Fig. 4.

Effects of anti-CD73 antibody on splenocytes of irradiated tumor bearing mice. CD73 mAb enhances IFN-γ production and cytotoxicity of splenocytes from irradiated mice. a, b Tumor bearing mice received 3 fractions of 4 Gy local radiation therapy (RT) together with intraperitoneal injection of 200 μg anti-CD73 mAb or Rat IgG2a isotype control on days 12, 14, 16, and sacrificed at day 18. The splenocytes were cultured in RPMI-1640 + 10% FCS in the presence of brefeldin A and then fixed, permeabilized and stained with PE-conjugated IFN-γ or isotype control and APC-conjugated anti-CD3 and BV421-conjugated anti-CD4 mAb and FITC-conjugated anti-CD8 mAb as well as FVS780 for dead cell exclusion. The ratio of IFN-γ positive cells were calculated in CD3 (+) CD4 (+) or CD3 (+) CD8a (+) gated area. c The splenocytes were cultured with irradiated LuM1 in 2 ml 10% FCS+ RPMI-1640 medium supplemented with 20 ng/ml mouse recombinant IL-2 for 12 days, and then incubated with LuM-1 cells at an E/T ratio of 20:1. After 4 h incubation, all cells were stained with FITC-conjugated Annexin-V, 7-AAD and APC-conjugated anti-CD45 mAb, and ratios of 7-AAD positive dead cells calculated in LuM-1 population defined in FSC/SCC and CD45 (−) gated area. P value was calculated with the Mann-Whitney test and * showed p < 0.05