Advertisement

A Flow Cytometry-Based Approach to Unravel Viral Interference with the MHC Class I Antigen Processing and Presentation Pathway

  • Patrique Praest
  • Hendrik de Buhr
  • Emmanuel J. H. J. WiertzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1988)

Abstract

MHC class I molecules are an important component of the cell-mediated immune defense, presenting peptides to surveilling CD8+ cytotoxic T cells. During viral infection, MHC class I molecules carry and display viral peptides at the cell surface. CD8+ T cells that recognize these peptides will eliminate the virus-infected cells. Viruses counteract this highly sophisticated host detection system by downregulating cell surface expression of MHC class I molecules.

In this chapter, we describe a flow cytometry-based method that can be used for the identification of viral gene products potentially responsible for evasion from MHC class I-restricted antigen presentation. The gene(s) of interest are expressed constitutively through lentiviral transduction of cells. Subsequently, MHC I surface expression is monitored using MHC class I-specific antibodies. Once the viral gene product responsible for MHC I downregulation has been identified, the same cells can be used to elucidate the mechanism of action. The stage at which interference with antigen processing occurs can be identified using specific assays. An essential step frequently targeted by viruses is the translocation of peptides into the ER by the transporter associated with antigen processing, TAP. TAP function can be measured using a highly specific in vitro assay involving flow cytometric evaluation of the import of a fluorescent peptide substrate.

The protocol described in this chapter enables the identification of virus-encoded MHC class I inhibitors that hinder antigen processing and presentation. Subsequently, their mechanism of action can be unraveled; this knowledge may help to rectify their actions.

Key words

Immune evasion Major histocompatibility complex (MHC) class I Antigen presentation Antigen processing Lentiviral transduction Transfection Cell surface staining Flow cytometry Viral infection Transporter associated with antigen processing (TAP) 

Notes

Acknowledgments

This work was funded by the European Commission under the Horizon2020 program H2020 MSCA-ITN GA 675278 EDGE.

