Fig. 1
From: HDAC6 selective inhibition of melanoma patient T-cells augments anti-tumor characteristics
ACY-1215 and ACY-241 reduce Treg percentages and suppressive function. (a) CD4+ and (b) CD8+ melanoma patient T-cells were cultured for 72 h with 100IU/mL IL-2, plus ACY-1215 (circles), ACY-241 (squares), MS275 (triangles), Tubastatin A (inverted triangles), LBH589 (diamonds), or DMSO. Viability was assessed by DAPI. Representative experiment assessing three samples is shown; six patients were assessed over two experiments. (c) CD4 + CD127loCD25+ viability was assessed in T-cells cultured with 100IU/mL IL-2 for 72 h, plus ACY-1215 (circles) or ACY-241 (squares). Representative experiment assessing three patient samples is shown; eight patients were assessed over three experiments. (a-c) Y-axis displays the mean viability (±SEM) relative to DMSO; X-axis indicates inhibitor doses. (d) FOXP3 expression was assessed in CD4 + CD127loCD25+ Tregs by flow cytometry for ACY-1215 or ACY-241 treatments (500nM). Representative contour plots for one sample are shown; four patients were assessed. (e) FOXP3 expression was evaluated in additional four samples over two experiments, each denoted by a color, treated with the indicated nhibitors. (f) DMSO (black histogram) or ACY-1215 500nM (orange histogram) pre-treated nTregs from one patient were co-cultured with autologous CD8+ Tcons (1:5 Treg:CD8+). Ki67 expression in CD8+ T-cells was assessed after five days. Control (only CD8+, no nTregs) is shown in grey. (g) nTreg suppression was assessed in three samples in an independent experiment, using CD4+ Tcons. The percentage of Ki67 expressing CD4 + FOXP3- Tcon cells is shown. (h-i) CD4+ naïve-derived iTregs from seven patient samples were expanded and treated with DMSO or 500nM ACY-1215 over four experiments. (h) Relative viability and (i) cell counts were assessed by Trypan Blue seven days after culture. (j) iTregs from three patients were expanded and treated with DMSO or 500nM ACY-1215 over three experiments. Autologous CD8+ T-cells were co-cultured with iTregs, activated for 48-72h, and assessed for Ki67 expression. Proliferation percentages relative to control (Tcons only) are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001