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TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming

Nature Immunology volume 22, pages 497–509 (2021)Cite this article

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Abstract

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor–regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER–Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment–dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.

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Fig. 1: MHC-I molecules are retained in the ERGIC of murine DCs upon TAP deficiency.
Fig. 2: MHC-I molecules are retained in the ERGIC of human DCs upon TAP blockade by virus or TAP inhibitor.
Fig. 3: Intact cross-presentation occurs independently of TAP function and despite ERC depletion of MHC-I.
Fig. 4: Altered subcellular MHC-I localization in the absence of TAP bypasses TLR regulation of phagocytic antigen cross-presentation.
Fig. 5: Noncanonical cross-presentation in the absence of TAP is independent of TLR signal-dependent phosphorylation of phagosomal SNAP23.
Fig. 6: Noncanonical cross-presentation is dependent on Sec22b-mediated trafficking of MHC-I to phagosomes.
Fig. 7: Noncanonical cross-presentation cell-autonomously counters TAP deficiency by presenting viral- but also cellular-derived antigens.
Fig. 8: TAP deficiency in hematopoietic cells does not preclude the generation of a protective CD8+ T cell response to viral infection.

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Data availability

Source data and uncropped immunoblot images are provided with this paper. All other data supporting the findings of the paper are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank V. Gillespie for expert pathology on mouse lung tissues. We thank S. Trombetta (Boehringer Ingelheim); K. Rock (University of Massachusetts); P. Cresswell (Yale University); and W. Li, T. M. Moran, D. B. Rubiov and A. Fernandez-Sesma (Icahn School of Medicine at Mount Sinai) for reagents and technical advice. We are grateful to current Blander laboratory members and to H. Gupta, M. A. Blander and S. J. Blander for discussions and support. The ISMMS-Microscopy Shared Resource Facility was supported by grants no. NIH 5R24 CA095823-04, no. S10 RR0 9145-01 and no. NSF DBI-9724504. This work was supported by NIH grants no. AI073899 and no. AI123284 to J.M.B.; an NIH/NIAID Center for Research on Influenza Pathogenesis contract as part of the CEIRS Network no. HHSN266200700010C to A.G.-S., grant nos. AI101820 and AI112318 to D.T., and grant no. AI143861 to K.M.K.; German Research Foundation grants no. SFB 807–Membrane Transport and Communication and no. TA157/7; and by the European Research Council (ERC Advanced Grant no. 789121) to R.T. Support to J.M.B. was also provided by NIH grants no. DK111862 and no. AI127658, the Burroughs Wellcome Fund, and the Leukemia and Lymphoma Society. G.B. has been supported by a fellowship and is currently supported by a career development award from the Crohn’s and Colitis Foundation. T.G. was supported by an American Heart Association pre-doctoral fellowship and NIH grant no. F32CA224438.

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Author notes

  1. Gaëtan Barbet

    Present address: The Child Health Institute of New Jersey, and Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA

  2. Priyanka Nair-Gupta

    Present address: Janssen Research and Development LLC, Spring House, PA, USA

  3. Stephen T. Yeung

    Present address: Division of Infectious Disease, Department of Medicine, Weill Cornell Medicine, New York, NY, USA

  4. Thomas Gardner

    Present address: ArsenalBio, San Francisco, CA, USA

  5. Angela Choi

    Present address: Moderna Inc., Cambridge, MA, USA

  6. These authors contributed equally: Gaëtan Barbet, Priyanka Nair-Gupta.

Authors and Affiliations

  1. The Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, Cornell University, New York, NY, USA

    Gaëtan Barbet, Julien Moretti & J. Magarian Blander

  2. Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA

    Gaëtan Barbet, Julien Moretti & J. Magarian Blander

  3. Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Priyanka Nair-Gupta

  4. Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Priyanka Nair-Gupta & Adolfo García-Sastre

  5. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Michael Schotsaert, Giorgi Metreveli, Angela Choi & Adolfo García-Sastre

  6. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Michael Schotsaert, Giorgi Metreveli, Angela Choi, Domenico Tortorella & Adolfo García-Sastre

