Fluorescent antigen-transfected target cell cytotoxic T lymphocyte assay for ex vivo detection of antigen-specific cell-mediated cytotoxicity

Carel A. Van Baalen, David Kwa, Esther J. Verschuren, Mariska L. Reedijk, Adrianus C.M. Boon, Gerrie De Mutsert, Guus F. Rimmelzwaan, Albert D.M.E. Osterhaus, Rob A. Gruters

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine production, degranulation, or perform contents of antigen-specific CDS* T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigen-transfected target cell-CTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were gen-erated by nucleofection with DNA vectors encoding antigen-green 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 virus-specific 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.

Original languageEnglish
Pages (from-to)1183-1190
Number of pages8
JournalJournal of Infectious Diseases
Volume192
Issue number7
DOIs
StatePublished - Sep 1 2005
Externally publishedYes

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