Altered expression of Foxp3 in Bcl11b-deficient T_reg cells of Bcl11b^F/F/CD4-Cre and Bcl11b^F/F/Foxp3-Cre mice and altered induction of Foxp3 during generation of iT_reg cells. (A and C) Evaluation of Foxp3 protein levels in T_reg cells from Bcl11b^F/F/CD4-Cre (A) and Bcl11b^F/F/Foxp3-Cre (C) versus control mice. Splenocytes were surface stained for CD4, fixed and permeabilized, and further stained with anti-Foxp3 antibodies. Numbers indicate the percentages of CD4⁺Foxp3⁺ cells among all splenocytes, and the ratio between KO and WT Foxp3 mean fluorescence intensity (MFI) is indicated below. The results are representative for four pairs of mice. (B and D) Relative Foxp3 mRNA levels in sorted GFP⁺ Bcl11b-deficient T_reg cells from Bcl11b^F/F/CD4-Cre/Foxp3-GFP (B) and Bcl11b^F/F/Foxp3-Cre (D) mice versus control levels evaluated by qRT-PCR. The data show one representative experiment of two with cells sorted from two to three mice, or multiple mice in the case of Bcl11b^F/F/CD4-Cre/Foxp3-GFP mice. Bcl11b^F/F/Foxp3-Cre mice and their controls were 5–6 mo of age. (E) ChIP assays with anti-Bcl11b or IgG antibodies. The immunoprecipitated DNA was amplified with primers corresponding to the indicated regulatory regions. Binding was normalized to the input, as described in Material and methods. Enrichment by Bcl11b is indicated as fold increase versus IgG. Data are derived from a representative experiment of two. (F) Foxp3-GFP⁻CD4⁺CD45rb^hi cells from Bcl11b^F/F/CD4-Cre/Foxp3-GFP⁺ and Foxp3-GFP⁺ WT mice were sorted and incubated in complete media in the presence of cross-linked anti-CD3e, 100 U IL-2, and 10 ng Tgf-β, for 3 d, and then stained for CD4 and Foxp3 and analyzed by FACS. Data are representative of three independent experiments.