RORγt⁺ Dendritic Cells Are a Distinct Lymphoid-Derived Lineage [CITE-seq]

How tolerogenic dendritic cell (DC) lineages are established to prevent inappropriate immune responses to commensals and food antigens remains unclear. We identify 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 kb enhancer, whose accessibility was ensured 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 (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.