Mechanism behind HDAC-i inhibition of CD154 expression.

(A) Previously activated PBL cultures were stimulated for 3 days with 3 × 28 beads, then cultured for 24 hours with 3 × 28 beads and 100 U/mL IL-2; 3 × 28 beads and 10 μg/mL blocking anti–IL-2 Ab; or 100 U/mL IL-2, 10 μg/mL blocking anti–IL-2 Ab, as indicated. Prior to stimulation, for the last 24 hours, cells were incubated without (black bars) or with 500 ng/mL cyclosporine (white bars) or with 20 ng/mL FR901228 (hatched bars). (B) Flow cytometric analysis of CD154 surface expression on mycosis fungoides cells and intracellular CD154 expression on Jurkat cells 24 hours after addition of vehicle control, 20 ng/mL FR901228, or 500 ng/mL cyclosporine, as indicated. Control stain indicates staining with IgG1-PE Ab. (C) Jurkat cells were transiently transfected with empty vector or CD154 promotor–GFP construct as described in “Materials and methods” and stimulated with or without anti-CD3 mAb for 24 hours. Figure shows the flow cytometric profile of GFP expression in the cells. (D) Jurkat cells were transiently transfected with CD154 promotor–GFP construct and stimulated with or without anti-CD3 Ab for 24 hours in the presence or absence of 20 ng/mL FR901228, 100 ng/mL trichostatin A, or 500 ng/mL cyclosporine. Figure shows CD154 promotor activity as MFI of gated GFP-expressing cells multiplied by the percentage of GFP-positive cells in order to take into account both the level and percentage of GFP expression. Error bars indicate SEM.