Decrypting MHC class I trafficking in cross-presentation

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Cross-presentation of antigens internalized by dendritic cells (DCs) is critical for priming of CD8+ T cell responses to pathogens and tumors, and for immune tolerance. Despite many published studies, the trafficking pathways and loading compartments for cross-presentation by MHC-I molecules remain unclear. Preliminary evidence suggests that cross-presentation may employ diverse MHC-I sources and trafficking pathways, including newly synthesized MHC-I molecules routed through conventional and non-conventional secretory and/or Golgi-to-endosome pathways, but also MHC-I stores in different subtypes of endosomes, and recycling MHC-I molecules, depending on the antigen entry route and the cell type investigated. This project will bring together two teams with documented strong expertise in studying trafficking of MHC-I molecules. The teams will employ their complementary expertise together with an array of cross-presentation assays to decipher the origin, loading compartments, and travel to the cell surface of MHC-I molecules loaded with cross-presented peptides derived from two antigens (ovalbumin and insulin) internalized by distinct mechanisms (phagocytoseis, mannose receptor endocytosis, Fc receptor endocytosis, pinocytosis). The development of novel assays using the RUSH (retention using selective hooks) and SpyTag/SpyCatcher systems will enable the teams to study synchronized intracellular trafficking as well as internalization of covalently labeled cell surface MHC-I molecules. If possible, the impact of key trafficking pathways will be assessed using conditional deletion models in the context of antitumor vaccination in vivo. Ultimately, the results obtained might be instrumental for pharmacological manipulation of cross-presentation with the objective to up-regulate antitumor immune responses or target antigens into specific processing pathways but also to down-regulate cross-presentation of self- antigens and consequently autoimmune inflammation.