Evidence of focusing the MHC class I immunopeptidome by tapasin

Major Histocompatibility Complex class I (MHC-I) molecules bind and present peptides to cytotoxic T cells, protecting against pathogens and cancer. MHC-I is highly polymorphic and each allotype is promiscuous, and capable of binding a unique and diverse repertoire of peptide ligands. The peptide editing chaperone tapasin optimizes this allotype specific repertoire of peptides, resulting in the selection of high affinity peptides. MHC-I allotypes differ in the extent they engage tapasin. This suggests that tapasin-dependent MHC-I allotypes should present a less diverse repertoire that is enriched in higher-affinity peptides, and which are present in higher abundance, than tapasin independent MHC-I allotypes, which should present a broader repertoire containing peptides with a lower average affinity. Experimental verification of this hypothesis has been confounded by the different peptide binding specificities of MHC-I allotypes. Here, we independently investigated the peptide focusing function of tapasin by introducing a point mutation into a tapasin independent MHC-I allotype that dramatically increased its tapasin dependence without substantially altering its peptide binding specificity. This allowed us to demonstrate ligand focusing by tapasin at both the repertoire level <i>in cellulo</i>, and by using an <i>in vitro</i> system in which tapasin was artificially tethered to MHC-I, at the individual peptide level. We found that tapasin had a greater influence on tapasin dependent MHC-I molecules, and that tapasin modulated peptide selection according to peptide-MHC-I complex stability, disfavoring short-lived peptide-MHC-I complexes. Thus, tapasin dependent MHC-I molecules experience greater tapasin filtering, resulting in less diverse MHC-I immunopeptidomes that are enriched in high affinity peptide-MHC-I complexes.