RORγt⁺ APCs require a distinct cis-regulatory element to instruct tolerance to dietary antigens

Oral tolerance represents a hallmark of intestinal mucosal immunity to prevent inflammatory responses to harmless natural antigens, such as dietary components or commensal organisms. According to recent studies, RORγt⁺ antigen-presenting cell (APC) contributes to intestinal homeostasis, including oral tolerance, through inducing microbiota- and dietary antigen-specific Tregs. Here we identify a cis transcriptional regulatory element that distinguishes RORγt⁺ APCs from other of RORγt⁺ cell types. This sequence within Rorc gene loci, OCR369 governs RORγt expression in ILC3s and other RORγt⁺ APCs, but not T cells, through interaction with RUNX3 and formation of chromatin loops. OCR369 deletion results in a significant reduction of RORγt⁺ APCs in mLN around the weaning period and ILC3s in mLN and intestines of adult mice, accompanied by a decrease in RORγt⁺ Tregs and spontaneous inflammation in the small intestine. Mechanistically, the reduction in RORγt⁺ APCs, including both DC-like cells and MHCII⁺ ILC3s, impairs the development of both dietary antigen-specific and microbiota-specific RORγt⁺ Tregs and results in the loss of oral tolerance, thereby increasing allergy susceptibility. Thus, our findings identify a specific regulatory mechanism for RORγt expression in RORγt⁺ APCs and underscore the pivotal role of these cell types in mediating oral tolerance and maintaining intestinal health.