Mechanism and in vitro reconstitution of tapasin-mediated peptide exchange

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Presentation of antigens to T cells is provided by proteins encoded by the Major Histocompatibility Complex (MHC). The alleles of this highly polymorphic gene locus lead to the expression of thousands of different MHC allotypes across the population. Each allotype is capable of binding to a manifold of different antigenic peptides. When displayed on the cell surface, they can be surveilled by T cells, potentially invoking the cellular arm of adaptive immunity. Of the two major classes of canonical MHC molecules, MHC class I (MHC-I), which are present on all nucleated cells, typically derive their antigens from the proteasome. Peptides of 9-15 amino acid in length after proteasomal cleavage enter the endoplasmic reticulum through the transporter associated with antigen processing, are further trimmed by the aminopeptidase ERAP, and are loaded onto the MHC-I molecules in the peptide loading complex (PLC). At the heart of the PLC, the exchange catalyst tapasin stabilizes the MHC-I molecules and acts to replace lower affine by higher affine peptides, thus influencing the MHC-I displayed immunopeptidomes and ultimately T cell reactivity. The binding and peptide exchange activity of tapasin strongly differs for the individual MHC-I allotypes and raises the question of the mechanism behind allotypic distinction. Here, this central question is first addressed by structural biology and biochemical methods. Several human and rodent MHC-I molecules will be investigated for their binding and exchange susceptibility to tapasin. Furthermore, an in vitro reconstituted system will be developed that comprises essential components of the MHC-I loading complex. Establishing such a minimalistic experimental system allows for the identification of MHC-I bound peptidomes derived from viral proteins or from tumor proteins featuring neo-antigens. The tapasin dependency of MHC-I-peptide display will be measured and combined with the mechanistic insights to derive allotype-specific features. This knowledge can either be used to make individualized predictions of MHC-I presentation of proteins from novel viruses or patient-specific tumor neo-antigens or it will allow to develop tools that can be used to visualize, enrich, or expand antigen-specific T cell clones.