Figure 3

Human PBLs transduced with the EpCAM-specific CAR cause cytotoxicity of PC3M tumor cells. A) PBLs were stimulated with OKT3 and anti-human CD28 antibodies for 2 d before being transduced with retroviruses encoding the EpCAM-specific CAR or an empty vector. Five days later, transduced cells were incubated with Hela-luc or PC3M-luc tumor cells at an E:T ratio of 2:1. After 24 h of incubation, luciferin was added and the cell viability was examined by luminescence imaging. Tumor cells alone were used as a control. Statistical analysis was performed using a Student’s t-test. B) PBLs were stimulated as in (A) and were transduced with the EpCAM-specific CAR. Five days later, the transduced cells were incubated with PC3M-luc cells at different E:T ratios, including 1:1, 2:1, 5:1, and 10:1. After 24 h of incubation, luciferin was added and cell viability was determined by luminescence imaging. PC3M tumor cells alone were used as controls. C) PBLs were stimulated as in (A) and transduced with the EpCAM-specific CAR or control vector. Five days later, the cells were labeled with CFSE and were co-cultured with or without PC3M cells at an E:T ratio of 2:1. Three days later, the cells were collected and stained with the anti-CD8 antibody and cell proliferation was examined by flow cytometry.