Cross-presentation — the display of internalized exogenous antigens on MHC class I molecules by dendritic cells — is thought to require that phagosomes acquire components from the endoplasmic reticulum (ER), including peptide-receptive ('open') MHC-I conformers and, on the prevailing but contested model, the SNARE protein Sec22b and the NADPH oxidase NOX2. The small GTPase Rab39a was identified as a promoter of ER-to-phagosome delivery, most clearly of MHC-I molecules, but the molecular machinery that executes it is unknown: the effectors that couple Rab39a nucleotide cycling to vesicle recruitment, the mechanisms by which unstable empty MHC-I molecules escape ER retention and survive until peptide loading on phagosomes, and the quantitative contribution of this intraphagosomal loading route relative to cytosol-to-ER routes in different dendritic cell subsets all remain open. This problem asks for the molecular identification of this trafficking pathway and the measurement of its contribution to T cell cross-priming across dendritic cell subsets.

Background

CD8 T cells detect infected or cancerous cells by recognizing peptides bound to MHC class I molecules. Normally these peptides derive from the cell's own cytosolic proteins: proteasome-generated peptides are imported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) and loaded onto MHC-I within the peptide-loading complex, a multi-protein assembly that includes the chaperones calreticulin, ERp57, and tapasin, which stabilize peptide-empty ('open') MHC-I conformers and retain improperly loaded molecules in the ER. Dendritic cells (DCs), the professional antigen-presenting cells that prime naive CD8 T cells, can additionally present antigens they have internalized by phagocytosis on MHC-I — a process called cross-presentation (XPT) — which is essential for immunity to tumors and viruses that do not directly infect DCs.

In the phagosome-to-cytosol-to-phagosome (P2C2P) route of cross-presentation, internalized antigen is exported from the phagosome to the cytosol, degraded by proteasomes, and the resulting peptides are transported back into the phagosome by phagosomal TAP, where they bind MHC-I molecules. How phagosomes acquire the needed ER-derived machinery (TAP, Sec22b, MHC-I itself) is incompletely understood. Rab proteins — small GTPases that switch between GDP- and GTP-bound states to direct vesicular transport — are candidate regulators, and membrane fusion is ultimately executed by SNARE proteins such as Sec22b, whose role in cross-presentation remains contested. NOX2, the phagosomal NADPH oxidase that alkalinizes the vacuole by consuming protons, helps preserve antigen and MHC-I from excessive acidification.

Cruz, Colbert and Rock (EMBO Journal, 2020) identified Rab39a in a forward genetic screen and showed that it is required for optimal cross-presentation in vitro and cross-priming — the activation of naive CD8 T cells through cross-presentation — in vivo: Rab39a promotes delivery of MHC-I molecules from the ER to phagosomes, raises the levels of peptide-empty MHC-I conformers capable of loading peptide inside phagosomes, and increases phagosomal Sec22b and NOX2. Unexpectedly, although CD8α+ conventional DCs (the subset that specializes in cross-presentation, purified via the XCR1 receptor) express the most Rab39a, cross-presentation by these cells was not significantly impaired by Rab39a loss, whereas CD8α−/DCIR2+ DCs (the other main classical DC subset, identified by the DCIR2 surface receptor) were partially impaired — a subset difference whose basis (differences in the levels of redundant mechanisms, adaptation to the loss of Rab39a, the relative activity of the P2C-to-ER versus P2C2P pathways, or the much greater MHC-II presentation activity of the CD8α− subset) was not resolved. A central puzzle the paper left open is physical: peptide-empty MHC-I conformers are thermally unstable and normally retained in the ER by quality control, so how they escape retention, survive transit, and remain loading-competent in phagosomes — whether through molecular carriers and chaperone associations or by other stabilizing mechanisms — is unknown. Related reviews (Cruz/Chan/Rock 2023; Blander 2023) document parallel MHC-I delivery routes to phagosomes, from a Toll-like receptor-regulated endosomal recycling compartment and from the ER-Golgi intermediate compartment (ERGIC), but the molecular carriers on the Rab39a-dependent route remain undefined.

Problem Statement

The problem is to determine, at molecular resolution and across dendritic cell subsets, how the Rab39a-dependent ER-to-phagosome delivery arm of the cross-presentation pathway is executed. Specifically: (1) identify the Rab39a effector protein(s), adaptors, and SNARE regulators that couple Rab39a nucleotide cycling to the recruitment of Sec22b-containing vesicles and NOX2 complexes, and to the delivery of ER/ERGIC cargo including MHC-I, to phagosomes; (2) determine how peptide-empty (open) MHC-I conformers escape ER retention, remain intact during transport, and stay peptide-receptive inside phagosomes until loading, including whether molecular carriers and specific chaperone-MHC-I interactions are involved or whether other stabilizing mechanisms (such as rapid delivery and the Rab39a-NOX2-dependent reduction in phagosomal proteolysis) suffice; (3) establish experimentally whether phagosomal delivery of TAP and other ER components depends causally on Rab39a, on Sec22b, or on other factors; and (4) quantify the relative contribution of Rab39a-dependent intraphagosomal peptide loading versus cytosol-to-ER classical loading to cross-presentation in defined dendritic cell subsets (including why Rab39a loss partially but significantly reduced cross-presentation by CD8α−/DCIR2+ DCs while not significantly reducing it in CD8α+ DCs) and in defined immunological contexts, in vitro and in vivo. The four components are complementary and must all be addressed; a literature-grounded molecular model is sufficient and de novo experimental data are not required — candidate effector(s) and adaptors, a proposed mechanism by which peptide-empty MHC-I conformers are stabilized in transit to and within phagosomes, and a proposed or literature-based estimate of the subset-specific contribution may all be proposed rather than experimentally established.

References

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