Figure 3. TGF-β signaling requirements in the generation of Th17 or iTreg cells.

FACS-sorted naïve T cells from B6 mice were activated under (A-B) Th17 (TGF-β + IL-6 + IL-23 + anti-IFN-γ + anti-IL-4) or (C) iTreg conditions, and a TGF-βRI kinase inhibitor (SB431542, 5μM) was added at different time points as indicated. Cells were assessed for IL-17 and IFN-γ production and Foxp3 expression after 4 days of stimulation using intracellular staining. (A, C) A representative dot plot graph is shown in the left panel, and the numbers in quadrants represent the percentages. In the right panel, the percentage of (A) IL-17⁺ or (C) Foxp3⁺ cells for six independent experiments are indicated. *, p<0.05, Wilcoxon signed rank test. (B) mRNA expression of indicated genes was analyzed by real-time RT-PCR. The data shown was normalized to expression of a reference gene Actb. The lowest expression for each gene was referred as 1. (D) Naïve T cells from Smad4^fl/flCD4-Cre^- (WT) or Smad4^fl/fl CD4-Cre⁺ (Smad4^-/-) mice were cultured with or without WT or Smad4^-/- CD4⁺CD25⁺ nTreg cells in triplicate wells with irradiated APCs and stimulated with 2 μg/ml of anti-CD3. Proliferation was assayed 72 h after treatment by adding [³H]-thymidine to the culture for the last 8 h. A representative example of three independent experiments is shown. (A-D) Graph shows means ± s.d. (E-F) Naïve Smad4-sufficient or -deficient T cells were activated under (E) iTreg or (F) Th17 conditions, and IL-17, IFN-γ and Foxp3 expression was analyzed by intracellular staining. Numbers in quadrants represent the percentages. The experiments were repeated three times with consistent results.