Cytokine-mediated activation of host immunity is central to the control of pathogens. Interferon-gamma (IFNγ) 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 IFNγ-induced MHCII is not well understood. Using a genome-wide CRISPR-Cas9 screen in murine macrophages, we identified genes that control MHCII surface expression. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase three beta (GSK3β) or the mediator complex subunit 16 (MED16). Both pathways control distinct aspects of the IFNγ response and are necessary for IFNγ-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.

Chronic bacterial infections are caused by pathogens that persist within their hosts and avoid clearance by the immune system. Treatment and/or detection of such pathogens is difficult, and the resulting pathologies are often deleterious or fatal. There is an urgent need to develop protective vaccines and host-directed therapies that synergize with antibiotics to prevent pathogen persistence and infection-associated pathologies. However, many persistent pathogens, such as Mycobacterium tuberculosis, actively target the very host pathways activated by vaccination. These immune evasion tactics blunt the effectiveness of immunization strategies and are impeding progress to control these infections throughout the world. Therefore, it is essential that M. tuberculosis immune evasion-related pathogen virulence strategies are considered to maximize the effectiveness of potential new treatments. In this review, we focus on how Mycobacterium tuberculosis infects antigen-presenting cells and evades effective immune clearance by the adaptive response through (i) manipulating antigen presentation, (ii) repressing T cell-activating costimulatory molecules, and (iii) inducing ligands that drive T cell exhaustion. In this context, we will examine the challenges that bacterial virulence strategies pose to developing new vaccines. We will then discuss new approaches that will help dissect M. tuberculosis immune evasion mechanisms and devise strategies to bypass them to promote long-term protection and prevent disease progression.

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The activation of macrophages by IFNγ induces transcriptional and metabolic changes that enable the direct control of pathogens and improves interactions with T cells. In particular, IFNγ induces the expression of antigen presentation machinery like MHCII. Despite its central role in host immunity, the regulation of IFNγ-induced MHCII is poorly understood. Using CRISPR Cas9 screens we examined regulators of distinct IFNγ-mediated mechanisms in macrophages including MHCII. One key regulator found was the multifunctional kinase, GSK3β. Not only was GSK3β required to induce transcriptional activation of MHCII, but RNAseq analysis suggests broad changes to the IFNγ response in the absence of GSK3β. Recent work in our group suggests that changes in metabolism are essential for macrophages to respond to IFNγ. While GSK3β is known to directly modulate metabolism, its role in controlling metabolic changes following IFNγ activation remains unknown. Given that GSK3β interfaces with metabolic pathways like mTORC1 and glycolysis we hypothesize that the loss of GSK3β inhibits important metabolic shifts required for macrophages to effectively respond to IFNγ. Using a Seahorse Analyzer we are now determining how genetic or chemical inhibition of GSK3β changes the rate of glycolysis or oxidative phosphorylation following IFNγ stimulation. We will then directly target metabolic networks in the presence and absence of GSK3β to determine how these impact IFNγ responses including the expression of MHCII. Given the broad impact of GSK3β on several disease states, such as inflammatory disease and tumor progression, understanding the regulation of GSK3β has the potential to contribute to several aspects of human health.

The IFNg-dependent induction of MHCII expression is critical for CD4+ T cell function. Dysregulation of MHCII is associated with autoimmunity, graft versus host disease, and increased susceptibility to cancers and chronic infections. Despite its central role in host immunity, the complex and dynamic regulation of IFNg-induced MHCII is poorly understood. We hypothesize that modulating MHCII regulatory pathways in macrophages will directly alter T cell effector function. Our goal is to dissect MHCII regulation to identify new therapeutic targets that will improve MHCII disease states, antigen presentation, and host immune responses against persistent infections. Using a CRISPR screen we identified new MHCII regulators including the multifunctional kinase GSK3 and the mediator complex subunit Med16. Mechanistic studies found that both GSK3 and Med16 are essential for IFNg-mediated Ciita induction and for macrophages to serve as antigen presenting cells for CD4+ T cells. Using the GSK3 inhibitor CHIR99021 in Med16 KO macrophages we observed further inhibition of MHCII expression suggesting GSK3 and Med16 function in parallel networks. To test this directly, RNAseq was used to globally profile the transcriptomes in Med16 and GSK3 KO cells in the presence and absence of IFNg. We found that Med16 and GSK3 control distinct transcriptional networks without disrupting canonical IFNg transcription factors like Stat1, Stat3 and IRF1. The RNAseq also suggested IFNg dependent control of cytokines are disrupted in the absence of GSK3 and Med16 which we confirmed using Mycobacterium tuberculosis infection. Thus, GSK3 and Med16 are parallel regulators that balance IFNg responses in macrophages and modulate adaptive immune responses.