During MHC class I cross-presentation, dendritic cells transfer internalized exogenous antigens from endosomes into the cytosol for proteasomal processing, yet the molecule that mediates this endosome-to-cytosol translocation step has remained unidentified and contested for over a decade. Zehner and colleagues proposed that the ER translocon Sec61 is recruited to antigen-containing endosomes in a TLR/TRIF-dependent manner and there forms the export channel, while leaving open whether Hrd1/ERAD components act instead or additionally, which TRIF-independent signals recruit Sec61, which route (ERGIC/Sec22b-dependent transport versus ER-endosome membrane contacts) delivers Sec61 and other ER proteins to endosomes, and how Sec61 would function at the endosomal membrane. Subsequent work has both supported and challenged each element: pharmacologic Sec61 blockade by mycolactone inhibits cross-presentation without blocking export, DC-specific Sec22b knockouts contradict knockdown-based claims, and non-channel mechanisms (aquaporin-3-dependent lipid peroxidation, perforin-2 pores, redox-controlled membrane permeabilization) have entered the field without a decisive winner. The problem is to establish, by direct experimental evidence, the identity and mechanism of the machinery that exports antigen across the endosomal membrane, how that machinery is recruited to antigen-containing endosomes, and how its action is regulated during dendritic-cell activation.

Background

Cytotoxic CD8+ T cells recognize peptides of 8-10 amino acids displayed on MHC class I molecules (the cell-surface glycoproteins that present intracellular protein fragments to T cells). Because MHC class I normally presents peptides from the cell's own cytosolic proteins, the discovery that dendritic cells (the antigen-presenting cells that initiate T-cell responses) can also display internalized extracellular antigen on MHC class I — called cross-presentation — explained how the immune system primes CD8+ T cells against tumors and viruses that do not infect dendritic cells themselves. In the cytosolic route of cross-presentation, internalized antigen must cross the endosomal membrane into the cytosol, where it is tagged with ubiquitin (a small protein whose attachment marks proteins for destruction), degraded by the proteasome (the cellular protein-degradation machine), and the resulting peptides are imported by TAP (the transporter associated with antigen processing) into the endoplasmic reticulum (ER) or into endosomes for loading onto MHC class I. The physical step of antigen translocation across the endosomal membrane is the least understood part of this pathway, and its molecular identity has been controversial ever since it was proposed that components of ER-associated degradation (ERAD, the quality-control pathway that retrotranslocates misfolded ER proteins back to the cytosol) might be co-opted at the endosome. Candidate ERAD factors include the Sec61 translocon (the protein channel that normally imports nascent secretory proteins into the ER), the E3 ubiquitin ligase Hrd1 (the enzyme that attaches ubiquitin tags to target proteins), the adaptor Derlin-1, and the ATPase p97/VCP that pulls ERAD substrates into the cytosol. Sec61 itself had both supporters and dissenters: cross-presentation of synthetic long peptides by human dendritic cells was reported to require p97/VCP but not Sec61 or Derlin-1 (Ménager et al., 2014), while Zehner et al. (Immunity 2015) provided the most direct evidence in favor of Sec61. Using a Sec61-specific intrabody (an antibody fragment expressed inside the cell) that trapped Sec61 in the ER and prevented its recruitment to endosomes without perturbing ER function, they showed that endosomal recruitment of Sec61 is required for antigen translocation and cross-presentation, and that recruitment depends on dendritic-cell activation via Toll-like receptor (TLR) ligands acting through the adaptor TRIF (TIR-domain-containing adapter inducing interferon, the signaling adaptor for TLR3 and TLR4). Their study raised four linked open points that define the current problem: (i) whether Hrd1 or other ERAD proteins are alternative or complementary exporters (Hrd1 knockdown reduced cross-presentation but also altered MHC class II presentation — the related pathway that displays internalized antigen to CD4+ helper T cells — leaving direct versus indirect effects unresolved); (ii) which TRIF-independent innate signals account for the residual Sec61 recruitment and cross-presentation; (iii) which cell-biological route — vesicular transport via the ER-Golgi intermediate compartment (ERGIC) mediated