FIGURE 6.

Role of programmed death‐ligand 1 (PD‐L1) and NKG2D ligands on pemetrexed (PEM)‐treated cancer cells in mediating sensitivity to cytotoxic immune cells. (A) Activated T cells were stained with APC‐conjugated anti–CD8, PE‐conjugated anti–CD4, FITC‐conjugated anti–PD‐1 or FITC‐conjugated anti–NKG2D antibody. As a control, these cells were stained with FITC‐conjugated mouse IgG. The bold line indicates staining with FITC‐conjugated anti–PD‐L1 antibody, and the gray background indicates staining with isotype‐matched FITC‐conjugated mouse IgG. The number represents the mean fluorescence intensity (MFI) ratio. (B) Similarly, purified natural killer (NK) cells were stained with PE‐conjugated anti–CD56 antibody and analyzed by flow cytometry. The number represents the MFI ratio. (C) Cancer cells that were cultured with PEM (2 µmol/L) for 2 d were cultured with activated T cells in the presence of the indicated antibodies in 96‐well plates for 6 h. After harvesting, annexin V‐FITC positive cells were examined as shown in Figure 2. (D) Similarly, PEM‐treated cancer cells were cultured with in vitro NK cells in the presence of the indicated antibodies in 96‐well plates for 12 h. After harvesting, annexin V‐FITC positive cells were examined as shown in Figure 2D and E. *P < 0.05. **P < 0.01. ***P < 0.005