Fig. 4

CAR T cells exhibit an increased cytotoxicity against colon cancer cells. a EGFR expression in DLD-1 and HCT-116 cells measured by flow cytometry. Cells were treated with 30 ng/ml IFN-γ for 24 h before incubated with antibodies against EGFR or an IgG control (control). b The EGFRvIII-CAR transgene structure. c Expression of EGFRvIII-CAR molecules in primary T cells as measured by flow cytometry. d qPCR analysis of EGFRvIII-CAR expression in T cells. qPCR was performed using two pairs of oligonucleotides. The mRNA levels of the CAR were determined using GAPDH as a reference. ND not detectable. e Cytokine production of WT T and CAR T cells co-cultured with HCT-116 cells as measured by ELISA (n = 3). f CFSE-based cell proliferation assays of WT and CAR T cells co-cultured with HCT-116 cells. X-axis is for the CFSE intensity; Y-axis is for the events based on side-scattered light (cell numbers). g Apoptosis analyses of HCT-116 or DLD-1 cells co-cultured with WT T or CAR T cells. Green florescence represents the release of caspase 3/7. h CAR T cell-mediated killing of tumor cells. HCT-116 or DLD-1 cells carrying firefly luciferase gene were co-cultured with WT or CAR T cells at different ratios (E:T effector T cells to target tumor cells). Luminescence was measured as a percentage of tumor cell death. The green line denotes CAR T cells, and orange denotes WT T cells. *P ≤ 0.05; **P ≤ 0.01