A Flow Cytometry-Based Approach to Unravel Viral Interference with the MHC Class I Antigen Processing and Presentation Pathway
| Autor: | Hendrik de Buhr, Patrique Praest, Emmanuel J. H. J. Wiertz |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Antigen presentation Lentiviral transduction Transfection 03 medical and health sciences 0302 clinical medicine MHC class I Transporter associated with antigen processing (TAP) Genetics Cytotoxic T cell Flow cytometry Viral Interference Molecular Biology biology Immune evasion Antigen processing MHC class I antigen Transporter associated with antigen processing Major histocompatibility complex (MHC) class I Cell biology 030104 developmental biology Viral infection biology.protein CD8 Cell surface staining 030215 immunology |
| Zdroj: | Antigen Processing ISBN: 9781493994496 Antigen Processing-Methods and Protocols Methods in Molecular Biology Methods in Molecular Biology-Antigen Processing Methods in Molecular Biology, 188(2), 187. Humana Press Inc. |
| ISSN: | 1064-3745 1940-6029 |
| DOI: | 10.1007/978-1-4939-9450-2_14 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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