FIGURE 4.

Reduced MOG-specific Th1 and Th17 responses, but normal Treg cell compartment, in Def6^−/− mice. A and B, Splenocytes (A) or dLN cells (B) from WT and KO mice isolated 8 days postimmunization were challenged in vitro with the indicated concentrations of MOG_35–55 peptide, and culture supernatants collected after 72 h were analyzed for IFN-γ (left panel) and IL-17 (right panel) concentration by ELISA. Statistical differences were determined as in Fig. 1. C, Cytokine production by CNS-derived, MOG_35–55-stimulated CD4⁺ T cells. CD4⁺ T cells were isolated from CNS mononuclear cells 16 days after disease induction. The cells were stimulated for 5 h in vitro with PMA plus ionomycin in the presence of GolgiStop and analyzed for IFN-γ and IL-17 expression by ICS. D, Absolute number of IFN-γ- or IL-17-producing CD4⁺ T cells in CNS on day 16 after MOG/CFA immunization. For C and D, cells from three mice per group were pooled for analysis. Data are representative of three experiments. E, Suppressive activity of WT and KO Treg cells. CD4⁺CD25⁺ cells (Tregs) were purified from the spleens and LNs of WT or KO mice and were added (or not) to cultures of purified CD4⁺CD25⁻ WT T cells (responders). Proliferation of WT responder T cells stimulated by anti-CD3 and anti-CD28 mAbs (5 µg/ml each) was determined by [³H]TdR incorporation, and cultures of Tregs alone served as a control. Data are means ± SD of triplicate wells. The results are representative of two independent experiments. F and G, Flow cytometric analysis (F) and calculated number (G) CD4⁺CD25⁺FoxP3⁺ Treg cells in WT and KO mice before (naive) and 15 days after MOG/CFA immunization. dLN cells were stained for CD4, CD25, and intracellular FoxP3 expression. The right panel in F shows that CD4⁺CD25⁺ cells are mostly FoxP3⁺, whereas CD4⁺CD25⁻ and CD4⁻CD25⁻ cells are FoxP3⁻.