T_reg cells of Bcl11b^F/F/CD4-Cre and Bcl11b^F/F/Foxp3-Cre mice show altered suppressor activity in vitro. (A and B) Evaluation of absolute numbers of T_reg cells in the colon of Bcl11b^F/F/CD4-Cre/Foxp3-GFP mice. Cells were isolated from the colon LP and epithelial compartment (IEL) stained for CD4 and analyzed by flow cytometry, and the percentage of Foxp3-GFP⁺ cells and their absolute numbers were evaluated. Four to five mice were used for each group. Asterisks indicate statistical significance determined by unpaired two-tailed Student’s t test (*, P = 0.04 in A; *, P = 0.02 in B). Horizontal bars indicate the mean. (C and D) Sorted CD4⁺CD45Rb^hiFoxp3-GFP⁻ CFSE-labeled WT T cells were activated and co-cultured with sorted Foxp3-GFP⁺ T_reg cells from Bcl11b^F/F/CD4-Cre/Foxp3-GFP⁺ or Foxp3-GFP⁺ mice (C) or from Bcl11b^F/F/Foxp3-Cre or Foxp3-Cre mice (D) at the indicated ratios of conventional T cells to WT or KO T_reg cells (conventional/T_reg cells). After 72 h, cells were stained with anti–CD4-APC-Cy7 and anti–Foxp3-APC antibodies and analyzed by flow cytometry. The number of cell divisions was evaluated in the CD4⁺/Foxp3⁻ population based on gradual loss of CFSE fluorescence. The maintenance of Bcl11b-deficient and WT T_reg cells during the suppression assay is shown in Fig. S4 B. The results are representative of three independent experiments, each with one pair of mice.