A genome-wide screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation

Abstract

Cytokine-mediated activation of host immunity is central to the control of pathogens. A key cytokine in protective immunity is interferon-gamma (IFNγ), which is a potent activator of antimicrobial and immunomodulatory effectors within the host. A major role of IFNγ is to induce major histocompatibility complex class II molecules (MHCII) on the surface of cells, which is required for CD4+ T cell activation. Despite its central role in host immunity, the complex and dynamic regulation of IFNγ-induced MHCII is not well understood. Here, we integrated functional genomics and transcriptomics to comprehensively define the genetic control of IFNγ-mediated MHCII surface expression in macrophages. Using a genome-wide CRISPR-Cas9 library we identified genes that control MHCII surface expression, many of which have yet to be associated with MHCII. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase 3 beta (GSK3β) or the mediator complex subunit MED16. Both pathways are necessary for IFNγ-mediated induction of the MHCII transactivator CIITA, MHCII expression, and CD4+ T cell activation. Using transcriptomic analysis, we defined the regulons controlled by GSK3β and MED16 in the presence and absence of IFNγ and identified unique networks of the IFNγ-mediated transcriptional landscape that are controlled by each gene. Our analysis suggests GSK3β and MED16 control distinct aspects of the IFNγ-response and are critical for macrophages to respond appropriately to IFNγ. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses by macrophages. These discoveries will aid in our basic understanding of macrophage-mediated immunity and will shed light on mechanisms of failed adaptive responses pervasive in infectious disease, autoimmunity, and cancer.

Mitochondrial respiration contributes to the interferon gamma response in antigen-presenting cells

“…To investigate the diverse regulatory pathways underlying the IFNγ response, we examined the expression of three functionally distinct cell surface markers that are induced by IFNγ. These studies utilized a J2 virus transformed bone marrow derived macrophage line that expressed Cas9 (75). Stimulation of these macrophages with IFNγ for 24 hours resulted in the upregulation of T cell stimulatory molecules, major histocompatibility complex class II (MHCII) and CD40, and the inhibitory ligand PD-L1 (Cd274), on the cell surface (Figure 1A).…”

Mitochondrial respiration contributes to the interferon gamma response in antigen presenting cells

“…For example, genes previously shown to specifically control MHCII transcription, such as Ciita , Rfx5 , Rfxap , Rfxank , and Creb1 (8, 76–78) were found in Cluster 4 along with several novel regulators that appear to be specifically required for this pathway. MHCII-specific factors are reported in an accompanying study (79).…”

“…A clonal macrophage cell line stably expressing Cas9 (L3) was established as described elsewhere(79). A plasmid library of sgRNAs targeting all protein coding genes in the mouse genome (Brie Knockout library, Addgene 73633) was packaged into lentivirus using HEK293T cells.…”

“…Sequence reads were trimmed to remove adapter sequence and to adjust for staggered forward (p5) primer using Cutadapt v2.9. Raw sgRNA counts for each sorted and unsorted (input library) population was quantified using bowtie2 via MAGeCK to map reads to the sgRNA library index (no mismatch allowed); a sgRNAindex was modified to reflect genes transcribed by our macrophage cell line either basally or upon stimulation with IFNγ as previously published(79). Counts for sgRNAs were median normalized to account for variable sequencing depth.…”

“…Briefly, annealed oligos containing the sgRNA targeting sequence were phosphorylated and cloned into a dephosphorylated and BsmBI (New England Biolabs) digested SgOpti (Addgene#85681) which contains a modified sgRNA scaffold for improved sgRNA-Cas9 complexing. Use of sgOpti derivatives for delivery of multiple sgRNAs was performed as detailed elsewhere(79). The sgRNA targeting sequences used for cloning were as follows:…”