Evaluation of viral interference with MHC class I-restricted antigen processing and presentation using a flow
Daniëlle Horst1, Maaike E Ressing1, Arend Mulder2
1Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
Insights
Viruses evade immune detection by downregulating MHC class I molecules. This study presents a flow cytometry method to identify viral proteins that inhibit antigen presentation, aiding in understanding virus-host interactions.
Area of Science:
- Immunology
- Virology
- Molecular Biology
Background:
- CD8(+) cytotoxic T cells survey cell surface MHC class I molecules presenting peptides.
- Viral infections trigger MHC class I presentation of viral peptides, enabling T cell recognition and elimination of infected cells.
- Viruses have evolved mechanisms to downregulate MHC class I expression, evading T cell surveillance.
Purpose of the Study:
- To describe a flow cytometry-based method for identifying viral gene products that evade MHC class I-restricted antigen presentation.
- To characterize the mechanisms of action of identified viral proteins responsible for MHC class I downregulation.
Main Methods:
- Transient cell transfection using polyethylenimine (PEI).
- Cell surface staining with MHC class I-specific monoclonal antibodies.
- Flow cytometry analysis to detect MHC class I expression levels.
Main Results:
- Identification of specific viral gene products that downregulate cell surface MHC class I expression.
- Characterization of the mechanisms by which these viral proteins inhibit antigen presentation.
Conclusions:
- The developed method effectively identifies viral factors involved in MHC class I evasion.
- Understanding these viral inhibitors enhances knowledge of virus-host interactions, antigen processing, and presentation pathways.
Abstract:
The peptide content of MHC class I molecules present at the cell surface is monitored by surveilling CD8(+) cytotoxic T cells. In case of a viral infection, a proportion of the MHC class I molecules will carry peptides derived from viral proteins. This allows the CD8(+) T cells to recognize and eliminate virus-infected cells. This highly sensitive detection system of the host is counteracted by viruses, which have acquired functions to downregulate cell surface expression of MHC class I molecules. In this chapter, we describe a flow cytometry-based method to identify viral gene product(s) responsible for evasion from MHC class I-restricted antigen presentation. To this end, cells are transiently transfected using polyethylenimine (PEI) as a transfection reagent, followed by cell surface staining with MHC class I-specific monoclonal antibodies. Once viral proteins responsible for MHC class I downregulation have been identified, their mechanism of action can be characterized. Identification and characterization of virus-encoded MHC class I inhibitors augments our understanding of virus-host interactions and often provides new insights into antigen processing and presentation pathways, including related cellular processes such as protein trafficking and degradation.
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