Time course of induction of GFP in SIN-(NFAT)_x-GFP transduced Jurkat cells and primary T-cell bulk cultures.

(A-C) Unsorted, SIN-(NFAT)₃-GFP– (open symbols and bars) or SIN-(NFAT)₆-GFP– (filled symbols and bars) transduced, Jurkat T cells (panel B; panel A, squares) and bulk T cells from a healthy donor (H.S.P.) (panel C; panel A, circles) were stimulated with PMA in combination with ionomycin. The percentage of GFP expressing cells, relative to the maximal level, which varied between 20% to 40%, and the MFI of GFP positive cells are plotted against the time in hours after initial stimulation. Shown are representative data from 3 independent experiments. (D) GFP expression in SIN-(NFAT)₆-GFP–transduced cells is blocked by CsA and by FK506. T cells transduced with the SIN-(NFAT)₆-GFP construct were incubated overnight in medium containing PMA and ionomycin. The percentage of GFP positive cells reached was set to 100%. Next, CsA or FK506 (final concentrations are shown), which are inhibitors of calcineurin and thus of expression of GFP, were added 30 minutes before the addition of PMA and ionomycin. Indicated is the percentage of GFP positive cells. Bars represent the following: bulk T-cell blasts (black), sorted CD4⁺ T cells (hatched), and sorted CD8⁺ T cells (dotted) all derived from donor K.W.E., and bulk T-cell blasts from donor H.S.P. (striped). Shown are representative data from 3 independent experiments.