Helios⁺ and RORγt⁺ Treg populations are differentially regulated by MHCII, CD28, and ICOS to shape the intestinal...

Foxp3⁺ regulatory T cells (Tregs) are essential for intestinal homeostasis. Tregs in the small intestine include Helios⁺ thymus-derived Tregs (tTregs) and RORγt⁺ Tregs that differentiate in the periphery after antigenic stimulation (pTregs). TCR and costimulatory signals sustain Tregs with effector phenotypes, including those in the intestine, but it is unknown if tTregs and pTregs have similar requirements for these pathways. We previously used mice lacking peripheral expression of MHCII to demonstrate that the small intestine sustains tTregs independently of peripheral antigen. Here, we show that the effector phenotype and tissue-resident signature of tTregs are also MHCII-independent. Using this model, we define the distinct costimulatory requirements of intestinal tTregs and pTregs. Helios⁺ effector tTregs proliferate through CD28 and require neither ICOS nor MHCII for maintenance. In contrast, RORγt⁺ pTregs use CD28 and ICOS. Notably, the differential costimulatory utilization allows tTregs and pTregs to dynamically respond to perturbations to support a fixed number of intestinal Tregs. This suggests that the environmental regulation of costimulatory ligands might shape the subpopulations of intestinal Tregs and promote effective homeostasis and defense. Our data reveal new complexity in effector Treg biology and costimulatory signaling of tTregs and pTregs and highlight the importance of analyzing both subpopulations.