A distal Foxp3 enhancer enables interleukin-2 dependent thymic Treg cell lineage commitment for robust immune tolerance

STAT5 transcription factor activation downstream of the Interleukin-2 receptor (IL-2R) induces expression of Foxp3, a critical step in the differentiation of regulatory T (Treg) cells. Due to the pleiotropic outputs of IL-2R signaling, it is unclear whether STAT5 acts directly on the Foxp3 locus to promote its expression . Here, we report that IL-2 – STAT5 signaling converged on an enhancer (CNS4) during Foxp3 induction. CNS4 facilitated and sustained the IL-2 dependent CD25+Foxp3– precursor to Treg cell transition in the thymus. Its deficiency resulted in markedly impaired Treg cell generation in neonates, which was partially mitigated with age. While the thymic Treg cell paucity caused by CNS4 deficiency did not result in autoimmunity on its own, it exacerbated autoimmune manifestations caused by disruption of the Aire gene. Thus, CNS4 enhancer activity ensures robust Treg cell differentiation early in postnatal life and cooperatively with other tolerance mechanisms, minimizes autoimmunity. Overall design: Treg cells (CD4+Foxp3-GFP+) were sorted from the SLOs of 8-week-old Cd4-cre(tg+)CNS4FL (experimental) and Cd4-cre(tg–)CNS4FL (control) littermates. There were 2 biological replicates per cell population with 40,000 to 50,000 cells. Immediately after sorting cells were processed to generate ATAC libraries as previously described with minor modifications (Buenrostro et al., 2015).