1. Histone deacetylase inhibitors: a new class of immunosuppressors targeting a novel signal pathway essential for CD154 expression
by Søren Skov, Klaus Rieneck, Lone Frier Bovin, Kresten Skak, Søren Tomra, Birgitte K. Michelsen, and Niels Ødum
Blood
Volume 101(4):
February 15, 2003
©2003 by American Society of Hematology

2. HDAC-i inhibits proliferation of T cells
HDAC-i inhibits proliferation of T cells.PBL cultures (A-D,F) or purified CD4 T cells (E) were treated without or with the indicated amount of FR (A-E) or trichostatin A (F) for 2 hours prior to stimulation with 3 × 28 beads (A,E,F), ionomycin/PMA (B)...
HDAC-i inhibits proliferation of T cells.PBL cultures (A-D,F) or purified CD4 T cells (E) were treated without or with the indicated amount of FR (A-E) or trichostatin A (F) for 2 hours prior to stimulation with 3 × 28 beads (A,E,F), ionomycin/PMA (B), or allogeneic dendritic cells (C). Proliferation was measured by MTT assay after 3 (A-B) or 6 (C) days of stimulation (stim). Panel D shows a flow-cytometric profile of CFSE-labeled PBL cultures 6 days after stimulation with 3 × 28 beads in the presence or absence of 20 ng/mL FR901228, added before or after stimulation as indicated.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

3. HDAC-i influence on viability, cell cycle, and apoptosis
HDAC-i influence on viability, cell cycle, and apoptosis.PBL cultures were treated with or without the indicated amount of FR and stimulated for 24 hours with 3 × 28 beads.
HDAC-i influence on viability, cell cycle, and apoptosis.PBL cultures were treated with or without the indicated amount of FR and stimulated for 24 hours with 3 × 28 beads. Viability was measured as exclusion of PI by flow cytometry (A). Cell cycle distribution and apoptosis was measured by intracellular PI staining (B) as described in “Materials and methods.”
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

4. Calcium measurement after FR pretreatment of CD3-activated PBL cultures.Increase in intracellular calcium content was measured after anti-CD3 stimulation of Fura-2–loaded cells preincubated with or without 20 ng/mL FR
Calcium measurement after FR pretreatment of CD3-activated PBL cultures.Increase in intracellular calcium content was measured after anti-CD3 stimulation of Fura-2–loaded cells preincubated with or without 20 ng/mL FR Thick line represents anti-CD3 stimulation; thin line, control stimulation with PBS. Stimulation was initiated 2 minutes after start of recording and is indicated by arrow.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

5. Expression of activation markers on activated CD4 T cells treated with HDAC-i.CD25 (A), CD154 (B), and CD69 (C) were measured by flow cytometry 24 hours after stimulation with 3 × 28 beads with or without the indicated amount of FR
Expression of activation markers on activated CD4 T cells treated with HDAC-i.CD25 (A), CD154 (B), and CD69 (C) were measured by flow cytometry 24 hours after stimulation with 3 × 28 beads with or without the indicated amount of FR Results are shown as mean fluorescence intensity (MFI) of gated CD4 T cells.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

6. HDAC-i influence on aggregation and expression of adhesion molecules after T-cell activation.PBL cultures were preincubated with or without 20 ng/mL FR or 100 ng/mL trichostatin A and stimulated for 24 hours with 3 × 28 beads or uncoupled beads.
HDAC-i influence on aggregation and expression of adhesion molecules after T-cell activation.PBL cultures were preincubated with or without 20 ng/mL FR or 100 ng/mL trichostatin A and stimulated for 24 hours with 3 × 28 beads or uncoupled beads. (A) A characteristic example of the HDAC-I–mediated inhibition of aggregation; similar results were always observed upon visual inspection of stimulated cells treated with HDAC-i. Original magnification, × 20. (B) CD11a and CD54 were measured by flow cytometry 24 hours after stimulation with control (−) or 3 × 28 beads (+) without (white bars) or with (black bars) 20 ng/mL FR or 100 ng/mL trichostatin A (hatched bars). Results are shown as MFI of gated CD4 T cells.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

7. HDAC-i influence on IL-2 production from activated CD4 T cells
HDAC-i influence on IL-2 production from activated CD4 T cells.PBL cultures were preincubated with or without 20 ng/mL FR or 100 ng/mL trichostatin A (TSA) and stimulated for 24 hours with 3 × 28 beads.
HDAC-i influence on IL-2 production from activated CD4 T cells.PBL cultures were preincubated with or without 20 ng/mL FR or 100 ng/mL trichostatin A (TSA) and stimulated for 24 hours with 3 × 28 beads. (A) IL-2 was measured in the supernatant by ELISA. (B) Intracellular IL-2 was measured by flow cytometry on gated CD4 cells.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

8. Mechanism behind HDAC-i inhibition of CD154 expression
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 10...
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 FR (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.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

9. Involvement of c-Myc in CD154 expression
Involvement of c-Myc in CD154 expression.(A) Western blot of c-Myc expression in PBL or Jurkat T cells 18 hours after addition of the indicated amount (ng/mL) of FR
Involvement of c-Myc in CD154 expression.(A) Western blot of c-Myc expression in PBL or Jurkat T cells 18 hours after addition of the indicated amount (ng/mL) of FR PBLs were stimulated (stim) with or without 3 × 28 beads as indicated. (B) PBLs were incubated with the indicated phosphothioate oligo probes, 10 μM, for 6 hours prior to stimulation with or without 3 × 28 beads as indicated. CD154 expression was measured after 18 hours by flow cytometry on gated CD4 T cells. (C) Jurkat cells were transiently transfected, in a 1:3 ratio, with CD154 promotor–GFP construct in combination with empty vector, c-Myc vector, or Madmyc vector as indicated. Cells were then stimulated with (black bars) or without (white bars) anti-CD3 Ab for 24 hours. 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.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology

10. In vivo effect of FR901228 on glucose level in diabetic NOD mice
In vivo effect of FR on glucose level in diabetic NOD mice.Development in blood glucose level of NOD mice treated with vehicle control (▪) or FR (○) twice a week after their blood glucose level exceeded 10 mM.
In vivo effect of FR on glucose level in diabetic NOD mice.Development in blood glucose level of NOD mice treated with vehicle control (▪) or FR (○) twice a week after their blood glucose level exceeded 10 mM.
Søren Skov et al. Blood 2003;101:
©2003 by American Society of Hematology