Figure 7

Transcriptional regulation of various genes by TSA (A) Gene expression profile of TSA-treated and non-treated CD4⁺ T-cells. Naïve CD4⁺ CD62L⁺ CD44^low cells were isolated from C57BL/6 spleen cell preparations, cultured in the presence of IL-2 (10 U/ml) for 24 hours and Atlas Mouse cDNA Expression Arrays were used to analyse the differential gene expression caused by exposure of these cells to 100 nM TSA for 4 hours. Shown are all genes that varied in expression by greater than 2-fold in all experiments. Upward black arrows indicate upregulated genes, downward grey arrows denote downregulated genes. Data are based on two independent preparations of mRNA, for each experiment. Two membranes were used per sample. (B) Verification of several of the genes affected by TSA by semi-quantitative RT-PCR analysis. CD4⁺ T-cells were isolated by magnetic cell separation from pre-activated splenocytes (48 h in the presence of soluble anti-CD3 antibody). Cells were then stimulated with immobilized anti-CD3 mAb or cultured in the presence of IL-2 (10 U/ml), and harvested after 1, 4, or 20 hours in the presence of 100 nM TSA. Expression of 18S rRNA was used as normalizing control (C) Flow cytometric analysis of CD3ε and CD3 MC (molecular complex) expression in CD4⁺ T-cells exposed to TSA. CD4⁺ T-cells were cultured in the presence of immobilized anti-CD3 antibody (2:1 beads to cells ratio) or IL-2 (10 U/ml) and exposed to 100 nM TSA for 4 hours (open black histogram) and 20 hours (bold open black histogram), or to DMSO alone for 4 hours (open grey histogram) or 20 hours (open bold grey histogram). Data are representative of three independent experiments.