Figure 3: Effects of treatment with decitabine, ionizing radiation (IR), or their combination on activity of T cells against target tumor cells.

T cells were co-cultured with decitabine, IR or both treated tumor cells (A549, HCT-116, and HepG2 cells) for 5days for MLR assay. Also, T cells were co-cultured with decitabine, IR or both treated tumor cells at various ratios for 4 h for cytotoxicity assay. For blockade experiments, T cells were co-cultured with combination-treated target tumor cells in the presence of 10 ug/ml anti-CD40, CD80, and/or HLA-A,B,C antibody. T cell proliferation (a) or inhibition (b) was assessed using Cell Counting Kit-8 (CCK8), and T cell cytotoxicity (c) or inhibition of cytotoxicity (d) was performed by flow cytometry. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. Results express the average T cell proliferation or T cell-mediated cytotoxicity ± SD in A549, HCT-116, and HepG2 cells. Experiments were independently performed from five healthy donors. The assay was performed in triplicated each donor. The statistical significance was determined using a one-way ANOVA. ^#P < 0.05, ^##P < 0.005, ^###P < 0.0005 (^#DAC 0 versus other groups, mIgG versus other groups). *P < 0.05, **P < 0.005, ***P < 0.005 (*DAC 5 versus other groups, αCD40 versus other groups). ^**P < 0.05, P < 0.005, P < 0.0005 (^**IR 8 Gy versus DAC 5 + IR 8 Gy, αCD80 versus αCD40 + αCD80). ^@P < 0.05, ^@@P < 0.005 (^@αCD40 + αCD80 versus αCD40 + αCD80 + αHLA-ABC).