by the SNARE protein Sec22b (a membrane-fusion regulator found on ERGIC vesicles), or direct ER-endosome membrane contacts — delivers Sec61, TAP, and calnexin (an ER folding chaperone) but not Derlin-1 or ERAP (ER aminopeptidase, which trims peptides for MHC loading) to antigen-containing endosomes; and (iv) how Sec61 is inserted and gated in the endosomal membrane and whether translocation there resembles canonical ERAD dislocation. The subsequent decade transformed this from a two-horse race into an open field. Mycolactone, a natural product that blocks the Sec61 channel, inhibited cross-presentation without preventing endosome-to-cytosol export, arguing the drug's effects arise from failed ER biogenesis of MHC class I molecules rather than from blocking an endosomal Sec61 channel (Grotzke et al., 2017). The Sec22b route collapsed into an explicit controversy: knockdown experiments and in vivo tumor experiments supported a critical role (Alloatti et al., 2017), but dendritic-cell-specific Sec22b knockout mice cross-presented normally and the knockdown phenotype was attributed to off-target effects (Wu et al., 2017), a conflict extended by Sec22b-independent Toxoplasma antigen presentation (Buaillon et al., 2017), negative knockout results in antigen-presenting cell lines (Tondeur et al., 2023), and positive in vivo results during Trypanosoma cruzi infection (Biscari et al., 2023), while mechanistic work recast Sec22b as a regulator of ER-phagosome lipid exchange and of the endosomal proteome. Meanwhile, genetic and pharmacological screens proposed export mechanisms that require no dedicated protein channel at all: aquaporin-3, a hydrogen-peroxide channel, promotes endosomal lipid peroxidation and antigen release (Nalle et al., 2020); lysosomally trapped small molecules permeabilize endosomes and enhance import (Kozik et al., 2020); the pore-forming protein perforin-2 promotes cross-presentation (Zha et al., 2026); and mitochondrial complex I activity controls endosomal escape through NAD+-dependent redox metabolism and lipid peroxidation (Khouili et al., 2026). Authoritative reviews in 2023 continued to treat the cytosolic-access step of cross-presentation as unsettled. The question posed here is therefore the mechanistic core of the field: what, molecularly, moves exogenous antigen across the endosomal membrane of the dendritic cell, and how is that machinery recruited and regulated.

Problem Statement

Determine the molecular mechanism by which internalized exogenous antigen crosses the endosomal membrane into the cytosol of dendritic cells for MHC class I cross-presentation — the open question raised by Zehner et al. (Immunity 2015) and still undecided. A complete answer must establish: (1) the identity of the export machinery — whether the Sec61 translocon directly mediates translocation at the endosomal membrane, whether ERAD components such as the E3 ligase Hrd1 act as alternative or complementary exporters (and their effects are direct rather than secondary perturbations of ER function or MHC trafficking), or whether export instead proceeds through a non-channel mechanism such as aquaporin-3-dependent lipid peroxidation, pore formation by perforin-2, or redox-controlled membrane permeabilization; (2) the cell-biological route and sorting determinants that relocate the relevant ER proteins (Sec61, TAP, calnexin, but not Derlin-1 or ERAP) to antigen-containing endosomes — Sec22b/ERGIC-dependent vesicular transport, direct ER-endosome membrane contacts, or another route — resolving the conflicting knockout and knockdown evidence on Sec22b; (3) the signals that recruit the machinery, including which innate pathways (TRIF-dependent and the unexplained TRIF-independent components) drive recruitment to antigen-containing endosomes; and (4) the mechanistic mode of action at the endosomal membrane (orientation, accessory factors, gating/regulation of pore opening at non-ER sites) and whether the translocation event differs mechanistically from canonical ERAD dislocation. These four deliverables jointly support one objective — a causally tested molecular account of the endosome-to-cytosol antigen export step — and an answer is complete when it identifies the best-supported machinery and grounds it in the direct, on-target loss-of-function evidence the literature provides (reconstitution evidence where the literature supplies it), reconciles the evidence that conflicts with it, connects the identified machinery to antigen export and to functional cross-presentation to CD8+ T cells, and proposes the decisive discriminating experiment where the contest remains open.

