TSA represses IL-2 gene expression. Spleen cells from C57BL/6 mice were cultured for 48 h in the presence of soluble anti-CD3 antibody (0.5 μg/ml), whereafter CD4+ T-cells were isolated by magnetic cell separation as described in the materials and methods section. Cells were subsequently stimulated with the indicated immobilized antibodies and in the presence of the indicated inhibitors. (A) Inhibitors or DMSO alone (vehicle) were added to the medium – TSA (final concentration of 10 nM or 100 nM). After 8 hours the supernatant was harvested and IL-2 levels quantified by ELISA. (B) Purified CD4+ T-cells stimulated with immobilized anti-CD3 mAb were harvested after 4, 8, or 20 hours in the presence of 100 nM TSA, and levels of IL-2 mRNA quantified by relative RT-PCR as described (materials and methods section). Primers for 18S rRNA were used as control. (C) CD4+ T-cells stimulated with immobilized anti-CD3 mAb and cultured in growing concentrations of TSA were harvested after 8 hours and intracellular levels of IL-2 analysed by flow cytometry. Dead cells are excluded from the analysis. The data are representative of three independent experiments.