Normal Treg homeostasis and suppressive function require both FOXP1 and FOXP4.

FOXP3+ Treg cells are critical for immune tolerance. Genetic deletion of the Forkhead domain-containing proteins of the FOXP-subfamily member FOXP1 from Tregs results in impaired function associated with reduced CD25 expression and IL-2 signaling, but to date the only other FOXP family member expressed in Tregs, FOXP4, has been minimally studied. To investigate the potential functional interactions among FOXP family members in Tregs, we specifically deleted Foxp1, Foxp4, or both in FOXP3+ committed Tregs in mice. Our findings show that mice with combined, but not individual, deficiency in FOXP1 and FOXP4 exhibit lymphoproliferation, inflammation, autoimmunity, and early lethality. The combined absence of FOXP1 and FOXP4 in Tregs results in an activated/effector-like phenotype with compromised suppressive function in peripheral lymphoid organs, an enhanced germinal center response, and proinflammatory cytokine production. We further show that FOXP1 and FOXP4 bind to Il2ra promoter regions to regulate CD25 expression in Tregs. Through pairwise comparison among mouse strains with Treg-specific deletion of Foxp1, Foxp4, or both, our findings indicate a nonredundant but insufficient role of FOXP4 in Treg function.