FIGURE 4.

GSK3β inactivation leads to enhanced cellular levels of c-Jun in TCR-activated memory CD4⁺ T cells. A and B, Memory CD4⁺ T cells were pretreated for 2 h with DMSO (0.1%) or with SB216763 (6 μM) and then transferred to plates precoated with anti-CD3 (3 μg/ml). On the indicated day, whole-cell lysates were prepared and analyzed for phospho-STAT3 (T705), phospho-CREB (S133), c-Jun, or β-actin. C, Human memory CD4⁺ T cells were transfected with a nontargeting pool of siRNA or a pool of GSK3β siRNA and the levels of endogenous GSK3β were assessed by flow cytometry 48 h posttransfection. D, Memory CD4⁺ T cells were transfected with equal amounts of nontargeting siRNA GSK3α-specific siRNA or GSK3β-specific siRNA. On day 3, transfected cells were transferred to anti-CD3-coated plates (3 μg/ml) and analyzed for cellular levels of c-Jun 48 or 72 h postactivation by flow cytometry. E, Memory CD4⁺ T cells were pretreated in NaCl (6 mM) or LiCl (6 mM) for 2 h and then transferred to plates coated with anti-CD3 (3 μg/ml). Monensin was added during the last 6 h of a 72-h culture and cells were analyzed for intracellular c-Jun and IL-10 by flow cytometry. Results are representative of three individual experiments.