A genetic screen in macrophages identifies new regulators of IFNg-inducible MHCII that contribute to T cell activation

Cytokine-mediated activation of host immunity is central to the control of pathogens. Interferon-gamma (IFNg) is a key cytokine in protective immunity that induces major histocompatibility complex class II molecules (MHCII) to amplify CD4+ T cell activation and effector function. Despite its central role, the dynamic regulation of IFNg-induced MHCII is not well understood. Using a genome-wide CRISPR-Cas9 screen in murine macrophages we globally identified genes that control MHCII surface expression. Mechanistic studies uncovered two parallel pathways of IFNg-mediated MHCII control that require the multifunctional glycogen synthase kinase 3 beta (GSK3b) or the mediator complex subunit MED16. Both pathways control distinct aspects of the IFNg response and are necessary for IFNg-mediated induction of the MHCII transactivator CIITA, MHCII expression, and CD4+ T cell activation. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses. Overall design: RNAseq analysis in murine macrophages of CRISPR mutants that regulate IFN gamma responses. We report RNAseq analysis on control (non-targeted) immortalized macrophages or knockout (sgGSK3b or sgMed16) immortalized macrophages generated using CRISPR in the presence or absence of IFN-gamma stimulation. Total RNA was isolated 24 hours following IFN-gamma stimulation. We generated CRISPR targeted cell lines in immortalized C57bl/6 macrophages that were either non-targeting or targeting Med16 or GSK3b. Knockouts were confirmed by TIDE analysis. We examined the differential expression of genes between genotypes and cytokine activation after 24 hours of IFN-gamma treatment. There are biological triplicates for each genotype and condition resulting in 6 samples per genotype with 3 samples without IFN gamma treatment and 3 samples with IFN gamma Treatment. Total RNA was isolated 24 hours after treatment and processed for RNAseq analysis.