CRISPR-based identification of factors influencing IL-22 and IL-17 expression in type III innate lymphoid cells

Abstract

Abstract Cytokine regulation of immune responses is influenced by multiple factors, including the target cell type and its microenvironment. Type III innate lymphoid cells (ILC3s) rapidly produce cytokines, including interleukin (IL)-22 and IL-17, in response to pathogens or injury. Dysregulation of IL-22 or IL-17 contributes to numerous pathologies, including autoimmune diseases and malignancies. Thus, a deeper understanding of the factors that regulate productive expression of IL-17/22 by ILCs or by their type III helper T cell counterparts is warranted. We have used a genome-wide CRISPR interference screen to elucidate genes that drive IL-22/17 expression by ILC3s. We engineered the murine ILC3-like line, MNK3, to inducibly express catalytically dead Cas9 (dCas9) linked to the transcriptional repressor KRAB. Following lentiviral transduction of a sgRNA library, cells were stimulated with IL-23 and IL-1b. Guides from cell populations sorted by levels of IL-17/22 expression were sequenced and analyzed for depletion (target may be necessary for cytokine expression, e.g., il17, jak3, and il23r) and enrichment (target’s suppression may promote cytokine expression). We are currently validating “hits” that are components of several biological pathways, including transcriptional regulation, chromatin modification, notch/wnt and cadherin signaling, and RNA splicing. Results will also be presented for a complementary CRISPR activation screen. These data will contribute new insights into the biological processes necessary for proper expression of type 3 cytokines by innate and adaptive lymphoid cells.