CD4⁺CD25⁺ T cells were purified from splenocytes of GKO and WT mice at day 16 after immunization and used as inhibitor. The resulting CD4⁺CD25⁺ T cells were assayed for inhibitory activity on the proliferation of CD4⁺CD25^– T cells (responder) purified from WT EAE mice in the presence of the MOG peptide (5 μg/ml) and APCs. Data are presented as mean cpm ± SD at a fixed ratio of inhibitor (CD25⁺) to responder (CD25^–) of 1 (A) or percentage of inhibition at the indicated ratio of inhibitor to responder (B). (C) Splenocytes and purified CD4⁺ and CD4⁺CD25⁺ T cells were obtained from GKO (IFN-γ^–/–) and WT (IFN-γ^+/+) mice at day 16 after immunization and subject to real-time PCR analysis for the expression of Foxp3. (D) mRNA expression of Foxp3 in CD4⁺ T cells purified from CNS tissue of GKO and WT EAE mice. Data are presented as relative expression of Foxp3 in reference to β-actin. (E) Splenocytes were derived from WT (dotted lines) or GKO (solid lines) EAE mice on day 0 and day 16 after immunization. Cells were analyzed for intracellular Foxp3 staining and surface expression of the indicated cellular markers in the gated T cell populations by flow cytometry. Plots shown are representative of 6 independent experiments. Asterisks represent statistical differences between groups; *P < 0.05.