Role of CBFβ in the induction of Foxp3
CBFβ, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences. To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene. Retinoic acid and TGF-β synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007). To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFβ, naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-β. After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-γ expression. TGF-β induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells. Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-β and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-β doses. The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-β induction of Foxp3 in T reg cells (Fig. 5 A). There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-γ were neutralized (Fig. S6).
Figure 5.  Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, ± 10 nM retinoic acid (RA), and increasing concentrations of TGF-β. After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-γ expression. One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 × 106 cells per transfer). 6 wk later, TCRβ+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression. Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown. The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.   The importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined. Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2−/− mice. 6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B). There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C). These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.