Abstract Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory, and malignant diseases 1–8 . Here, we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that are specific for the microbiota and express the transcription factor RORγt. We profiled all RORγt + immune cells at single cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of Tregs and lymphoid tissue-induced (LTi)-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s have interconverting potential with RORγt + extrathymic Aire-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt + Tregs and prevent their expansion as inflammatory T helper (Th)17 cells. This occurs through ILC3-mediated antigen-presentation, interleukin-2 gradients, and α v integrin. Finally, single-cell analyses demonstrate that ILC3 and RORγt + Treg interactions are impaired in inflammatory bowel disease. Our results define a novel paradigm whereby ILC3s positively select for antigen specific RORγt + Tregs, and against Th17 cells, to establish immune tolerance to the microbiota and intestinal health.

ILC3s select for RORγt+ Tregs and establish tolerance to intestinal microbiota