Single-cell and bulk RNA-Seq analysis of WT and Bach2-deficient Foxp3⁺ Treg cells. (A) tSNE representation clustered by gene expression profiles of single EGFP⁺ Treg cells isolated from the spleens of tamoxifen-treated Bach2^+/+Foxp3^{EGFP-Cre-ERT2} (WT) mice and Bach2^fl/flFoxp3^{EGFP-Cre-ERT2} (CKO) mice. Three biological replicate cell populations per genotype were subjected to scRNA-Seq. (B) Heatmap showing the expression of the top 10 differentially expressed genes in each cluster identified in A. (C) The relative frequency of Treg cells of indicated genotypes within each cluster normalized to their average ratio among WT replicates. (D) Graph showing the fold change in the frequency of cluster 4 versus cluster 0 cells expressing the indicated differentially expressed genes (P_adj < 0.005 and log₂ fold-change > 1 in cluster 4 versus cluster 0). Wald test with Benjamini–Hochberg adjustment. (E and F) Experimental schema (E) and results of GSEA analysis (F) demonstrating enrichment of activated Treg cell genes in CKO versus WT cells isolated from spleens of tamoxifen-treated Bach2^+/+Foxp3^{EGFP-Cre-ERT2} and Bach2^fl/flFoxp3^{EGFP-Cre-ERT2} animals. (A–F) Data from three independent biological replicates per genotype are shown. *, P < 0.05; **, P < 0.01; unpaired two-tailed Student’s t test (C). Bars and error show mean and SEM.