References

  1. 1.
    Hansen TH, Bouvier M (2009) MHC class I antigen presentation: learning from viral evasion strategies. Nat Rev Immunol 9(7):503–513CrossRefGoogle Scholar
  2. 2.
    Horst D, Ressing ME, Wiertz EJHJ (2011) Exploiting human herpesvirus immune evasion for therapeutic gain: potential and pitfalls. Immunol Cell Biol 89(3):359–366CrossRefGoogle Scholar
  3. 3.
    Horst D, Verweij MC, Davison AJ, Ressing ME, Wiertz EJHJ (2011) Viral evasion of T cell immunity: ancient mechanisms offering new applications. Curr Opin Immunol 23(1):96–103CrossRefGoogle Scholar
  4. 4.
    Schuren AB, Costa AI, Wiertz EJ (2016) Recent advances in viral evasion of the MHC class I processing pathway. Curr Opin Immunol 40:43–50CrossRefGoogle Scholar
  5. 5.
    Verweij MC, Horst D, Griffin BD, Luteijn RD, Davison AJ, Ressing ME, Wiertz EJHJ (2015) Viral inhibition of the transporter associated with antigen processing (TAP): a striking example of functional convergent evolution. PLoS Pathog 11(4):e1004743CrossRefGoogle Scholar
  6. 6.
    Praest P, Liaci AM, Förster F, Wiertz EJHJ (2018) New insights into the structure of the MHC class I peptide-loading complex and mechanisms of TAP inhibition by viral immune evasion proteins. Mol Immunol pii:S0161-5890(18)30099-3Google Scholar
  7. 7.
    Kim TK, Eberwine JH (2010) Mammalian cell transfection: the present and the future. Anal Bioanal Chem 397(8):3173–3178CrossRefGoogle Scholar
  8. 8.
    Doom CM, Hill AB (2008) MHC class I immune evasion in MCMV infection. Med Microbiol Immunol 197(2):191–204CrossRefGoogle Scholar
  9. 9.
    Reusch U, Muranyi W, Lucin P, Burgert HG, Hengel H, Koszinowski UH (1999) A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation. EMBO J 18(4):1081–1091CrossRefGoogle Scholar
  10. 10.
    Wiertz EJHJ, Tortorella D, Bogyo M, Yu J, Mothes W, Jones TR, Rapoport TA, Ploegh HL (1996) Sec6l-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 384(6608):432–438CrossRefGoogle Scholar
  11. 11.
    Wiertz EJ, Jones TR, Sun L, Bogyo M, Geuze HJ, Ploegh HL (1996) The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84(5):769–779CrossRefGoogle Scholar
  12. 12.
    Griffin BD, Verweij MC, Wiertz EJHJ (2010) Herpesviruses and immunity: the art of evasion. Vet Microbiol 143(1):89–100CrossRefGoogle Scholar
  13. 13.
    Fischbach H, Döring M, Nikles D, Lehnert E, Baldauf C, Kalinke U, Tampé R (2015) Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets. Nat Commun 6:6199CrossRefGoogle Scholar
  14. 14.
    Koppers-Lalic D, Reits EAJ, Ressing ME, Lipinska AD, Abele R, Koch J, Rezende MM, Admiraal P, van Leeuwen D, Bienkowska-Szewczyk K, Mettenleiter TC, Rijsewijk FAM, Tampe R, Neefjes J, Wiertz EJHJ (2005) Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing. Proc Natl Acad Sci 102(14):5144–5149CrossRefGoogle Scholar
  15. 15.
    Hislop AD, Ressing ME, van Leeuwen D, Pudney VA, Horst D, Koppers-Lalic D, Croft NP, Neefjes JJ, Rickinson AB, Wiertz EJHJ (2007) A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates. J Exp Med 204(8):1863–1873CrossRefGoogle Scholar
  16. 16.
    Koppers-Lalic D, Verweij MC, Lipińska AD, Wang Y, Quinten E, Reits EA, Koch J, Loch S, Marcondes Rezende M, Daus F, Bieńkowska-Szewczyk K, Osterrieder N, Mettenleiter TC, Heemskerk MHM, Tampé R, Neefjes JJ, Chowdhury SI, Ressing ME, Rijsewijk FAM, Wiertz EJHJ (2008) Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP. PLoS Pathog 4(5):e1000080CrossRefGoogle Scholar
  17. 17.
    Matschulla T, Berry R, Gerke C, Döring M, Busch J, Paijo J, Kalinke U, Momburg F, Hengel H, Halenius A (2017) A highly conserved sequence of the viral TAP inhibitor ICP47 is required for freezing of the peptide transport cycle. Sci Rep 7(1):2933CrossRefGoogle Scholar
  18. 18.
    Wycisk AI, Lin J, Loch S, Hobohm K, Funke J, Wieneke R, Koch J, Skach WR, Mayerhofer PU, Tampe R (2011) Epstein-Barr viral BNLF2a protein hijacks the tail-anchored protein insertion machinery to block antigen processing by the transport complex TAP. J Biol Chem 286(48):41402–41412CrossRefGoogle Scholar
  19. 19.
    van Ham SM, Tjin EP, Lillemeier BF, Grüneberg U, van Meijgaarden KE, Pastoors L, Verwoerd D, Tulp A, Canas B, Rahman D, Ottenhoff TH, Pappin DJ, Trowsdale J, Neefjes J (Dec. 1997) HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading. Curr Biol 7(12):950–957CrossRefGoogle Scholar
  20. 20.
    Ressing ME, van Leeuwen D, Verreck FAW, Gomez R, Heemskerk B, Toebes M, Mullen MM, Jardetzky TS, Longnecker R, Schilham MW, Ottenhoff THM, Neefjes J, Schumacher TN, Hutt-Fletcher LM, Wiertz EJHJ (2003) Interference with T cell receptor-HLA-DR interactions by Epstein-Barr virus gp42 results in reduced T helper cell recognition. Proc Natl Acad Sci U S A 100(20):11583–11588CrossRefGoogle Scholar
  21. 21.
    Sena-Esteves M, Gao G (2018) Production of high-titer retrovirus and lentivirus vectors. Cold Spring Harb Protoc 2018(4):pdb.prot095687CrossRefGoogle Scholar
  22. 22.
    van de Weijer ML, Bassik MC, Luteijn RD, Voorburg CM, Lohuis MAM, Kremmer E, Hoeben RC, LeProust EM, Chen S, Hoelen H, Ressing ME, Patena W, Weissman JS, McManus MT, Wiertz EJHJ, Lebbink RJ (2014) A high-coverage shRNA screen identifies TMEM129 as an E3 ligase involved in ER-associated protein degradation. Nat Commun 5:3832CrossRefGoogle Scholar
  23. 23.
    Barnstable CJ, Bodmer WF, Brown G, Galfre G, Milstein C, Williams AF, Ziegler A (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell 14(1):9–20CrossRefGoogle Scholar
  24. 24.
    Aubin RJ, Weinfeld M, Paterson MC (1988) Factors influencing efficiency and reproducibility of polybrene-assisted gene transfer. Somat Cell Mol Genet 14(2):155–167CrossRefGoogle Scholar
  25. 25.
    van Gent M, Griffin BD, Berkhoff EG, van Leeuwen D, Boer IGJ, Buisson M, Hartgers FC, Burmeister WP, Wiertz EJ, Ressing ME (2011) EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection. J Immunol 186(3):1694–1702CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Patrique Praest
    • 1
  • Hendrik de Buhr
    • 1
  • Emmanuel J. H. J. Wiertz
    • 1
    Email author
  1. 1.Department of Medical MicrobiologyUniversity Medical Center UtrechtUtrechtThe Netherlands

Personalised recommendations