RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-beta stimulation in the presence of IL-2. Real-time PCR of human naive CD4+ T cells after 48 h of culture. Bars show the mean +/- SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-beta in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown. Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-beta and IL-2. Bars show the mean +/- SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3. Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d. Bars show the mean +/- SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown. Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-beta in the presence of IL-2. CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h. One of three independent experiments is shown. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01. 
