Reciprocal Transcription Factor Networks Govern Tissue-Resident ILC3 Subset Function and Identity

Innate lymphoid cells (ILCs) play important roles in health and tissue homeostasis. While the transcriptional networks that drive their development have been defined, the transcriptional requirements that support the phenotypes and functions of mature tissue-resident populations remain poorly understood. Here we employed combinatorial inducible deletion of core transcription factors (TFs) to test how co-operative and antagonistic networks operate to control function and identity. Utilizing single cell sequencing and epigenetic profiling we dissect the complex interplay between ROR family member TFs and T-bet in determining the function and phenotype of ILC3 and NCR-lineage ILCs, defining the ontogeny and surface phenotype across a continuum of ILC3s and mature ILC1s in the small intestine, alongside the differential requirements for ROR family TFs in the maintenance of effector functions and phenotype in mature ILCs. Combined, our data demonstrates how TF networks integrate in vivo to sustain effector functions and restrict alternative differentiation programmes. Overall design: Comparison of chromatin accessibility in small intestinal innate lymphoid cells expressing or lacking core lineage-defining transcription factors (TFs). Gene encoding core TFs are flanked with LoxP sites and can be deleted by the Cre recombinase (inducible action by administration of tamoxifen). The experiment contain biological replicates (2 or 3), each of them containing samples from 1 or 2/3 pooled mouse intestines.