Foxp3+ T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation

Foxp3 (forkhead box P3 transcription factor)-expressing regulatory T cells (Tregs) are essential for immunological tolerance, best illustrated by uncontrolled effector T-cell responses and autoimmunity upon loss of Foxp3 expression. Tregs can adopt specific effector phenotypes upon activation, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3(+)CD4(+) T cells coexpressing retinoic acid-related orphan receptor-gamma t (ROR gamma t), the master transcription factor for T helper type 17 (Th17) cells, represent a stable effector Treg lineage. Transcriptomic and epigenetic profiling revealed that Foxp3(+)ROR gamma t(+) T cells display signatures of both Tregs and Th17 cells, although the degree of similarity was higher to Foxp3(+)ROR gamma t(-) Tregs than to Foxp3(-)ROR gamma t(+) T cells. Importantly, Foxp3(+)ROR gamma t(+) T cells were significantly demethylated at Treg-specific epigenetic signature genes such as Foxp3, Ctla-4, Gitr, Eos, and Helios, suggesting that these cells have a stable regulatory rather than inflammatory function. Indeed, adoptive transfer of Foxp3(+)ROR gamma t(+) Tcells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and revealed an enhanced suppressive capacity as compared with Foxp3(+)ROR gamma t(-) Tregs. Thus, our data suggest that ROR gamma t expression in Tregs contributes to an optimal suppressive capacity during gut-specific immune responses, rendering Foxp3(+)ROR gamma t(+) T cells as an important effector Treg subset in the intestinal system.