Dendritic cells can cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules via a non-canonical route in which intact, active proteasomes reside inside phagosomes and endolysosomes, locally generating peptides that load onto recycling MHC-I molecules without the transporter associated with antigen processing (TAP). This problem asks for the molecular mechanism of this pathway — the machinery that delivers proteasomes into endocytic lumina (substantially advanced by 2024 in vitro evidence for a route dependent on LC3A/B, the lipidated light-chain-3 marker of autophagosome membranes, with recruitment signals and in vivo operation still undefined), the luminal peptide-handling components (any transporters, editors, or trimming enzymes required for intraphagosomal MHC-I loading), and the quantitative contribution of this route relative to the cytosolic TAP-dependent and canonical vacuolar protease routes to cross-priming of CD8-positive T cells in vivo, in the pathogen and tumor settings that the source highlights. The problem originates from Sengupta, Graham, Liu and Cresswell (EMBO Journal, 2019) and its follow-up literature.

Background

Cytotoxic CD8-positive T lymphocytes recognize peptides displayed on MHC class I molecules (heterotrimeric cell-surface complexes of a heavy chain, beta-2 microglobulin, and a bound peptide). Conventionally, these peptides are produced in the cytosol by the proteasome (the ATP-dependent multisubunit protease complex), translocated into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP, an ABC heterodimer of TAP1 and TAP2), trimmed by ER aminopeptidases (ERAP1/ERAP2 in humans, ERAAP in mice), and loaded onto nascent MHC-I with the help of tapasin and its homologue TAPBPR, peptide editors within the peptide-loading complex. Dendritic cells (DCs), the professional antigen-presenting cells that activate naive T cells, can additionally display peptides derived from internalized exogenous proteins on MHC-I — a process called cross-presentation, which when it primes naive CD8 T cells in vivo is termed cross-priming. Cross-presentation is essential for immunity to tumors and many viruses and is executed mainly by the type 1 conventional dendritic cell subset (cDC1). Two long-debated mechanisms dominate the field: a cytosolic route, in which internalized antigen escapes from phagosomes/endosomes into the cytosol for proteasomal degradation followed by TAP-dependent ER loading, and a vacuolar route, in which antigens are processed by lysosomal proteases such as cathepsins within endocytic compartments and loaded onto MHC-I there. Sengupta, Graham, Liu and Cresswell (EMBO Journal, 2019) described a third, hybrid variant of the vacuolar route: active proteasomes are delivered into the lumen of phagosomes and endolysosomes of cross-presenting mouse bone marrow-derived DCs, where they degrade internalized antigen and generate peptides that load onto lumenal MHC-I molecules — internalized, recycling MHC-I returning through endocytic compartments — independently of TAP. Key evidence included proteasome activity and subunit detection inside endocytic compartments, enhancement of TAP-independent cross-presentation by human beta-2 microglobulin expression (which stabilizes peptide-receptive MHC-I), and augmentation by mutants of Rab GTPases (small regulatory GTP-binding proteins that orchestrate vesicle trafficking) that restrain phagosome maturation. Because epitopes generated this way match those made by conventional cytosolic processing in infected or tumor cells, the route could explain how cross-presentation yields complexes identical to those on target cells. The authors' discussion left the mechanism and significance open: the cellular route delivering proteasomes into endocytic lumina (candidates: macroautophagy, the delivery of cytosolic material to endolysosomes via double-membrane autophagosomes; chaperone-mediated autophagy, the direct translocation of specific cytosolic proteins across the lysosomal membrane; or inward budding of intraluminal vesicles driven by ESCRT, the endosomal sorting complexes required for transport), the possible roles of the peptide editors tapasin and TAPBPR and the endosome-resident aminopeptidase IRAP (insulin-regulated aminopeptidase) and ERAP-like enzymes in intraphagosomal loading, and the relative in vivo roles of this pathway versus cytosolic translocation and canonical vacuolar proteolysis in initiating CD8 T-cell responses to pathogens and tumors. Note that in the source model proteasomes and MHC-I face the same lumen; the source nonetheless records that an alternate peptide transporter had been speculated to drive these pathways and that its data "certainly do not eliminate the possibility of such a transporter working in tandem with luminal proteasomes", naming TAP-Like (TAP-L), a TAP homologue localized to lysosomal membranes, as a suggested candidate while noting that BMDCs derived from TAP-L knockout mice do not exhibit impaired cross-presentation — so whether any luminal peptide transporter is required at all is itself part of the question. Subsequent work has advanced parts of this agenda: Sengupta et al. (Journal of Immunology, 2024) showed that phagosome-associated autophagosome-like structures positive for LC3A/B (microtubule-associated proteins 1A/1B light chain 3A/B, ubiquitin-like markers conjugated onto autophagosome membranes when autophagy is activated) and containing both antigen and proteasomes, together with canonical autophagy dependent on ULK1 (unc-51-like kinase 1, the autophagy-initiating kinase) and on LC3 lipidation, drive TAP-independent cross-presentation in vitro; and Postoak et al. (PNAS, 2025) showed that WDFY4 (a large BEACH-domain scaffolding protein strictly required in vivo for cross-presentation of cell-associated antigens by cDC1) operates through a vacuolar antigen-processing route, and reinterpreted the TAP dependence of cDC1 cross-presentation as a requirement for MHC-I trafficking rather than for peptide transport into the ER. Nevertheless, authoritative reviews (Ohara and Murphy 2023; Luri-Rey et al. 2025) state that the processing route used by cDC1 in vivo remains unresolved and that assigning relative contributions across antigens and settings is an open agenda.

