Control of Th17 differentiation by PPARγ. (a) MOG-EAE was induced in PIO or vehicle-treated wild-type mice (n = 6 per group, 3 experiments), and the clinical disease score was assessed daily. (b) In a separate experiment, mice were sacrificed at the peak of disease (day 18), CD4⁺ T cells were isolated from the CNS, restimulated with PMA/ionomycin, and analyzed by flow cytometry gated on CD4⁺ T cells; representative dot plots and mean results ± SEM from four animals per group are shown; data are from two experiments. (c) CD4⁺ T cells from the CNS were restimulated with MOG_35-55-loaded DCs, and numbers of IL-17 and of IFN-γ–producing cells per 3 × 10⁴ CD4⁺ T cells were determined by ELISpot analysis. Graphs denote mean ± SEM of all animals (n = 6 per group, 2 experiments). (d) Purified CD4⁺ T cells from CD4-PPARγ^KO mice or WT littermates were treated with PIO or the endogenous PPARγ agonist 13s-HODE, and Th17 differentiation was induced by stimulation for 72 h (top row). Alternatively, Th1 differentiation was induced for 72 h (bottom row). Cytokine-producing cells were determined by flow cytometry after PMA/ionomycin restimulation. Only living cells were analyzed by using LIVE/DEAD stain and exclusion of autofluorescence (x axis). Numbers denote mean percentage ± SEM. (e) Th17 differentiation was induced as above and TNF, IL-17A, and IL-22 expression were determined by flow cytometry. Numbers denote mean percentage ± SEM. (f) Th17 differentiation was induced as above; after 72 h expression of IL-17A, IL-17F, IL-21, and IL-23R were measured by quantitative real-time RT-PCR normalized to β-actin levels. (g) Additionally, CCR6-expression was determined by flow cytometry; CCL20-release was assessed by ELISA. (d–g) One out of at least three independent experiments is shown.