The role of RUNX1 and RUNX3 transcription factors in TGF-beta-mediated iT reg cell generation
To investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells. Stimulation with anti-CD2/3/28 mAbs or TGF-beta alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A). In contrast, the combination of both TGF-beta and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells. This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction. 
To test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B). Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-beta-mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA. The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B). 
The influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated. Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed. We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells. On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C). 
The effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry. FOXP3 was only slightly reduced after RUNX1 silencing. Transfection of siRNA for RUNX3 had a stronger effect. The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D). Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2). The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3. In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-gamma in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-beta did not show any significant difference (Fig. S3). 
To determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127- CD25high T reg cells and compared them with CD4+ CD127+ CD25- T cells. Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25- cells. As expected, IL-10, TGF-beta, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A). There was no difference in RUNX1 mRNA expression between these two cell subsets. We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006). Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B). 
