Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 459-466
NFAT-controlled expression of GFP permits visualization and
isolation of antigen-stimulated primary human T cells
Erik Hooijberg,
Arjen Q. Bakker,
Janneke J. Ruizendaal, and
Hergen Spits
From the Department of Immunology, The Netherlands Cancer
Institute/Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.
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.