  7. The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Stephen T. Yeung & Adolfo García-Sastre

  8. Perlmutter Cancer Center, New York University Langone Health, New York, NY, USA

    Fabian Seyffer & Kamal M. Khanna

  9. Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, New York, NY, USA

    Thomas Gardner

  10. Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany

    Robert Tampé

  11. Department of Microbiology, New York University School of Medicine, New York, NY, USA

    Kamal M. Khanna

  12. Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA

    J. Magarian Blander

  13. Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA

    J. Magarian Blander

  14. Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY, USA

    J. Magarian Blander

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Contributions

G.B., P.N.-G. and J.M.B. designed experiments, directed the study and wrote the manuscript. P.N.-G. and G.B. performed most in vitro and in vivo experiments, respectively, and curated data. M.S., G.M., A.C. and A.G.-S. provided influenza A virus animal model expertise and management and conducted animal weight loss and lung viral titer measurements. F.S. and R.T. prepared and provided recombinant soluble TAP inhibitor US6. J.M. and G.B. performed immunoblots for knockdown validation. T.G. and D.T. provided reagents and methodology for HCMV infections. S.T.Y. and K.M.K. sectioned, stained and imaged lung tissues by confocal microscopy. J.M.B. supervised and conceived of the study.

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Correspondence to J. Magarian Blander.

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The authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Malini Raghavan, Scheherazade Sadegh-Nasseri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Steady state residual expression of MHC-I at the plasma membrane of Tap–/– DC.

a, Confocal micrographs of WT or Tap−⁄− resting DCs stained for cholera toxin B subunit (CTB), H2-Kb and ERGIC-53. b, FACS analyses of WT or Tap−⁄− DCs for surface H2-Kb. Scale bars represent 10 µm. Data represent at least three independent experiments.

Extended Data Fig. 2 HCMV protein expression is detected only in DC treated with live and not UV-inactivated virus.

Confocal micrographs of human DCs stained for immediate early (IE) viral protein along with CTB, 48h post infection with either HCMVTB40/E or its UV-irradiated counterpart at a MOI=10. Scale bars represent 10 µm. Data represent at least three independent experiments.

Extended Data Fig. 3 Impaired TAP function blocks classic MHC-I presentation but not cross-presentation.

a, Classic MHC-I presentation by WT DCs of SIINFEKL derived from influenza PR/8-OTI virus to OVA-specific CD8+ T cells. DCs were infected with influenza PR/8-OT-I virus for 5h and incubated in the presence of active or inactive US6. b, c, WT and Tap−⁄− DC cross-presentation of SIINFEKL from E. coli-OVA at several time points (b), or at 4.5h post-phagocytosis of either E. coli-OVA or 5h post-infection with influenza PR/8-OTI virus (c). Data represent at least three independent experiments except for (b) depicting two independent experiments. The mean ± SEM are presented and each symbol represents biological replicate. **P < 0.01; N.S.= non statistically significant (P>0.05) using an unpaired two-tailed t-test.

Source data

Extended Data Fig. 4 Classic MHC-I presentation of viral antigen by Wt DC is not affected by Sec22b or Rab11a inhibition.

a, Immunoblots for the expression of β-actin, Sec22b and Rab11a on whole cell extracts prepared from DCs seven days post-differentiation of bone marrow progenitors that had been transduced with lentiviruses encoding for control shRNA or shRNA targeting either Rab11a or Sec22b. Immunoblots were cropped to show indicated proteins. b, WT DC progenitors were transduced with recombinant lentiviruses expressing scrambled, Sec22b or Rab11a specific shRNA. Classic MHC-I presentation of SIINFEKL at 5h after infection with recombinant SIINFEKL-expressing Influenza-OTI (PR/8-OTI) (left panels, no secondary treatment) or cross-presentation of SIINFEKL from heat-inactivated Influenza-OTI given to DCs at 3h following infection by Influenza-OTI virus. Cross-presentation was assessed 2h later. Data represent at least three independent experiments. The mean ± SEM are presented and each symbol represents biological replicate. N.S.= non statistically significant (P>0.05) using an unpaired two-tailed t-test.

