Figure 3

MEDI-565/T-cell-induced apoptotic cell death of a CEA+ tumour cell line. (A) MEDI-565 induced cytotoxic capacity of T cells derived from colorectal cancer patients compared with normal healthy donor T cells. T cells were isolated from the PBMCs derived from normal healthy donors (left panel) or colorectal cancer patients (right panel) using T-cell-negative isolation kit. Isolated T cells were co-cultured with AsPC-1 tumour cells in a 5 : 1 effector-to-target ratio in the presence of various concentrations of MEDI-565 (0.01∼10 000 ng ml⁻¹) in 96-well U-bottom plates as described in Materials and Methods. Cells were incubated for 2 days, and the cytotoxicity was analysed using flow cytometry with 7-AAD staining. No significant difference (P>0.05 for Student's t-test, see text) in MEDI-565-induced T-cell cytotoxicity was observed between T cells from cancer patients and T cells from normal donors when compared at each concentration of MEDI-565. (B) Carcinoembryonic antigen (CEA) specificity of MEDI-565/T-cell mediated killing of tumour cells. The CEA+ tumour cell lines (Ls174T and AsPC-1) and CEA-negative tumour cell lines (SW480 and HCT116) were incubated in a 96-well U-bottom plate with T cells negatively isolated from the PBMCs of normal donors (triangle symbols) or cancer patients (square symbols). MEDI-565 or Cont BiTE was put into the culture at the indicated concentrations. After 2 days of incubation, cells were harvested from the plates with 0.05% trypsin/EDTA and stained with FITC-labelled anti-lineage marker and 7-AAD. The 7-AAD-positive cells in lineage marker negative cells are analysed and shown in each line graph. (C, D) Effects of time and concentration of MEDI-565 on T-cell-induced apoptosis of CEA+ tumour cells. Both the AsPC-1 cells (1 × 10⁵ cells per well) and normal donor's T cells (5 × 10⁵ cells per well) were put into a 96-well U-bottom plate with indicated concentrations of MEDI-565 or Cont BiTE. After 4 days of incubation, cells were harvested with 0.05% trypsin/EDTA and labelled with biotin-labelled Annexin V, and then stained with FITC-anti-lineage mixture, PE-anti-CEA, 7-AAD and streptavidin-APC. Lineage marker-negative and CEA+ tumour cells were analysed for their 7-AAD/Annexin V staining. (C) The AsPC-1 cells were incubated with T cells in the presence of MEDI-565 or Cont BiTE (100 ng ml⁻¹) for 1, 2 or 4 days and stained as described in (B). Percentages of annexin V-positive cells (including 7-AAD negative and 7-AAD positive) in CEA+ tumour cells are shown in each line graph.