A Flow Cytometry-Based Approach to Unravel Viral Interference with the MHC Class I Antigen Processing and Presentation Pathway
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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 wordsImmune 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)
This work was funded by the European Commission under the Horizon2020 program H2020 MSCA-ITN GA 675278 EDGE.
- 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
- 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
- 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
- 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.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.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
- 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