency and number of IFN-γ⁺CD4⁺ Th1 and IL-17⁺CD4⁺ Th17 cells were determined by intracellular cytokine staining in response to PMA + ionomycin stimulation of splenocytes or brain leukocytes prepared from Ccr5^+/+ and Ccr5^−/− mice. Values in representative dot-plots are the average percentage of Foxp3⁺ cells, IFN-γ⁺ and IL-17⁺ in CD4⁺ T cells. e Expression of transcription factors by CNS-infiltrated CD4⁺ T cells. After vigorous heart perfusion, sorted CD4⁺ T cells from CNS-infiltrated leukocytes were briefly stimulated with PMA plus ionomycin for 3 h. The expression of transcription factors of CD4⁺ Th1, Th2, Th17, and Tregs was determined by real-time qRT-PCR using total RNA extracted from stimulated CD4⁺ T cells. 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