Defining IL22 regulatory logic in type 3 lymphoid cells

Abstract

Abstract The cytokine IL-22 is a major effector expressed by type 3 immune cells (T helper 17, Th17 and type 3 innate lymphoid cells, ILC3s), and has diverse roles in maintaining intestinal homeostasis, defense against pathogens, cell proliferation, and tissue regeneration. Perturbations in the homeostatic levels of IL-22 have been associated with various inflammatory disorders and tumors. However, significant gaps remain in our understanding about the regulatory mechanisms controlling IL-22 expression by type 3 cells in health and disease. In this regard, we generated a stable clone of Mnk3, a mouse ILC3-like cell line, which harbored a doxycycline-inducible version of catalytically dead Cas9 nuclease (dCas9) fused with KRAB repressor domain. We performed a CRISPRi screen in this dCas9-KRAB Mnk3 line by infecting it with a lentiviral library of sgRNAs targeting IL22 regulatory space. Our CRISPRi screen identified a set of candidate enhancers important for IL22 expression in type 3 cells. Chromatin profiling data suggested a differential pattern of activities for these enhancer elements in immune cell subsets. We have now deleted several enhancers from the germline of mice and will present results describing the impact of these mutations on IL-22 expression in type 3 lymphoid cells from the innate and adaptive immune systems. Collectively, this study defines the cis-regulatory circuits controlling IL22 in type 3 immune cells and thus will open new avenues for therapeutic intervention in IL-22 pathologies.