High-throughput screening for class I peptide MHC binding via yeast surface display

T cells rely on short peptides presented by highly polymorphic major histocompatibility complexes (MHCs) to selectively initiate adaptive immune responses. Despite its importance, few techniques can systematically evaluate stable peptide presentation across diverse MHC alleles. Here, we describe a yeast display pipeline that can be deployed to rapidly screen peptides to identify class I pMHC binders across many alleles. Through this, we isolate unique biological phenomena such as alteration of the peptide presentation of HLA-B57 via interaction with the antiviral small molecule abacavir. We apply this approach to multiple pathogen proteomes (<i><i>Mycobacterium tuberculosis</i></i> Type VII secretion substrates, SARS-CoV-2, Dengue, and Zika) to create a comprehensive list of potential T cell antigens. Altogether, this platform acts as a flexible tool to generate large unbiased datasets for class I peptide binding at a speed and scale competitive with the biological systems they represent.