TSA induces apoptosis and cell cycle arrest in CD4
+ T-cells. (A) CD4+ T-cells were treated for 24 hours with increasing concentrations of TSA (■). Cell death was determined by flow cytometric analysis of DNA content by PI staining of nuclei. Error bars show SD. (B) CD4+ T-cells were treated for 8 or 20 hours with various concentrations of TSA or DMSO alone as a negative control (-TSA). Cells were subsequently stained with Annexin V and PI. Inset in the bottom right quadrant of each plot are the percentage of cells that gated to the various quadrants (UL – upper left; UR – upper right; LL – lower left; and LR – lower right). The numbers for LR correspond to cells in early apoptosis (Annexin V positive and PI negative). (C) Effect of TSA on cell cycle progression of T cells. CD4+ T-cells were incubated for 24 h in the presence of medium containing IL-2 (10 U/ml), or anti-CD3 coupled beads (2:1 beads to cells ratio) after which either TSA (final concentration of 10 nM) or DMSO alone was added. After 20 hours the cells were analysed for cell cycle distribution by flow cytometry. Data are plotted as DNA content (propidium iodide fluorescence) vs relative live cell number (cells gated to R1). The total percentages of cells in the G0/G1 (region M1), S (region M2), and G2/M (region M3) phases of the cell cycle are shown. (D) Live TSA-treated cells (20 h with 10 nM TSA) were isolated by centrifugation in Ficoll Paque, washed extensively, labelled with CFSE and incubated in the presence of anti-CD3 coated beads (2:1 bead to cell ratio). Proliferation status of T cells was evaluated after 24 h (gray shaded histogram), 72 h (black open histogram), and 8 days (open gray histogram). Arrows indicate CFSE division peaks. For simplicity dead cells were excluded from the analysis. The data are representative of three independent experiments.