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Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen-Specific Cell-Mediated Cytotoxicity

Identifieur interne : 000A50 ( Istex/Checkpoint ); précédent : 000A49; suivant : 000A51

Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen-Specific Cell-Mediated Cytotoxicity

Auteurs : Carel A. Van Baalen [Pays-Bas] ; David Kwa [Pays-Bas] ; Esther J. Verschuren [Pays-Bas] ; Mariska L. Reedijk [Pays-Bas] ; Adrianus C. M. Boon [Pays-Bas] ; Gerrie De Mutsert [Pays-Bas] ; Guus F. Rimmelzwaan [Pays-Bas] ; Albert D. M. E. Osterhaus [Pays-Bas] ; Rob A. Gruters [Pays-Bas]

Source :

The Journal of Infectious Diseases [ 0022-1899 ] ; 2005.

RBID : ISTEX:0751D90D016305325EF2BDA3C255F2198B03D61D

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 antigen–transfected target cell–CTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated 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

Url:

https://api.istex.fr/ark:/67375/HXZ-94R7CGL2-M/fulltext.pdf

DOI: 10.1086/444546

Affiliations:

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ISTEX:0751D90D016305325EF2BDA3C255F2198B03D61D

Le document en format XML

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<front><div type="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 antigen–transfected target cell–CTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated 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</div>
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Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen-Specific Cell-Mediated Cytotoxicity

Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen-Specific Cell-Mediated Cytotoxicity

Source :

Abstract

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