FIGURE 2.

Twist1 suppresses cytokine production in Th17 cells. A, naïve CD4⁺ T cells were isolated from wild type mice and differentiated under Th17 culture conditions. On day 2, cells were transduced with either control or Twist1-GFP (Twist1)-expressing retrovirus. On day 5, cells were stimulated with PMA and ionomycin for 6 h before intracellular staining (ICS) for cytokine production. Data are gated on GFP⁺ cells. B, differentiated wild type and Twist1-deficient Th17 cells were stimulated with PMA and ionomycin for 6 h before ICS analysis. C and D, naïve wild type and Twist1-deficient CD4⁺ T cells were cultured under Th17 polarizing conditions with or without TGF-β. On day 5, cells were left unstimulated for gene expression analysis by qRT-PCR (C) or reactivated with anti-CD3 for 24 h to assess cytokine production by ELISA (D). E, naïve CD4⁺ T cells were isolated from PBMCs and differentiated under Th17 culture conditions. On day 5, cells were transfected with control or siRNA targeting TWIST1, rested overnight, and stimulated with anti-CD3 to assess gene expression by qRT-PCR. F and G, differentiated wild type and Twist1-deficient Th17 cells were used for gene expression analysis by qRT-PCR before (Rorc, Batf, and Maf) or after (Il17a) 6 h anti-CD3 stimulation (F) and ChIP analysis using STAT3 antibody (G). Data are mean of four to five independent experiments ± S.D (A–D) or are mean of replicate samples ± S.D. and representative of three independent experiments with similar results (E–G). *, p < 0.05; **, p < 0.01. ND, not detectable.