Fig. 4.

Protein kinase B glycogen synthase kinase 3β (AKT/GSK-3β) phosphorylation and cytokine/CD40L gene expression in CD2-activated CD45RO⁺ CD4⁺ peripheral blood T lymphocytes (PBT) and total CD4⁺ PBT. (a) CD4⁺ PBT and CD45RO⁺ PBT were cultured in the presence or absence of CD2 monoclonal antibody (mAb) M1 (1 μg/ml), M2 (1 μg/ml) and 3PT (0·33 μg/ml) for the indicated time-periods. The phosphorylation state of GSK-3β (Ser9) and AKT (Ser473) was determined by immunoblotting of whole cell extracts. The phosphorylation index of AKT (Ser473 and Thr308; upper panels) and GSK-3β (Ser9; lower panel) was determined as described in Materials and methods. Data are expressed as mean ± standard deviation (n = 3). (b) Isolated CD4⁺ PBT and CD45RO⁺ PBT were activated by CD2 mAb M1 (1 μg/ml), M2 (1 μg/ml) and 3PT (0·33 μg/ml). After 4 h, IL-2 mRNA expression was determined by quantitative reverse transcribed polymerase chain reaction. Culture supernatant was harvested after 24 h and the IL-2 content was determined by enzyme-linked immunosorbent assay (detection limit 16 pg/ml). Results are representative of three independent experiments.