Rôle du compartiment IRAP dans la présentation-croisée : décryptage des interactions moléculaires et du trafic du CMH-I

Cross-presentation is an essential mechanism for initiating CD8+ T cell responses against certain pathogens and tumours, and for maintaining immune tolerance to self. The process involves the uptake of extracellular antigens by professional antigen presenting cells before their processing and presentation on MHC-I molecules to CD8+ T cells. The insulin regulated aminopeptidase (IRAP) typically resides in regulated endosomes and plays an important role in trimming N-terminal extensions of antigenic peptides to the optimal length for the loading on MHC-I molecules. The interaction between IRAP and MHC-I molecules occurs within an endosomal compartment in dendritic cells (DCs), highlighting its pivotal function in cross-presentation. The dynamic molecular composition of this compartment is still poorly understood but may be fundamental for optimal loading of MHC-I molecules with cross-presented peptides. Additionally, the origin of MHC-I molecules within the IRAP+ compartment and their specific contribution to the cross-presentation of distinct antigenic forms remain to be fully elucidated. To map IRAP proximal proteins, we employed TurboID, a biotin-based proximity labeling method, in MuTuDC1940 cells, a model of conventional type 1 DCs. We generated a stable cell line expressing IRAP-TurboID and validated its subcellular localization by immunofluorescence. IRAP-TurboID co-localized most strongly with Syntaxin-6 and the Transferrin Receptor (TfR), and less with KDEL receptor, GM130, MR, and EEA1, faithfully recapitulating endogenous IRAP distribution. Biotin incubation for 6 hours resulted in robust labeling of IRAP proximal proteins. Proteomic analysis under steady-state conditions revealed a marked enrichment of ER-associated proteins, suggesting functional communication between IRAP+ endosomes and the ER in the absence of stimulation. To explore how cross-presentation affects IRAP+ endosome remodeling, we stimulated IRAP-TurboID-expressing MuTuDCs with immunogenic yeast cells. Proteomic analysis identified a strong enrichment of antigen-presenting machinery components, including MHC-I, TAP1/2, tapasin, and WDFY4, within IRAP+ endosomes. Notably, this enrichment was partially dependent on Sec22b, as knockdown of this SNARE protein reduced the proteomic recruitment of these proteins, highlighting its key role in regulating endosomal trafficking during cross-presentation. Stimulation with apoptotic cells, non-inflammatory antigens lacking TLR ligands and thought to trigger tolerogenic responses, elicited significantly weaker biotinylation of IRAP proximal proteins. Most antigen presentation-related proteins enriched under yeast stimulation were absent, underscoring a stimulus-dependent modulation of IRAP+ endosomes. Interestingly, specific proteins such as CD84 and PLD4 were uniquely enriched upon apoptotic cell uptake, potentially reflecting roles in immune regulation or tolerance. In conclusion, our study reveals that the antigenic context, immunogenic vs. tolerogenic, shapes the molecular composition of IRAP+ endosomes. These findings advance our understanding of how DCs modulate the proteomic composition of cross-presentation compartments based on the nature of the internalized antigen, with implications for immunity and tolerance. Furthermore, we employed the Retention Using Selective Hooks (RUSH) system to investigate the functional role of MHC-I molecules localized within the IRAP compartment during cross-presentation. In this system, MHC-I was fused to a Streptavidin Binding Peptide (SBP) and co-expressed in MuTuDC1940 cells with IRAP fused to streptavidin, allowing synchronized retention and biotin-dependent release of MHC-I from IRAP+ endosomes. Although this project is still in its early stages, it holds significant potential to clarify the specific contribution of MHC-I molecules trafficking through the IRAP compartment to the cross-presentation of distinct antigen forms.