Foxp3+ T Cells Expressing RORγt Represent a Stable Regulatory T Cell Effector Lineage with Enhanced Suppressive Capacity during Intestinal Inflammation

Foxp3-expressing regulatory T cells (Treg) are essential for immunological tolerance, best illustrated by uncontrolled effector T cell responses and clinical presentation of systemic autoimmunity in mice and humans upon loss of Foxp3 expression. The vast majority of Foxp3⁺ Treg is already generated within the thymus, however, Treg can adopt specific effector phenotypes upon activation in the periphery, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3⁺CD4⁺ T cells coexpressing RORγt, the master transcription factor for inflammatory Th17 cells, represent a stable effector Treg lineage that is specifically enriched in the gut of mice possessing a complex microbial microflora. Transcriptomic and epigenetic profiling revealed that Foxp3⁺RORγt⁺ T cells display signatures of both Treg and Th17 cells, albeit the degree of similarity was higher to Foxp3⁺RORγt^- Treg than to Foxp3^-RORγt⁺ T cells. Importantly, Foxp3⁺RORγt⁺ 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⁺RORγt⁺ T cells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and in addition revealed a significantly enhanced suppressive capacity as compared to Foxp3⁺RORγt^- Treg. Thus, our data suggest that RORγt expression in Treg contributes to an optimal suppressive capacity during gut-specific immune responses, which renders Foxp3⁺RORγt⁺ T cells an important effector Treg subset in the intestinal system.