ROR?t? Dendritic Cells Are a Distinct Lymphoid-Derived Lineage

How tolerogenic dendritic cell (DC) lineages are established to prevent inappropriate immune responses to commensals and food antigens remains unclear. We identify ROR?t? DCs in mice as a distinct lymphoid-derived lineage to safeguard intestinal tolerance. Using lineage tracing and single-cell transcriptomics, we unveiled bone marrow–resident Rorc(t)? progenitors, which include a ROR?t? innate lymphoid progenitor (RILP) that generates both ILC3s and ROR?t? DCs, and a pre-ROR?t? DC precursor committed exclusively to the ROR?t? DC lineage. ROR?t? DC development required the Rorc +7 kb enhancer, whose accessibility was ensured by the repressors REV-ERBa and REV-ERBß, and depended on the transcription factors PRDM16 and PU.1 for lineage commitment. Loss of any of these regulators abrogated ROR?t? DC differentiation, reduced peripheral regulatory T (Treg) cell induction, and skewed toward T helper 2 responses. Together, these findings define murine ROR?t? DCs as a lymphoid-derived lineage whose enhancer- and transcription factor–driven development is essential for peripheral Treg cell-mediated immune homeostasis. Overall design: We performed single-cell RNA sequencing on B220– CD3– Ly6C– tdTomato+ hCD2+ cells (ILC3s and ROR?t+ DCs) isolated from bone marrow cultures of 15-day-old Gm38411 iCre-hCD2 R26-tdTomato mice after 8 days of culture.