BACH2 restrains aTreg differentiation in a cell-intrinsic manner and is required for long-term maintenance of Treg cell populations. (A) Graphical representation of genetic heterogeneity of Treg cells from tamoxifen-treated Foxp3^{EGFP-Cre-ERT2/+}Bach2^fl/fl animals due to random X-inactivation. (B and C) Representative flow cytometry plots (B) and replicate measurements (C) of cell surface CD62L, CD44, and ICOS expression on EGFP⁺ Treg cells isolated from spleens and inguinal lymph nodes (iLN) of heterozygous female Foxp3^{EGFP-Cre-ERT2/+}Bach2^+/+ or Foxp3^{EGFP-Cre-ERT2/+}Bach2^fl/fl mice administered tamoxifen for 2 wk. (D) Representative flow cytometry showing the frequency of EGFP⁺ Treg cells of total CD4⁺ T cells in the blood of animals at indicated time points after commencement of tamoxifen treatment. (E) Replicate measurements of EGFP⁺ Treg cells shown in D, normalized to their mean frequency in Foxp3^{EGFP-Cre-ERT2/+}Bach2^+/+ control mice. Data are representative of two to three independently repeated experiments with five to seven (B and C) and four to five (D and E) mice per group. **, P < 0.01; unpaired two-tailed Student’s t test (C and E). Numbers in gates show percentages. Bars and error show mean and SEM.