References

  1. Matthias Zehner, Andrea L. Marschall, Erik Bos, Jan-Gero Schloetel, Christoph Kreer, Dagmar Fehrenschild, Andreas Limmer, Ferry Ossendorp, Thorsten Lang, Abraham J. Koster, Stefan Dübel, Sven Burgdorf. 'The Translocon Protein Sec61 Mediates Antigen Transport from Endosomes in the Cytosol for Cross-Presentation to CD8+ T Cells.' Immunity 42(5):850-863, 2015. DOI: 10.1016/j.immuni.2015.04.008. https://doi.org/10.1016/j.immuni.2015.04.008
  2. Jérémie Ménager, Frédéric Ebstein, Romain Oger, Philippe Hulin, Steven Nedellec, Eric Duverger, Andrea Lehmann, Peter-Michael Kloetzel, Francine Jotereau, Yannick Guilloux. 'Cross-Presentation of Synthetic Long Peptides by Human Dendritic Cells: A Process Dependent on ERAD Component p97/VCP but Not sec61 and/or Derlin-1.' PLoS ONE 9(2):e89897, 2014. DOI: 10.1371/journal.pone.0089897. https://doi.org/10.1371/journal.pone.0089897
  3. Ignacio Cebrian, Geraldine Visentin, Nicolas Blanchard, Mabel Jouve, Alexandre Bobard, Catarina Moita, Jost Enninga, Luis F. Moita, Sebastian Amigorena, Ariel Savina. 'Sec22b Regulates Phagosomal Maturation and Antigen Crosspresentation by Dendritic Cells.' Cell 147(6):1355-1368, 2011. DOI: 10.1016/j.cell.2011.11.021. https://doi.org/10.1016/j.cell.2011.11.021
  4. Jeff E. Grotzke, Patrycja Kozik, Jean-David Morel, Francis Impens, Natalia Pietrosemoli, Peter Cresswell, Sebastian Amigorena, Caroline Demangel. 'Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export.' Proceedings of the National Academy of Sciences USA 114(29):E5910-E5919, 2017. DOI: 10.1073/pnas.1705242114. https://doi.org/10.1073/pnas.1705242114
  5. Andrés Alloatti, Derek C. Rookhuizen, Leonel Joannas, Jean-Marie Carpier, Salvador Iborra, Joao G. Magalhaes, Nader Yatim, Patrycja Kozik, David Sancho, Matthew L. Albert, Sebastian Amigorena. 'Critical role for Sec22b-dependent antigen cross-presentation in antitumor immunity.' Journal of Experimental Medicine 214(8):2231-2241, 2017. DOI: 10.1084/jem.20170229. https://doi.org/10.1084/jem.20170229
  6. S. Julia Wu, Yashar S. Niknafs, Stephanie H. Kim, Katherine Oravecz-Wilson, Cynthia Zajac, Tomomi Toubai, Yaping Sun, Jayendra Prasad, Daniel Peltier, Hideaki Fujiwara, Israel Hedig, Nathan D. Mathewson, Rami Khoriaty, David Ginsburg, Pavan Reddy. 'A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation.' Cell Reports 19(13):2645-2656, 2017. DOI: 10.1016/j.celrep.2017.06.013. https://doi.org/10.1016/j.celrep.2017.06.013
  7. Célia Buaillon, Nestor A. Guerrero, Ignacio Cebrian, Sophie Blanié, Jodie Lopez, Emilie Bassot, Virginie Vasseur, Julien Santi-Rocca, Nicolas Blanchard. 'MHC I presentation of Toxoplasma gondii immunodominant antigen does not require Sec22b and is regulated by antigen orientation at the vacuole membrane.' European Journal of Immunology 47(7):1160-1170, 2017. DOI: 10.1002/eji.201646859. https://doi.org/10.1002/eji.201646859
  8. Sam C. Nalle, Rosa Barreira da Silva, Hua Zhang, Markus Decker, Cecile Chalouni, Min Xu, George Posthuma, Ann de Mazière, Judith Klumperman, Adriana Baz Morelli, Sebastian J. Fleire, Alan S. Verkman, E. Sergio Trombetta, Matthew L. Albert, Ira Mellman. 'Aquaporin-3 regulates endosome-to-cytosol transfer via lipid peroxidation for cross presentation.' PLoS ONE 15(11):e0238484, 2020. DOI: 10.1371/journal.pone.0238484. https://doi.org/10.1371/journal.pone.0238484
  9. Patrycja Kozik, Marine Gros, Daniel N. Itzhak, Leonel Joannas, Sandrine Heurtebise-Chrétien, Patrycja A. Krawczyk, Pablo Rodríguez-Silvestre, Andrés Alloatti, Joao Gamelas Magalhaes, Elaine Del Nery, Georg H. H. Borner, Sebastian Amigorena. 'Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity.' Cell Reports 32(2):107905, 2020. DOI: 10.1016/j.celrep.2020.107905. https://doi.org/10.1016/j.celrep.2020.107905
