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 (Treg 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 Treg cell-mediated immune homeostasis.