A hierarchical Rorc(γt) cis-regulatory cascade orchestrates differentiation of RORγt⁺ innate immune cells

Beyond their well-established roles in type 3 immunity, RORγt⁺ innate immune cells are also essential for secondary lymphoid organ (SLO) formation and gut homeostasis. However, the transcriptional mechanisms governing RORγt expression in these cells, including group 3 innate lymphoid cells (ILC3s), lymphoid tissue inducer (LTi) cells, and antigen-presenting cells (APCs), remain largely unresolved. Here, we identified two key cis-regulatory elements within conserved non-coding sequences (CNS)9 and 11 in the Rorc locus, which were sequentially utilized during differentiation. Initially, Runx-binding sites in CNS11 established chromatin accessibility as early as the hematopoietic stem cell (HSC) stage. Disruption of this chromatin priming prevented subsequent transcriptional activation, thereby abolishing the initial induction of RORγt in these cells. At later stages, CNS9 played a critical role, particularly in the development of RORγt⁺ APCs, contributing to colonic peripheral Treg (pTreg) cell induction. This hierarchical transcriptional regulation was essential for SLO formation and postnatal type 3 immunity and for restraining excessive intestinal type 2 immune responses through pTreg cell induction.