ERAP1-dependent extreme antigen processing efficacy can govern MHC class I expression hierarchy

Peptide presentation by major histocompatibility complex (MHC) class I molecules enables CD8+ T lymphocytes to monitor the intracellular proteome of tissue cells. CD8+ T cell priming and acquisition of effector functions is affected by cognate peptide-MHC-I complex density on the cell surface, which partly depends on the efficacy of intracellular proteolytic peptide generation. Peptide generation frequently requires final trimming by the human aminopeptidases ERAP1, ERAP2, and IRAP. All display genetic polymorphism associated with the risk of multiple autoimmune diseases but also some cancers. This finding has prompted interest in the development of small molecule inhibitors to enhance antitumor or conversely attenuate autoreactive T cell responses. However, efficient assays for assessment of inhibitor effects are wanting. We describe the development of an assay for quantitative assessment by flow cytometry of selective inhibitor effects on peptide trimming both in the endogenous MHC-I processing pathway and in cross-presentation. We use the assay to identify a selective ERAP2 inhibitor and show that inhibitor effects can be read out not only through assessing a specific peptide-MHC complexes but also by measuring cell surface levels of bulk MHC-I molecules. Next to its practical interest as tool for inhibitor testing, our assay highlights how ERAP1-dependent immunodominance of a single epitope processed with exceptional efficacy can have a massive effect on the immunopeptidomic identity of cells presented to CD8+ T cells. We propose that ERAP effects on the presentation of such rare and exceptionally immunodominant epitopes may underlie the epistatic genetic associations of ERAP polymorphism with HLA class I-linked autoimmune diseases.