Fluorescent antigen-transfected target cell cytotoxic T lymphocyte assay for ex vivo detection of antigen-specific cell-mediated cytotoxicity.
SourceThe Journal of Infectious Diseases, 192, 7, (2005), pp. 1183-90
Article / Letter to editor
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The Journal of Infectious Diseases
SubjectUMCN 4.1: Microbial pathogenesis and host defense
Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigentransfected target cellCTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection with DNA vectors encoding antigengreen fluorescent protein (GFP) fusion proteins. After coculture at various effector : target (E : T) cell ratios, viable and dead GFP-positive cells were quantified by flow cytometry, and antigen-specific target-cell elimination was calculated. The assay was validated with human immunodeficiency virus (HIV) and influenza virusspecific CTL clones and revealed cytotoxicity at lower E : T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex vivo within 1 day in peripheral blood mononuclear cells from HIV-infected individuals. The FATT-CTL assay provides a versatile tool that will advance our understanding of cell-mediated immunity.
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