Figure 4. Foxp3 inhibits Th17 cell cytokine induction by antagonizing RORγt function.

(A) FACS-sorted naïve OT-II T cells were activated under Th17 conditions and infected with an IRES-GFP-containing bicistronic retrovirus expressing Foxp3 or a vector control virus. IL-17- and Foxp3-expressing cells were measured by intracellular staining on the GFP^- and GFP⁺ population. The experiments were repeated at least three times with similar results. (B) GFP^- and GFP⁺ cells were sorted from (A) and restimulated for 4 hours with anti-CD3. mRNA expression of indicated genes was analyzed by real-time RT-PCR. The data shown were normalized to expression of a reference gene Actb. The lowest expression for each gene was referred as 1. *, p<0.05, t test. (C-F) EL-4 cells were transfected with a vector containing the firefly luciferase gene under the control of the Il17a promoter-CNS2 region, a vector expressing Renilla luciferase, and IRES-GFP-containing bicistronic vectors expressing RORγt, Foxp3 wild-type (WT) or various Foxp3 mutants, or vector alone. Luciferase activity was determined and normalized to Renilla luciferase. Values were also normalized to vector alone. The data represent at least four independent experiments with consistent results. *, p<0.05, t test. (B-F) Graph shows means ± s.d. (G) Naïve OT-II T cells were activated under Th17 conditions and infected with indicated viruses. IL-17 expression was analyzed by intracellular staining on either GFP⁺ or GFP^- gate. (H) Naïve WT or Scurfy OT-II T cells were stimulated with the indicated cytokines and neutralizing antibodies. Four days later, cells were assessed for IFN-γ and IL-17 production by intracellular staining. The data represent at least three independent experiments with consistent results.