Ror?t-positive dendritic cells are required for the induction of peripheral regulatory T cells in response to oral antigens

The intestinal immune system maintains tolerance to harmless food proteins and gut microbiota through peripherally-derived ROR?t+Tregs (pTregs), which prevent food intolerance and inflammatory bowel disease. Recent studies suggested that ROR?t+ antigen-presenting cells (APCs), which encompass rare dendritic cell (DC) subsets and type 3 innate lymphoid cells (ILC3s), are key to pTregs induction. Here, we developed a mouse with reduced ROR?t+APCs by deleting a specific cis-regulatory element of Rorc encoding ROR?t. Single-cell RNA-sequencing and flow cytometry analyses confirmed the depletion of a ROR?t+DC subset and ILC3s. These mice showed a secondary reduction in pTregs, impaired tolerance to oral antigens and increase in Th2 cells. Conversely, newly and previously generated ILC3-deficient mice showed no pTregs or Th2 cells abnormalities. Lineage tracing revealed that ROR?t+DCs share a lymphoid origin with ILC3s, consistent with their similar phenotypic traits. These findings highlight a unique role of lymphoid ROR?t+DCs in maintaining intestinal immune balance and preventing conditions like food allergies. Overall design: We performed single-cell RNA sequencing on sort purified CD3-B220-CD11c+MHC-II+ DCs as well as CD3-B220-CD11c-MHC-II+ CCR6+ ILC3 extracted from mLNs of 15days old Rorc+7kbE WT/WT and Rorc+7kbE KO/KO mice. Each line included four biological replicates, uniquely identified by the labeling of four distinct Hashtag antibodies.