RORγt⁺ 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 identified RORγt⁺ 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 RORγt⁺ innate lymphoid progenitor (RILP) that generates both ILC3s and RORγt⁺ DCs, and a pre-RORγt⁺ DC precursor committed exclusively to the RORγt⁺ DC lineage. RORγt⁺ DC development required the Rorc +7-kilobase enhancer, whose accessibility was regulated by the repressors REV-ERBα and REV-ERBβ, and depended on the transcription factors PRDM16 and PU.1 for lineage commitment. Loss of any of these regulators abrogated RORγt⁺ DC differentiation, reduced peripheral regulatory T cell (T_reg cell) induction, and skewed toward T helper 2 responses. Together, these findings define murine RORγt⁺ DCs as a lymphoid-derived lineage whose enhancer- and transcription factor-driven development is essential for peripheral T_reg cell-mediated immune homeostasis.