Fig. 2　Nivolumab treatment enhances the tumoricidal activity of NKT cells. (A, B) Purified NKT cells treated with nivolumab or isotype hIgG4 were further treated with nivolumab or hIgG4, and then co-cultured with CTV-labeled K562 or A549 cells for 4 h. The cells were stained with annexinV and PI, and determined for tumor cell-specific apoptosis by gating on CTV labeling. (A) Data for CTV⁺ K562 or A549 cells co-cultured at an E/T ratio of 10:1. (B) Cytotoxicity against K562 cells calculated for E/T ratios of 10:1, 5:1, and 2.5:1. The black circles indicate the cytotoxic activity of NKT cells treated with Nivolumab, while the white circles indicate the cytotoxic activity of NKT cells treated with an isotype control. The results of three healthy donors against K562 and A549 cells at an E/T ratio of 10:1 are shown in (C) . (D) Purified NKT cells treated with nivolumab or isotype hIgG4 were further treated with nivolumab or hIgG4, and then co-cultured with K562 cells for 4 h. The cells were subjected to real-time PCR to assess the expression levels of Granzyme B, Perforin, TNF-α, TRAIL, and FASL, normalized with HPRT1. Bars and error bars indicate the mean and SD of biological triplicate, respectively (n = 3) . The data are representative of the results from two independent experiments. P-values were calculated by an unpaired t-test. *P<0.05, **P<0.01, ***P<0.001.