NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells.
We have developed a new method that allows detection and isolation of viable, antigen-specific, human T cells from a heterogeneous pool of T cells. We have engineered a self-inactivating retroviral vector containing multiple (3 or 6) nuclear factor of activated T-cell (NFAT)-binding sites, followed by the minimal IL2 promoter and the reporter gene GFP. Jurkat cells, primary T-cell blasts, and T-cell clones were transduced with high efficiency (20%-40%). Stimulation of the transduced cells with phorbol myristate acetate (PMA) and ionomycin resulted in a high level expression of GFP that was maximal after 12 to 14 hours and remained stable for another 12 hours. Activation of T cells carrying the construct containing 6 NFAT-binding sites resulted in the highest mean fluorescence intensity. Cyclosporin-A and FK506 were able to block the activation-dependent GFP expression. Activation of transduced T-cell blasts with the superantigen staphylococcal enterotoxin B or of transduced antigen-specific T-cell clones with cognate antigen resulted in GFP expression. After an overnight stimulation of a heterogeneous T-cell bulk culture with an HLA mismatched stimulator cell (JY), the GFP expressing cells were cloned. As expected, the cloning frequency of the antigen-specific GFP(+) cells was considerably higher than that of the total T-cell population. Most of the T-cell clones were either cytolytic, or proliferative toward JY stimulator cells. Interestingly, we also isolated T-cell clones that were noncytolytic and nonproliferative toward JY cells, but specifically up-regulated GFP after an overnight stimulation with JY. (Blood. 2000;96:459-466)
Hooijberg
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Department of Immunology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.
Bakker
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AQ
Ruizendaal
J J
JJ
Spits
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D000941:Antigens / Q000276:immunology; D001665:Binding Sites; D002469:Cell Separation; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics*; D008213:Lymphocyte Activation; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D016559:Tacrolimus / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000941:Antigens; D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D008164:Luminescent Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D016572:Cyclosporine; D013755:Tetradecanoylphorbol Acetate; D016559:Tacrolimus