Evidence of focusing the MHC class I immunopeptidome by tapasin

Rachel Darley¹Patricia T Illing²Patrick Duriez³Alistair Bailey¹Anthony Wayne Purcell²Andy van Hateren¹Tim Elliott^4,5*

• ¹Institute for Life Sciences and Centre for Cancer Immunology, University of Southampton, Southampton, Hampshire, United Kingdom
• ²Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing & Health Sciences, Monash University, Clayton, Victoria, Australia
• ³Cancer Research UK Protein Core Facility, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
• ⁴Nuffield Department of Medicine, Medical Sciences Division, University of Oxford, Oxford, England, United Kingdom
• ⁵Centre for Immuno-Oncology and CAMS-Oxford Institute, Oxford, United Kingdom

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 optimises 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 in cellulo, and by using an in vitro 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, disfavouring 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.