There are no changes of T_reg differentiation and IL-10 production in iNOS^−/− mice. (A) Naive CD4⁺ T cells from WT or iNOS^−/− mice were differentiated under T_H0 and T_reg polarizing conditions for 3 d. Cells were then restimulated with PMA/ionomycin for 5 h, stained for intracellular FOXP3, and analyzed by flow cytometry. Representative FACS dot plots gated on CD4⁺ cells and the percentages of FOXP3-positive CD4⁺ cells are shown. Each represents mean ± SD from three independent experiments. (B) Naive CD4⁺ T cells from WT or iNOS^−/− mice were differentiated under T_H0 and T_H17 polarizing conditions for 3 d. Cells were then restimulated with PMA/ionomycin for 24 h and the supernatants were analyzed for IL-10 by ELISA. Data represent mean ± SD from three independent experiments.