IRAPDC

MHC class I molecules present peptides produced by intracellular degradation of antigenic proteins to CD8+ T lymphocytes, which can mount cytotoxic responses against cells displaying non-self peptides derived from pathogens or tumour-associated proteins. While most cell types display exclusively peptides derived from endogenous proteins, dendritic cells (DCs) are endowed with an additional pathway that allows them to process internalized proteins for MHC class I presentation, a phenomenon referred to as cross-presentation. Cross-presentation is thought to play an important role in several contexts, including priming of CD8+ cells against pathogens that do not directly infect DCs, and in maintaining self-tolerance to engulfed apoptotic cells. In addition, the cross-presentation pathway is important for uptake and processing of DNA and protein vaccines. Therefore, understanding the cell biological and molecular workings of this pathway has direct biomedical and biotechnological relevance. However, despite substantial efforts in many laboratories, the underlying mechanisms remain unclear. One significant obstacle to deciphering these mechanisms is the lack of a marker that would clearly distinguish the cross-presentation from the endogenous MHC class I processing pathway. Thus, all proteases and chaperones presumably involved in cross-presentation also act in the endogenous pathway. We have recently found that insulin-regulated aminopeptidase (IRAP), an enzyme localised exclusively in a specific endosomal storage compartment, plays an important role in cross-presentation of antigens internalised via phagocytosis or receptor-mediated endocytosis by DCs, but not in endogenous MHC class I presentation (Saveanu et al., in revision for Science). IRAP is an efficient trimming peptidase for precursors of MHC class I ligands that is strongly recruited to early phagosomes, co-localises with internalised MHC class I molecules but not with various markers of the endoplasmic reticulum (ER) or endolysosomes, and interacts physically with MHC class I molecules. Therefore, IRAP is a specific marker for an endosomal compartment previously not implicated in cross-presentation, that is distinct from the 'vacuolar compartment' and may represent a site not only for final peptide trimming, but also for MHC class I loading with peptides produced in the proteasome-dependent cross-presentation pathway. In this project, we will exploit the specific localisation and function of IRAP as a tool for characterisation of the cross-presentation compartment as well as for studying the global impact of cross-presentation on immune repertoires and responses. The project is organised in four Specific Aims and will be carried out by six scientific workers: the principal investigator, a staff scientist INSERM, two already recruited PhD students, one post-doctoral scientist that remains to be recruited and a technician. Task 1. Functional and structural characterisation of MHC class I ligand precursor trimming by IRAP. Here we will undertake a functional and structural analysis of the aminopeptidase activity of IRAP towards precursors of MHC class I ligands. This includes analysis of the specificity of IRAP, as well as examination of the composition and function of the complexes formed between IRAP and MHC class I molecules. Task 2. Characterising IRAP expression and regulation, and its role in cross-presentation. We will examine the expression of IRAP in different types of antigen presenting and other cells, and study regulation of IRAP expression and intracellular localisation as well as the relationship between IRAP and MHC class I trafficking, and cross-presentation. We hypothesise that dynamic regulation of the IRAP+ compartment may be coordinated with activation and maturation in DCs. Task 3. The role of IRAP in the generation of peptide and T cell repertoires, and anti-infectious responses. We intend to explore the global impact of IRAP on the repertoire of peptides presented by DCs, on the repertoire of CD8+ T cells, and on the efficacy and specificity of anti-infectious immune responses. These studies, in which collaboration with two European groups will play an important role, are designed to provide insight into the overall impact of cross-presented peptides on the normal functioning of the adaptive immune system. Task 4. Cell biological characterisation of the IRAP+ compartment in DCs. This Task concerns the cell biological characterisation of the IRAP+ compartment. Using purified IRAP+ vesicles, we will determine the protein composition of the compartment; search for the presence of proteins with a role in pH regulation; check for the presence of phagocytosed or receptor-internalized antigens in it; and study the origin as well peptide loading of class I molecules in/from it. We will also study the possible relationship of the IRAP+ compartment and its dynamics to the endosomal compartment containing TLR3/7/9 and Unc93b.