Cross-presentation is the process by which dendritic cells display peptides from internalized (extracellular) proteins on MHC class I molecules to prime CD8+ T cells. Singh and Cresswell (Science, 2010) showed that gamma-interferon-inducible lysosomal thiol reductase (GILT, encoded by Ifi30) is required in mice for cross-presentation of disulfide-bond-rich viral glycoprotein antigens such as HSV-1 gB and influenza hemagglutinin and neuraminidase: GILT-mediated reduction in the phagosome, followed by partial proteolysis, cytosolic translocation, and proteasomal processing, feeds a 'cytosolic' cross-presentation route. This problem concerns the mechanistic questions that work left open, which remain unresolved as of 2026: (1) the molecular machinery and route by which the reduced, partially proteolyzed fragments cross the phagosomal membrane into the cytosol (Sec61 translocon-mediated export, other ERAD components, or membrane damage and leakage remain competing, mutually incompatible models); (2) the intracellular compartment where the final peptides load onto MHC class I molecules (endoplasmic reticulum, ER-derived phagosomal membranes, the ER–Golgi intermediate compartment (ERGIC), or recycling MHC I in endosomes); (3) the quantitative in vivo contributions of cross-priming versus direct presentation by infected antigen-presenting cells to the residual CD8+ T cell responses observed in Ifi30-deficient mice; and (4) whether CD4+ T cell epitope specificity and helper function differ in GILT-deficient mice in ways that shape CD8+ outcomes. No consensus resolution of these four elements is currently established.

Background

CD8+ T cells detect virus-infected cells by recognizing short peptides bound to major histocompatibility complex class I (MHC I) molecules on the cell surface. Peptides presented on MHC I normally originate from proteins degraded in the cytosol by the proteasome; the resulting peptides are transported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) and loaded onto newly synthesized MHC I molecules. Because viruses that do not infect professional antigen-presenting cells (APCs) would be invisible to this pathway, APCs — chiefly type 1 conventional dendritic cells (cDC1), the specialized dendritic-cell subset dedicated to this function — can also acquire extracellular antigen by phagocytosis of infected-cell debris and present it on MHC I. This process is called cross-presentation, and the T-cell priming it produces in vivo is called cross-priming, distinguishing it from 'direct presentation' of antigen synthesized within an infected APC itself.

The dominant 'cytosolic' route of cross-presentation requires that internalized proteins or their fragments leave the endocytic compartment: antigen must cross the phagosomal membrane into the cytosol, where proteasomes generate the final peptides, which are then handled by TAP and MHC I as in the classical pathway. How antigen crosses the phagosomal membrane is one of the longest-standing controversies in antigen-processing biology. Candidate mechanisms include protein channels related to ER-associated degradation (ERAD, the quality-control pathway that retrotranslocates misfolded ER proteins to the cytosol), in particular the Sec61 translocon (the channel that normally inserts nascent proteins into the ER) and the p97/VCP ATPase; recruitment of ER membrane and ERAD machinery to phagosomes, regulated in part by the SNARE membrane-fusion protein Sec22b; and non-channel mechanisms in which endomembrane damage lets luminal content leak out, with the ESCRT-III membrane-repair complex counteracting this leakage. A competing 'vacuolar' route bypasses the cytosol entirely: peptides are generated within endocytic organelles (in some cases by proteasomes delivered into phagosomes) and load onto recycling MHC I molecules inside those organelles.

GILT (gamma-interferon-inducible lysosomal thiol reductase, gene Ifi30) is the only known thiol reductase resident in lysosomes and phagosomes. Singh and Cresswell (2010) established that GILT is required for cross-presentation of disulfide-rich antigens: cross-presentation of the HSV-1 glycoprotein B (gB) epitope gB(498–505), and of most epitopes of influenza A virus hemagglutinin (HA) and neuraminidase (NA), was strongly impaired in Ifi30−/− mice, while disulfide-free antigens (the HSV-1 ICP6 epitope, influenza PA and NP epitopes, ovalbumin) were unaffected. A catalytically inactive GILT could not restore presentation, and a 'trapping' GILT mutant caught gB in a mixed disulfide, proving direct reduction of the intact substrate. In vitro, GILT-negative dendritic cells presented gB normally when directly infected with HSV-1, and cross-presentation remained dependent on phagosomal acidification, cathepsin proteases, TAP, and the proteasome. The work therefore defined an early, genetically separable step — reductive unfolding in the phagosome — of the cytosolic cross-presentation route, and showed that cross-priming contributes substantially to anti-HSV-1 and anti-influenza CD8+ T cell responses in vivo.

