title: Fluorescent antigen-transfected target cell cytotoxic T lymphocyte assay for ex vivo detection of antigen-specific cell-mediated cytotoxicity.
abstract: 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.
authors.affiliation: Department of Virology, Erasmus MC, University Medical Center and Postgraduate School of Molecular Medicine, Rotterdam, The Netherlands.
authors.forename: Carel A
authors.initials: CA
authors.lastname: van Baalen
authors.forename: David
authors.initials: D
authors.lastname: Kwa
authors.forename: Esther J
authors.initials: EJ
authors.lastname: Verschuren
authors.forename: Mariska L
authors.initials: ML
authors.lastname: Reedijk
authors.forename: Adrianus C M
authors.initials: AC
authors.lastname: Boon
authors.forename: Gerrie
authors.initials: G
authors.lastname: de Mutsert
authors.forename: Guus F
authors.initials: GF
authors.lastname: Rimmelzwaan
authors.forename: Albert D M E
authors.initials: AD
authors.lastname: Osterhaus
authors.forename: Rob A
authors.initials: RA
authors.lastname: Gruters
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002461:Cell Line, Transformed; D002999:Clone Cells; D003601:Cytotoxicity Tests, Immunologic; D018984:Epitopes, T-Lymphocyte; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D015658:HIV Infections / Q000276:immunology; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D009980:Influenza A virus / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000821:virology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000821:virology; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000941:Antigens; D018984:Epitopes, T-Lymphocyte; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins