Proteomic Analysis of Regulated Dendritic Cell Endosomes Reveals Dynamic Adaptation to Antigen Uptake and Cross‐Presentation

MHC class I (MHC-I) cross-presentation involves final proteolytic peptide processing by the endosomal insulin-regulated aminopeptidase (IRAP). Reasoning that analysis of the IRAP-proximal proteome may inform about dynamic remodeling of a key cross-presentation compartment during antigen uptake, we developed a proximity biotinylation system by expressing an IRAP-TurboID fusion protein in MuTuDCs, a cell line resembling murine type 1 conventional dendritic cells. Analysis of luminal proteins associated with IRAP at steady state and during phagocytosis revealed a massive shift upon uptake of yeast but not apoptotic cells, favoring enrichment of antigen-processing machinery, MHC-I molecules, and proteins involved in ER-associated folding and trafficking. Importantly, Sec22b modulated this proteomic landscape, promoting the localization of MHC-I cross-presentation proteins (e.g., MHC-I, Tap1, Wdfy4, transferrin receptor) to the IRAP environment, while its absence favored MHC-II and ER-related proteins, suggesting a Sec22b-dependent dichotomy between pathways favoring cross-presentation versus antigen degradation. Intriguingly, uptake of apoptotic cells failed to promote cross-presentation but induced two proteins related to immune tolerance, suggesting potential adaptation of proteome modulation to the outcome of antigen presentation. These data suggest that IRAP⁺ endosomes serve as adaptive hubs integrating secretory and endocytic pathways, with Sec22b acting as a key determinant in tailoring this compartment for MHC-I-mediated cross-presentation.