exFoxp3 cells instruct differentiation of naive T cells to Th1 and Th17 in vitro. (A) 105 Socs1+/+, Ifnγ−/−Socs1+/+, or Ifnγ−/−Socs1−/− Treg cells/well were stimulated with anti-CD3/anti-CD28 beads, 10 ng/ml IL-2, and the indicated cytokines, and IL-17A was measured by ELISA. Data are representative of five independent experiments (***, P < 0.001). (B) Phosphorylation status of STAT1 and STAT3 in Treg cells with the indicated genotypes by flow cytometry (mean fluorescent intensity shown in bar graph). Data are representative of five independent experiments (**, P < 0.01). (C) 3 × 105 WT naive T cells/well were cultured for 5 d alone (top) or with untreated APCs (middle) or LPS-stimulated APCs (bottom ; 7 × 105 T cell–depleted spleen cells/well) plus supernatants from Socs1+/+, Socs1−/−, or Ifnγ−/−Socs1−/− Treg cells (2 × 105/well) cultured for 2 d in the presence of anti-CD3/anti-CD28 beads and 10 ng/ml IL-2. Flow cytometric analysis of IFN-γ and IL-17A on these cells is shown. Data are representative of three independent experiments. (D) 2 × 105 Socs1+/+, Socs1−/−, Ifnγ−/−Socs1+/+, or Ifnγ−/−Socs1−/− Treg cells were cotransferred with 4 × 105 Il17−/− naive T cells into Rag2−/− mice, and body weight is shown. Data are representative of three independent experiments (*, P < 0.05; **, P < 0.01). (A, B, and D) Values represent the mean ± SD.
