The phenotype of Foxp3⁺ Treg cells in Bach2^fl/flFoxp3^{EGFP-Cre-ERT2/+} mice. (A) Representative flow cytometry (left) and the normalized percentage (right) of EGFP⁺ Treg cells from thymi (gated on CD4SP cells) or spleens (gated on CD4⁺ T cells) of animals of the indicated genotypes fed tamoxifen for 8 wk. (B) Homozygous germline excision of the Bach2^fl allele results in a complete cell-intrinsic defect in the generation of Foxp3⁺ Treg cells. Representative flow cytometry (left) and replicate measurements (right) showing the frequency of Foxp3⁺ Treg cells within the CD45.2⁺ compartment of CD45.1⁺ (Ptprc^a/a) mice reconstituted following lethal irradiation with mixtures of WT CD45.1/2⁺ (Ptprc^a/b) BM cells in an ∼1:1 ratio with CD45.2⁺ BM cells from either Bach2^+/+, Bach2^−/−, or Bach2^ex/ex animals. A complete cell-intrinsic defect in generation of Foxp3⁺ cells is observed in either cells bearing the originally described Bach2 knockout alleles (Bach2^−/−) or those with germline excision of Bach2flox alleles, whereas Treg cells are produced by the WT (Bach2^+/+) BM compartments. Mice (Bach2^ex/ex) bearing germline excision of the Bach2^flox alleles were generated by transiently administering tamoxifen to male Bach2^fl/+Rosa26Cre-ERT2 mice which, after 2 mo, were used to breed with WT C57BL/6 female mice. F1 progeny bearing germline excision of the Bach2^fl allele were then backcrossed to generate Bach2^ex/ex animals, which were used to source cells for the BM chimera experiments described above. Data are representative of two independently repeated experiments with five (A) and six to seven (B) mice per group. *, P < 0.05; **, P < 0.01; ns, not significant; unpaired two-tailed Student’s t test (A). Numbers in gates show percentages. Bars and error show mean and SEM.