The same paper explicitly flagged what remained unknown, and these points define the present problem. First, the translocation step 'may involve components of the ERAD machinery', but the exact molecular machinery and route of phagosomal escape were (and remain) unproven; later work produced directly conflicting evidence — Sec61 intrabody experiments supporting Sec61-mediated export (Zehner et al. 2015), a Sec61 blocker (mycolactone) that inhibits cross-presentation without blocking export (Grotzke et al. 2017, from the same laboratory as Singh and Cresswell 2010), p97/VCP dependence without Sec61 or Derlin-1 dependence in human dendritic cells (Ménager et al. 2014), and ESCRT-III membrane-repair control of antigen leakage (Gros et al. 2022). Second, the site of final peptide loading — 'likely to occur in the ER, but could occur in phagosomes that have recruited ER membrane components, although this issue remains contentious' — is still framed as unresolved ('cytosolic, vacuolar, or both?' in a 2023 review by Ohara and Murphy), with Rab-family small GTPase Rab39a driving conversion of phagosomes into MHC-I loading compartments (Cruz et al. 2020) and an ER–Golgi intermediate compartment (ERGIC)-based noncanonical pathway under TAP dysfunction (Barbet et al. 2021) added to the options. Third, the residual gB-, HA- and NA-specific CD8+ T cell responses in Ifi30−/− mice 'may reflect priming by directly infected APCs', but the relative quantitative contributions of cross-priming and direct presentation have never been apportioned in vivo. Fourth, the epitope specificity of CD4+ T helper responses in Ifi30−/− mice 'may be different from wild type mice' even though total CD4+ T cell numbers are similar; adjacent work (limited effects of GILT on the MHC II self-peptidome, altered HLA-II peptide generation in GILT-expressing melanoma cells) touches this only indirectly, and the viral case is uncharacterized. Because GILT reduction is a defined entry step feeding substrates into the general cytosolic route, resolving these four questions completes the mechanistic and physiological description of how a disulfide-rich viral glycoprotein travels from an infected cell corpse to a peptide–MHC I complex on a dendritic cell — the prerequisite for rational targeting of this pathway by subunit vaccines and tumor-immunotherapy strategies, which currently remain limited by the poorly understood mechanism of cross-presentation engagement by clinically relevant adjuvants (Lee and Suresh 2022).

Problem Statement

For the GILT-dependent cytosolic route of MHC class I cross-presentation defined by Singh and Cresswell (2010), determine the following four linked, still-unresolved elements, without restricting the answer to a particular mechanism, antigen format, or compartment a priori:

  1. Antigen-export machinery and route. Identify the molecular machinery and mechanism by which partially proteolyzed, GILT-reduced fragments of disulfide-rich internalized antigens (e.g., HSV-1 gB) cross the phagosomal/endosomal membrane into the cytosol of dendritic cells — adjudicating among the competing channel/transporter models (Sec61 translocon recruitment to endosomes; other ERAD components such as the p97/VCP ATPase or Derlin retrotranslocation-channel proteins; Sec22b-dependent ER membrane recruitment to phagosomes) and non-channel mechanisms (constitutive or signaling-induced phagosomal membrane damage with luminal leakage, contained by ESCRT-III repair), including whether any single route is necessary and/or sufficient in primary cDC1 dendritic cells in vitro and in vivo.

  2. Loading compartment. Establish the intracellular compartment in which the final peptides derived from these GILT-dependent substrates are loaded onto MHC class I molecules — the endoplasmic reticulum via TAP, ER-derived phagosomal membranes that have recruited loading machinery (e.g., via Rab39a), the ER–Golgi intermediate compartment (ERGIC), or recycling MHC I molecules in endocytic compartments — and whether the answer depends on infection context such as viral TAP blockade.

  3. In vivo cross-priming versus direct priming. Quantify the relative contributions of cross-priming by uninfected dendritic cells and direct presentation by infected antigen-presenting cells to the residual, GILT-independent CD8+ T cell responses to GILT-dependent epitopes (HSV-1 gB(498–505); influenza A HA and NA epitopes) during viral infection in Ifi30−/− mice, using wild-type mice as the baseline — i.e., how much of that residual portion is attributable to direct presentation by infected APCs versus GILT-independent cross-presentation, under physiologically relevant infection conditions.

  4. CD4 helper and epitope-specificity effects. Determine whether the epitope specificity and helper function of CD4+ T cell responses in Ifi30−/− mice during HSV-1 or influenza A infection differ from wild type in ways that causally influence the magnitude or quality of the CD8+ T cell responses to GILT-dependent and GILT-independent epitopes.

All four elements are required and are held to the same completion standard. An element is complete when the answer commits to a definite resolution — selecting among the listed options (elements 1 and 2), or stating the apportionment (element 3) or the causal determination (element 4) — and states the evidence that discriminates it. Where the literature provides no discriminating evidence, the element is complete when the answer specifies a decisive experiment and the observation that would change the answer.

These four elements jointly complete the mechanistic and physiological description of the GILT-dependent cytosolic cross-presentation pathway — from reductive unfolding in the phagosome, through export and proteasomal processing, to the site of peptide loading and the resulting in vivo T-cell priming — as the basis for rational exploitation of this pathway in vaccine and immunotherapy design.

