Dif50c

T-cell supernatant in the co-culture system was collected, and the total protein was quantified using the BCA Kit (Pierce, Rockford). In the light of ELISA kit instruction, levels of interleukin (IL)-2 (D2050, R&D systems) and interferon (IFN)-γ (DIF50C, R&D systems) were determined. A microplate reader (Molecular Devices, California, USA) was adopted to evaluate the optical density (OD) value at 450 nm to quantify the levels of the above-mentioned cytokines.

Human CXCL10 (R&D Systems, catalog no. DIP100), human IFNγ (R&D Systems, catalog no. DIF50C), and human IL2 (R&D Systems, catalog no. D2050) ELISAs were performed according to the manufacturer's instructions. Conditioned media from each cell lines was collected after 24 or 72 hours culture.

The culture supernatants of SMMC-7721 cells infected with different adenoviruses were still used in the following experiments. 7721-PD-L1 cells were seeded into 96-well plates (1 × 10⁴ cells/well). The next day, PBMCs pretreated with IL-2 for 72 h were added at the effector: target (E:T) ratios of 5:1. Then, 100 uL of the supernatant was added simultaneously. The specific cytotoxicity toward 7721-PD-L1 cells was measured by the lactate dehydrogenase assay 16 h later using a CytoTox 96 nonradioactive cytotoxicity kit (#G1780, Promega, USA) following the manufacturer’s protocols. Then, the cells and the culture supernatant of every well were separated. The cells were stained with the APC-human CD3 antibody (#300312, Biolegend, USA) and the phycoerythrin (PE)-human CD69 antibody (#310906, Biolegend, USA) for 30 min at room temperature and measured using FACS to detect activated T cells. The levels of interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the supernatants were measured using the corresponding ELISA kits (D2050, DIF50C, and DTA00D, R&D, USA).

CAR T-cell effector function was determined in vitro in a coculture of CAR T cells and tumor cells by (i) measuring direct killing (cytotoxicity) of mesothelin-expressing tumor cells by the CAR T cells and (ii) quantifying the cytokines released by the CAR T cells. For cytotoxicity assays, 100 μL of complete RPMI media containing 5,000 tumor cells were seeded in a 96-well plate. Four to six hours after seeding the tumor cells, 50 μL of media containing CAR T cells or untransduced T cells at a range of effector-to-target (E:T) ratios were added to initiate the co-culture. Wells with tumor cells only, without the CAR T cells, were used as internal plate references. After 20- to 24-hour coculture, luciferase signal from the remaining live tumor cells was determined (Promega, Luciferase Assay System, catalog No. E1501). Percent killing was calculated using the formula: % killing = 100 × [1- relative light units (RLUs) from coculture wells/RLU from target-alone wells]. ELISA kits were used to quantify cytokines IL2, IFNγ, and TNFα (R&D Systems, catalog No. D2050, DIF50C, DTA00D) secreted by the stimulated CAR T cells in the culture supernatant after 20 to 24 hours of coculture with the tumor cells.