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
Part of the Methods in Molecular Biology book series (MIMB, volume 1988)


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) 



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


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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

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