  10. Gaëtan Barbet, Priyanka Nair-Gupta, Michael Schotsaert, Stephen T. Yeung, Julien Moretti, Fabian Seyffer, Giorgi Metreveli, Thomas Gardner, Angela Choi, Domenico Tortorella, Robert Tampé, Kamal M. Khanna, Adolfo García-Sastre, J. Magarian Blander. 'TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming.' Nature Immunology 22(4):497-509, 2021. DOI: 10.1038/s41590-021-00903-7. https://doi.org/10.1038/s41590-021-00903-7
  11. Emma G. M. Tondeur, Jane S. A. Voerman, Mitchell A. A. Geleijnse, Laure S. van Hofwegen, Anneloes van Krimpen, Julia Koerner, Gunja Mishra, Ziye Song, Christian Schliehe. 'Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro.' Journal of Immunology 211(8):1203-1215, 2023. DOI: 10.4049/jimmunol.2200473. https://doi.org/10.4049/jimmunol.2200473
  12. Lucía Biscari, Ma Carmen Maza, Cecilia Farré, Cintia Daniela Kaufman, Sebastian Amigorena, Manuel Fresno, Núria Gironès, Andrés Alloatti. 'Sec22b-dependent antigen cross-presentation is a significant contributor of T cell priming during infection with the parasite Trypanosoma cruzi.' Frontiers in Cell and Developmental Biology 11:1138571, 2023. DOI: 10.3389/fcell.2023.1138571. https://doi.org/10.3389/fcell.2023.1138571
  13. Nina Criado Santos, Samuel Bouvet, Maria Cruz Cobo, Marion Mandavit, Flavien Bermont, Cyril Castelbou, Farah Mansour, Maral Azam, Francesca Giordano, Paula Nunes-Hasler. 'Sec22b regulates phagosome maturation by promoting ORP8-mediated lipid exchange at endoplasmic reticulum-phagosome contact sites.' Communications Biology 6:1008, 2023. DOI: 10.1038/s42003-023-05382-0. https://doi.org/10.1038/s42003-023-05382-0
  14. Juliana M. Blander, Kevin J. Yee Mon, Aakanksha Jha, Dean Roycroft. 'The show and tell of cross-presentation.' Advances in Immunology 159:33-114, 2023. DOI: 10.1016/bs.ai.2023.08.002. https://doi.org/10.1016/bs.ai.2023.08.002
  15. Alice Senni, Louise Grumbach, Sara Ceccacci, Mathieu Frechin, Nicolas Goudin, Ida Chiara Guerrera, Barbara Bertocci, Peter van Endert. 'Proteomic Analysis of Regulated Dendritic Cell Endosomes Reveals Dynamic Adaptation to Antigen Uptake and Cross-Presentation.' European Journal of Immunology 56(2):e70133, 2026. DOI: 10.1002/eji.70133. https://doi.org/10.1002/eji.70133
  16. Zhi-Kai Zha, Cheng-Jie Deng, Ling-Jun Shen, Ling-Zhen Liu, Yun-Xiao Huang, Yan-Hong Li, Li Lv, Ke Zhang, Lin-Shuang Chen, Fei-Er Chen, Sheng-An Li. 'Perforin-2 enhances antigen-specific CTL immune response by promoting cross presentation.' Cell Death & Disease 17:485, 2026. DOI: 10.1038/s41419-026-08705-1. https://doi.org/10.1038/s41419-026-08705-1
  17. Sofía C. Khouili, Elena Priego, Ignacio Heras-Murillo, Gillian Dunphy, Annalaura Mastrangelo, Sarai Martínez-Cano, Vanessa Nuñez, Manuel Rodrigo-Tapias, Adrián Belinchón-García, Johan Garaude, Salvador Iborra, Navdeep S. Chandel, Patricia González-Rodríguez, Michel Enamorado, David Sancho. 'Mitochondrial complex I activity promotes antigen cross-presentation in dendritic cells.' Science Immunology 11(119):eaef0098, 2026. DOI: 10.1126/sciimmunol.aef0098. https://doi.org/10.1126/sciimmunol.aef0098
  18. Karin Römisch. 'A Case for Sec61 Channel Involvement in ERAD.' Trends in Biochemical Sciences 42(3):171-179, 2017. DOI: 10.1016/j.tibs.2016.10.005. https://doi.org/10.1016/j.tibs.2016.10.005
  19. Xiang You, Dan Dan Xu, Di Zhang, Jie Chen, Feng Guang Gao. 'PYR-41 and Thalidomide Impair Dendritic Cell Cross-Presentation by Inhibiting Myddosome Formation and Attenuating the Endosomal Recruitments of p97 and Sec61 via NF-κB Inactivation.' Journal of Immunology Research 2018:5070573, 2018. DOI: 10.1155/2018/5070573. https://doi.org/10.1155/2018/5070573
  20. Dan Dan Xu, Chun Fang Hu, Xiang You, Nan Nan Lu, Feng Guang Gao. 'Akt+ IKKα/β+ Rab5+ Signalosome Mediate the Endosomal Recruitment of Sec61 and Contribute to Cross-Presentation in Bone Marrow Precursor Cells.' Vaccines 8(3):539, 2020. DOI: 10.3390/vaccines8030539. https://doi.org/10.3390/vaccines8030539