BACH2 restrains TCR-driven aTreg differentiation from rTreg precursors. (A) Experimental rTreg activation assay schema. Resting CD62L⁺ EGFP⁺ Treg cells were purified by FACS from spleens of animals and cultured in the presence of the indicated conditions. (B) Representative flow cytometry plots showing cell surface CD62L and CD44 expression by cells of indicated genotypes without or with stimulation with plate-bound anti-CD3 and anti-CD28 monoclonal antibodies. (C) Replicate measurements of the phenotype of Treg cells of indicated genotypes at the end of the assay. (B and C) Data are representative of two independently repeated experiments with four replicates per genotype. ns, not significant; *, P < 0.05; ****, P < 0.001; unpaired two-tailed Student’s t test (C). Numbers in gates show percentages. Bars and error show mean and SEM.