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Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
Published on: October 22, 2012
Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood
X S He1, B Rehermann, J Boisvert
1Department of Medicine, Stanford University School of Medicine, California 94305-5187, USA. xiaosong@stanford.edu
Insights
This study introduces a novel assay for analyzing virus-specific CD8+ T cells directly from whole blood. The method efficiently identifies functional and non-functional T cells, crucial for understanding viral infections and immune responses.
Area of Science:
- Immunology
- Virology
- Cellular Biology
Background:
- The functional status of virus-specific CD8+ T cells significantly impacts viral infection outcomes and immunopathogenesis.
- Existing methods for T cell analysis often require extensive in vitro cultivation, which can alter cell function.
Purpose of the Study:
- To develop a rapid assay for the direct functional analysis of antigen-specific CD8+ T cells.
- To enable simultaneous detection of both functional and non-functional virus-specific CD8+ T cells.
Main Methods:
- Incubation of whole blood samples with peptide antigens for less than 5 hours.
- Staining with peptide-MHC tetramers to identify epitope-specific T cells.
- Simultaneous staining for activation markers (e.g., CD69) or cytokines (e.g., IFNγ, TNFα).
Main Results:
- The assay allows for direct functional analysis of antigen-specific CD8+ T cells without prolonged in vitro culture.
- It enables the simultaneous determination of the number of antigen-specific CD8+ T cells and their individual functional responses.
- This method can detect both functional and non-functional virus-specific CD8+ T cells under identical conditions.
Conclusions:
- This novel assay provides a direct and efficient method for assessing CD8+ T cell function in viral infections.
- The assay overcomes limitations of traditional methods like cytotoxicity assays, intracellular cytokine staining, and ELISPOT by allowing simultaneous detection of functional states.
- This advancement is critical for understanding T cell-mediated immunity and immunopathology in viral diseases.
Abstract:
The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.
