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. However, the underlying mechanisms governing oral tolerance remain incompletely understood. Recent studies have shown that RORγt⁺ antigen-presenting cells (APCs) contribute to intestinal homeostasis through inducing microbiota-specific Tregs. Whether RORγt⁺ APCs can regulate dietary antigen-specific Tregs and thereby mediating oral tolerance remains unclear. Here, by comparing ATAC-seq data within Rorc gene loci between RORγt⁺ cell types, we identified a distinct cis-regulatory element, OCR369, which specifically regulates RORγt expression in ILC3s and other RORγt⁺ APCs, but not T cells, through interaction with RUNX3 and formation of chromatin loops. OCR369 deletion resulted in a significant reduction of RORγt⁺ APCs in mLN around the weaning period and ILC3s in mLN and intestines of adult mice, accompanied by decreased RORγt⁺ Tregs and spontaneous inflammation in the small intestine. Mechanistically, the reduction in RORγt⁺ APCs, including both DC-like cells and MHCII⁺ ILC3s, impaired the development of both dietary antigen-specific and microbiota-specific RORγt⁺ Tregs and resulted in a 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.