Defining the four major routes of MHC class II antigen processing and presentation with influenza antigens - PROJECT SUMMARY CD4+ T cells (TCD4) orchestrate adaptive immune responses to infectious agents and cancers and instigate most autoimmune diseases. Thus, greater mechanistic insight into the drivers of TCD4 activation will be broadly bene- ficial. TCD4 are stimulated by antigen-derived peptides (“epitopes”) displayed at the surfaces of antigen-presenting cells (APCs) in combination with major histocompatibility complex class II (MHCII) molecules. By convention, peptide display entails internalization and proteolysis of extracellular antigen, loading of the resultant peptides onto MHCII in the late endosome, and transit of peptide:MHCII (p:MHCII) complexes to the cell surface. In truth, p:MHCII production is far more complex. The classical route is made up of many distinct pathways, and there are 3 multifaceted, nonclassical routes: 1) a recycling route: antigens that unfold in the early endosome are captured by MHCII in that compartment. 2) an endogenous network: antigens located to the APC cytoplasm, typically through biosynthesis, are converted to p:MHCII via an array of intracellular pathways, and 3) an “indi- rect” route: material from an infected non-APC is transferred to an uninfected APC. Despite their obscurity, these non-classical routes can play essential roles in TCD4 activation. Poor mechanistic understanding of all 4 routes, even the classical, has precluded their incorporation into many models of adaptive immunity or strategies to modulate TCD4+ responses. Here we propose to transform the MHCII processing and presentation landscape by testing the hypotheses that: (A) the degree to which cellular components contribute to epitope production and display is highly variable - from far reaching (impacting many epitopes) to highly focused (impacting only one epitope or a small set), and (B) processing and presentation pathways within one route or within functionally adjacent routes (e.g., classical and recycling), share more cellular components than disparate routes (e.g., clas- sical and endogenous). Drawing from our influenza (flu) antigen system, we have carried out an siRNA-based high throughput screen (HTS) for proteins involved in generation of one classical (“S1”) and one endogenous (“NA79”) epitope. Consistent with our hypothesis, hits are largely non-overlapping. Aim 1 of this project is to solidify this screen by validating key hits. Aim 2 is to expand the landscape by screening 6 flu epitopes that comprehensively encompass the 4 major routes. Instead of an siRNA screen, in Aim 2 we will use genome-wide CRISPR-Cas9 knockout and select hits via FACS using a panel of T cell receptor-like antibodies in concert with single-cell illumina sequencing. Outcomes from both aims will contribute to Gene Set Enrichment Analysis that will substantially revise the MHCII processing and presentation landscape with respect to both scope and reso- lution. Outcomes will launch many new avenues of investigation that explore: a) additional epitopes from flu and other pathogens, b) mechanisms underlying key hits and pathways, c) the impact on TCD4 activation of knocking out, in vivo, high priority hits, and d) development of therapeutics (e.g., small molecules) to enhance vaccine efficacy and anti-cancer immunity, and to diminish autoimmune diseases.
