Bach2 is highly expressed during Treg ontogeny but down-regulated in a subset of peripheral Treg cells in response to inflammation. (A) Representative flow cytometry of thymocytes from Foxp3^EGFP-DTRBach2^tdRFP/+ mice, showing gating of CD4 single-positive (CD4SP) cells (left) and expression of Bach2^tdRFP and Foxp3^EGFP-DTR in these cells (right). (B) Histograms showing representative Bach2^tdRFP expression (left) and replicate mean fluorescence intensity measurements (right) of indicated thymocyte populations from mice of indicated genotypes. (C) Representative gating of splenic EGFP⁺ Treg cells isolated from Foxp3^EGFP-DTRBach2^tdRFP/+ mice (left) and expression of Bach2^tdRFP and CD44 on gated cells (right). (D) Representative histograms (left) and frequency of Bach2^tdRFP-low cells (right) within indicated Nrp1⁺ and Nrp1⁻ subsets of EGFP⁺ Treg cells from spleens of Foxp3^EGFP-DTRBach2^tdRFP/+ mice. (E) Experimental schema of the acute inflammation model in Foxp3^EGFP-DTRBach2^tdRFP/+ mice. (F) Representative flow cytometry of splenic EGFP⁺ Treg cells from Foxp3^EGFP-DTRBach2^tdRFP/+ mice 11 d after i.p. injection with PBS or 50 µg/kg DTx, and their expression of Bach2^tdRFP and CD44. (G) Representative Bach2^tdRFP expression (top) and replicate measurements (bottom) of Bach2^tdRFP expression among EGFP⁺ Treg cells after treatment. (A–G) Data are representative of two independently repeated experiments with four mice per group. **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s adjustment for multiple testing (B) and unpaired two-tailed Student’s t test (D and G). Numbers in gates show percentages. Bars and error show mean and SEM.