The calcineurin inhibitors CsA and FK506 potentiate TCR-induced increase in LAT expression.

Jurkat T cells (A) or purified human normal resting T cells (B) were stimulated for 16 hours at 37°C with RPMI-5% FCS containing 1 μg/mL of anti-CD3 mAb in the absence (lane 2) or presence of the indicated concentrations of FK506 (lanes 3-5) or CsA (lanes 6-8). After incubation, the cells were immediately lysed with boiling 2× SDS-PAGE sample buffer. Proteins in WCL were separated by SDS-PAGE and then transferred to membranes and immunoblotted with anti-LAT mAb or anti-Csk Ab. This experiment was repeated 3 times with similar results. (C) Jurkat T cells were stimulated for 16 hours at 37°C with RPMI-5% FCS containing the indicated concentrations of anti-CD3 mAb in the absence (lanes 2-5) or presence (lanes 6-9) of FK506. Proteins were processed as described above. (D) Jurkat T cells were incubated for 16 hours at 37°C in RPMI-5% FCS containing the indicated concentrations of CsA (lanes 2 and 3) or FK506 (lanes 4 and 5). (E) IL-2 level in the supernatants of overnight cultures of Jurkat T cells stimulated for 16 hours at 37°C with 1 ng/mL of PMA or with the combination of PMA and 1 μM Ca⁺⁺ ionophore in the absence or presence of the indicated concentrations of CsA or FK506. IL-2 level was determined with a sandwich enzyme-linked immunosorbent assay technique using combinations of unlabeled and biotin-labeled antibodies, as described in “Materials and methods.” This experiment was repeated twice with similar results.