Foxp3 enhancers synergize to maximize regulatory T cell suppressive capacity [TCR-seq]

Treg cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How their robust function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of Treg lineage–specifying factor Foxp3. Foxp3 enhancers are known as distinct readers for environmental cues in promoting Treg cell induction or lineage stability. However, their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms determining Foxp3 expression and thereby Treg suppressive capacity uncertain. We examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize Treg cell induction, Foxp3 expression level, or lineage stability through distinct modes and that ablation of synergistic enhancers led to lethal autoimmunity in young mice. Thus, the induction and maintenance of a diverse, stable Treg cell repertoire rely on combinatorial Foxp3 enhancers, suggesting broad, stage-specific, synergistic activities of cell-intrinsic factors and -extrinsic cues in determining Treg suppressive capacity. Overall design: We examined the collective activities of Foxp3 enhancers and found CNS0 and CNS3 coordinate to maximize Treg cell induction. To characterize the impact of CNS0 and CNS3 single and double deficiencies on Treg cell TCR repertorie, we performed bulk TCR-sequencing of the total Treg cells isolated from the peripheral lymphoid organs of GFP, CNS0KO, CNS3KO, and CNS03DKO mice at 16-days of age. Treg cells sorted from spleen, skin draining lymph nodes, mesenteric lymph node were pooled for individual male mouse.