Problem Statement

Establish the molecular mechanism and physiological significance of the intraphagosomal proteasome-dependent, TAP-independent route of antigen cross-presentation described by Sengupta et al. (2019). Concretely: (1) identify the molecular machinery that delivers intact active proteasomes from the cytosol into the lumen of cross-presenting phagosomes/endolysosomes — 2024 in vitro work in bone marrow-derived DCs indicates that LC3A/B-positive autophagosome-related structures and canonical autophagy (ULK1- and LC3-lipidation-dependent) contribute to this delivery, although not every perturbation is concordant and the source's other candidate routes (ESCRT-dependent inward budding of intraluminal vesicles, chaperone-mediated autophagy) are not excluded; the open questions here include the recruitment signals and adaptors that target proteasome-containing autophagosomes to antigen-containing phagosomes, and whether this machinery operates in conventional type 1 dendritic cells in vivo; (2) determine which luminal components, if any, mediate peptide handling before MHC-I loading in this compartment — whether luminal peptide transfer requires any transporter, given that proteasomes and MHC-I face the same lumen, that BMDCs lacking the lysosomal TAP homologue TAP-L cross-present normally, and that the source's data nevertheless do not exclude another transporter working in tandem with luminal proteasomes — and whether the peptide editors tapasin and TAPBPR, the aminopeptidase IRAP, or ERAP-like trimming enzymes are recruited to and function in intraphagosomal loading; and (3) quantitatively determine the relative contribution of this pathway, compared with the cytosolic translocation/TAP-dependent route and the canonical vacuolar (lysosomal protease) route, to cross-priming of CD8-positive T cells in vivo, which the source poses as the relative roles of these pathways in the initiation of CD8-positive T-cell responses to a variety of pathogens and tumors, potentially differing between dendritic cell subsets. Because no currently available perturbation separates these routes uniquely, identifying a route-selective discriminant — a measurement or perturbation that attributes a cross-priming event to the intraphagosomal proteasome route rather than to the competing routes — is itself part of this target. Sub-goals (1), (2) and (3) are jointly required for a complete solution: resolving any one of them does not by itself resolve the task, and (3) is satisfied only by the quantitative in vivo attribution itself — identifying the route-selective discriminant is a necessary enabling step toward that attribution, not a substitute for it.

References

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