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

细胞因子介导的宿主免疫激活是病原体防控的核心环节。干扰素-γ（Interferon-gamma, IFNg）是保护性免疫中的关键细胞因子，可诱导主要组织相容性复合体Ⅱ类分子（major histocompatibility complex class II molecules, MHCII）表达，以放大CD4+ T细胞的激活与效应功能。尽管其作用至关重要，但IFNg诱导的MHCII的动态调控机制仍未得到充分阐明。我们在小鼠巨噬细胞中开展全基因组CRISPR-Cas9筛选，全局鉴定了调控MHCII表面表达的基因。机制研究揭示了两条平行的IFNg介导的MHCII调控通路，二者分别依赖多功能糖原合成酶激酶3β（glycogen synthase kinase 3 beta, GSK3b）或中介体复合物亚基MED16。两条通路分别调控IFNg应答的不同方面，且均为IFNg介导的MHCII反式激活因子CIITA的诱导、MHCII表达以及CD4+ T细胞激活所必需。我们的研究明确了此前未被认知的、调控CD4+ T细胞应答所需的MHCII表达调控机制。实验设计：对调控干扰素-γ应答的CRISPR突变体小鼠巨噬细胞进行RNA测序分析。本研究提交了对照组（非靶向）永生化巨噬细胞，或经CRISPR构建的敲除（sgGSK3b或sgMed16）永生化巨噬细胞在有或无干扰素-γ刺激条件下的RNA测序数据。于干扰素-γ刺激24小时后分离总RNA。我们在永生化C57BL/6巨噬细胞中构建了非靶向或靶向Med16、GSK3b的CRISPR靶向细胞系，敲除效果经TIDE分析（Tracking of Indels by Decomposition）验证。我们检测了经干扰素-γ处理24小时后，不同基因型细胞间的基因差异表达以及细胞因子激活情况。每种基因型与处理条件均设置生物学重复三次，因此每种基因型共获得6个样本：3个未经干扰素-γ处理的样本，3个经干扰素-γ处理的样本。于处理24小时后分离总RNA，并进行RNA测序分析。