Heterogeneous Bach2 expression distinguishes rTreg cells and aTreg cells. (A) Heatmap showing differentially expressed genes between Bach2^high and Bach2^low EGFP⁺ Treg cells purified by FACS from Foxp3^EGFP-DTRBach2^tdRFP/+ mice (fold-change > 2, FDR < 0.05; Wald test with Benjamini–Hochberg correction). Replicates and genes are hierarchically clustered on the x and y axes, respectively. Fragments per kilobase of transcript per million mapped reads values in the heatmap are normalized to row maxima. Data are from four independent biological replicates isolated on independent days. (B) GSEA of known differentially up-regulated genes in aTreg cells versus rTreg cells within the global transcriptional differences between Bach2^low and Bach2^high Treg cells. (C) GSEA of genes involved in apoptosis within the global transcriptional differences between Bach2^low and Bach2^high Treg cells. (D) Representative flow cytometry (left) and replicate frequencies (right) of the expression of CD62L and CD44 on indicated Treg cell subsets from Foxp3^EGFP-DTRBach2^tdRFP/+ mice. (E) Representative histograms (top) and replicate measurements (bottom) of the expression of the indicated proteins on the surface of Bach2^high and Bach2^low Treg cells. Data are representative of two individually repeated experiments with five (D) and four to five (E) mice per group. ns, not significant; ***, P < 0.001; ****, P < 0.0001; unpaired two-tailed Student’s t test (D and E). Numbers in gates show percentages. Bars and error show mean and SEM.