References

  1. Singh R, Cresswell P (2010). Defective Cross-Presentation of Viral Antigens in GILT-Free Mice. Science 328(5984):1394–1398. DOI: 10.1126/science.1189176. https://doi.org/10.1126/science.1189176
  2. Zehner M, Marschall AL, Bos E, Schloetel JG, Kreer C, Fehrenschild D, Limmer A, Ossendorp F, Lang T, Koster AJ, Dübel S, Burgdorf S (2015). The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells. Immunity 42(5):850–863. DOI: 10.1016/j.immuni.2015.04.008. https://doi.org/10.1016/j.immuni.2015.04.008
  3. Grotzke JE, Kozik P, Morel JD, Impens F, Pietrosemoli N, Cresswell P, Amigorena S, Demangel C (2017). 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. DOI: 10.1073/pnas.1705242114. https://doi.org/10.1073/pnas.1705242114
  4. Ménager J, Ebstein F, Oger R, Hulin P, Nedellec S, Duverger E, Lehmann A, Kloetzel PM, Jotereau F, Guilloux Y (2014). 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(3):e89897. DOI: 10.1371/journal.pone.0089897. https://doi.org/10.1371/journal.pone.0089897
  5. Gros M, Segura E, Rookhuizen DC, Baudon B, Heurtebise-Chrétien S, Burgdorf N, Maurin M, Kapp EA, Simpson RJ, Kozik P, Villadangos JA, Bertrand MJM, Burbage M, Amigorena S (2022). Endocytic membrane repair by ESCRT-III controls antigen export to the cytosol during antigen cross-presentation. Cell Reports 40(7):111205. DOI: 10.1016/j.celrep.2022.111205. https://doi.org/10.1016/j.celrep.2022.111205
  6. Sengupta D, Graham M, Liu X, Cresswell P (2019). Proteasomal degradation within endocytic organelles mediates antigen cross-presentation. The EMBO Journal 38(16):e99266. DOI: 10.15252/embj.201899266. https://doi.org/10.15252/embj.201899266
  7. Cruz FM, Colbert JD, Rock KL (2020). The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments. The EMBO Journal 39(2):e102020. DOI: 10.15252/embj.2019102020. https://doi.org/10.15252/embj.2019102020
  8. Barbet G, Nair-Gupta P, Schotsaert M, Yeung ST, Moretti J, Seyffer F, Metreveli G, Gardner T, Choi A, Tortorella D, Tampé R, Khanna KM, García-Sastre A, Blander JM (2021). TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming. Nature Immunology 22(4):497–509. DOI: 10.1038/s41590-021-00903-7. https://doi.org/10.1038/s41590-021-00903-7
  9. Ohara RA, Murphy KM (2023). Recent progress in type 1 classical dendritic cell cross-presentation — cytosolic, vacuolar, or both? Current Opinion in Immunology 83:102350. DOI: 10.1016/j.coi.2023.102350. https://doi.org/10.1016/j.coi.2023.102350
  10. West LC, Cresswell P (2013). Expanding roles for GILT in immunity. Current Opinion in Immunology 25(1):103–108. DOI: 10.1016/j.coi.2012.11.006. https://doi.org/10.1016/j.coi.2012.11.006
  11. Bogunovic B, Srinivasan P, Ueda Y, Tomita Y, Maric M (2010). Comparative quantitative mass spectrometry analysis of MHC class II-associated peptides reveals a role of GILT in formation of self-peptide repertoire. PLoS ONE 5(5):e10599. DOI: 10.1371/journal.pone.0010599. https://doi.org/10.1371/journal.pone.0010599
  12. Hathaway-Schrader JD, Norton D, Hastings K, Doonan BP, Fritz ST, Bethard JR, Blum JS, Haque A (2022). GILT Expression in Human Melanoma Cells Enhances Generation of Antigenic Peptides for HLA Class II-Mediated Immune Recognition. International Journal of Molecular Sciences 23(3):1066. DOI: 10.3390/ijms23031066. https://doi.org/10.3390/ijms23031066
  13. Tondeur EGM, Voerman JSA, Geleijnse MAA, van Hofwegen LS, van Krimpen A, Koerner J, Mishra G, Song Z, Schliehe C (2023). Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro. The Journal of Immunology 211(8):1203–1215. DOI: 10.4049/jimmunol.2200473. https://doi.org/10.4049/jimmunol.2200473
  14. Lee W, Suresh M (2022). Vaccine adjuvants to engage the cross-presentation pathway. Frontiers in Immunology 13:940047. DOI: 10.3389/fimmu.2022.940047. https://doi.org/10.3389/fimmu.2022.940047
  15. Nakamura T, Izumida M, Hans MB, Suzuki S, Takahashi K, Hayashi H, Ariyoshi K, Kubo Y (2024). Post-Transcriptional Induction of the Antiviral Host Factor GILT/IFI30 by Interferon Gamma. International Journal of Molecular Sciences 25(17):9663. DOI: 10.3390/ijms25179663. https://doi.org/10.3390/ijms25179663