Source data

Extended Data Fig. 5 Lung CD8 T cells during lethal challenge with influenza A virus.

a, Percent of influenza A specific CD8+ T cells in the lungs of WT→WT and Tap⎯/⎯→WT mice at days 3 and 5 post lethal challenge with 75 p.f.u. influenza A PR8 virus. Data show two different H-2Dd tetramers loaded with either a polymerase acidic protein epitope (SSLENFRAYV) or a nucleoprotein epitope (ASNENMETM). b, Representative FACS plot and % CD8+CD44+ cells in the lung of naïve chimeric mice. c, Flow cytometry plots showing CD8+ T cells in the lungs or spleens of WT mice on days 1 and 2 following injection with anti-CD8α antibody (clone 2.43) intranasally (i.n., 200µg), intraperitoneally (i.p., 100µg), or via both routes (i.p.+i.n.). Percent of CD3+CD8β+ T cells remaining are indicated in the blue gates. The mean ± SEM are presented and each symbol represents a mouse. N.S.= non statistically significant (P>0.05) using an unpaired two-tailed t-test.

Source data

Extended Data Fig. 6 CD8 T cells mediate protection of Tap⎯/⎯ chimeric mice against lethal challenge with influenza A virus.

a, Confocal micrographs at 20X magnification of lung sections from indicated anti-CD8α-treated mice stained for CD8α, CD11c, EpCAM and influenza A virus. Legend to the right shows the color code for each antibody. Scale bar represents 50 µm. b, Lung PR8 viral titers at days 3 and 5 in WT→WT and Tap⎯/⎯→WT chimeric mice. Each symbol is one mouse. c, Pathology scores of infected mice at different time points post lethal PR8 challenge in WT→WT and Tap⎯/⎯→WT mice. Slides were scored by a blinded pathologist for perivascular, bronchiolar or alveolar inflammation, epithelial degeneration or necrosis, and intraluminal debris or hemorrhage. Each symbol is one mouse. d, Hematoxylin-and-eosin staining of lung sections from WT→WT and Tap⎯/⎯→WT mice on days 3 or 5 post PR8 challenge. Scale bar represents 100 μm. The mean ± SEM are presented and each symbol represents a mouse. Data represent 2 experiments (for a total n of 140 mice).

Source data

Supplementary information

Supplementary Information

Supplementary gating strategy.

Reporting Summary

Supplementary Video 1

A schematic illustrating the cell biology of noncanonical cross-presentation based on the data presented in this study. The model phagocytic cargo shown here is a bacterium that engages TLR signaling. Based on previous work, two pathways of vesicular traffic contribute to the cross-presenting phagosome: TLR-regulated traffic from the ERC to the phagosome mediated by TLR–MyD88–IKK2-dependent phosphorylation of SNAP23 (not shown, refer to ref. 9) which delivers MHC-I molecules, and TLR-independent traffic from the ERGIC to phagosomes requiring the ER-SNARE Sec22b, which delivers the components of the peptide-loading complex, including TAP and Sec61 (refs. 9,24). Accumulation of MHC-I molecules in the ERGIC upon TAP dysfunction enables their Sec22b-dependent recruitment to phagosomes to prime CD8+ T cells through noncanonical cross-presentation.

Supplementary Video 2

3D reconstruction of confocal stacks showing CD11c+ cells with infected epithelial cell inclusions (white merge) in the lungs of WT → WT at day 3 post influenza A/PR8 challenge. Staining for CD11c is in red, influenza in green, CD8 in cyan and EpCAM in magenta.

Supplementary Video 3

3D reconstruction of confocal stacks showing CD11c+ cells with infected epithelial cell inclusions (white merge) in the lungs of Tap⎯/⎯ → WT at day 3 post influenza A/PR8 challenge. Staining for CD11c is in red, influenza in green, CD8 in cyan and EpCAM in magenta.

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Barbet, G., Nair-Gupta, P., Schotsaert, M. et al. TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming. Nat Immunol 22, 497–509 (2021). https://doi.org/10.1038/s41590-021-00903-7

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