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. 2016 Apr 29;7(23):34341–34355. doi: 10.18632/oncotarget.9114

Figure 2. CAR T cell effector function.

Figure 2

(A) Viability of skrc59 CAIX+/PD-L1+ cells or (B) Skrc52 CAIX-/PD-L1- cells incubated ON with each indicated CAR T cells. These CAR T cells were used 4 days after lentiviral transduction. The cell viability was evaluated by MTT (Molecular Probes). *P < 0.05 for all CAR T cells compared to anti-BCMA CAR/anti-SARS IgG1. (C) IL2 released by the CAR T cells after overnight contact with skrc59 CAIX+/PD-L1+ cells or (D) Skrc52 CAIX-/PD-L1- cells. The IL-2 secretion was evaluated using the Human IL-2 ELISA Ready-SET-Go Kit (eBioscience). *P < 0.001 for all CAR T cells compared to anti-BCMA CAR/anti-SARS IgG1. (E) IFNγ released by the CAR T cells after overnight contact with skrc59 CAIX+/PD-L1+ or (F) Skrc52 CAIX-/PD-L1- cells. The IFNγ secretion was evaluated using the Human IFNγ ELISA Ready-SET-Go Kit (eBioscience). *P < 0.05 for all CAR T cells compared to anti-BCMA CAR/anti-SARS IgG1. (G) Ab-dependent cell-mediated cytotoxicity (ADCC) of skrc59 CAIX+/PD-L1+ cells or (H) skrc52 CAIX-/PD-L1- cells after incubation with the supernatant (SN) of the CAR T cells containing 500 ng/mL of the anti-PD-L1 IgG1, anti-PD-L1 IgG4 or the anti-SARS IgG1. NK cells were purified using an EasySep™ Human NK Cell Enrichment Kit (StemCell™ Technologies) from PBMC. RCC cell lines skrc59 CAIX+/PD-L1+ and skrc52 CAIX-/PD-L1- were used as target cells and plated at 1.5 × 103/well in a 96-well plate. RCC cells were incubated with 50 μL of the CAR T cells supernatant adjusted for 500 ng/mL of the respective Ab by 1 hour. After the incubation, the cells were washed with medium and incubated with 12.5:1, 25:1 or 50:1 NK cells:tumor cells for 4 h, 37°C. Culture supernatants were harvested by centrifugation and lactate dehydrogenase (LDH) measured in the supernatant by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) at 490 nm. These results represent the average ± SD of three donors in duplicate.