FIGURE 1.

Twist1 is regulated by STAT3-activating cytokines in Th17 cells. A, naive wild type and Twist1-deficient CD4⁺ T cells were cultured under Th1, Th2, Th9, Th17, and Treg cell polarizing conditions. Th1, Th2, Th9, and Th17 cells were restimulated with anti-CD3 for 24 h to access cytokine production by ELISA. B, percentage of Foxp3 expression in Treg cells following in vitro differentiation. C and D, on day 5, differentiated wild type Th17 cells generated as described in A were rested or stimulated with IL-6, IL-23, or IL-12 for 2 h before gene expression analysis by qRT-PCR (C) and Twist1 expression by immunoblot (IB) with densitometry normalized against β-actin (D). E, naïve wild type and Stat3-deficient CD4⁺ T cells were activated with anti-CD3 and anti-CD28 in the presence or absence of IL-6, TGF-β, or IL-12 and gene expression was analyzed by qRT-PCR after 3 days. F, schematic of Twist1 promoter containing STAT3 binding sites. G, cells prepared as described in C were used for ChIP analysis using STAT3 antibody. Data are mean of four independent experiments ± S.E. (A and B), or are mean of replicate samples ± S.D. and representative of three independent experiments with similar results (C–G).**, p < 0.01. unstim, unstimulated.