CTL-Mediated Cytolysis of Tumor Cells

CTL lysis of PDA30364/OVA cells was assessed in vitro using the impedance-based xCELLigence Real-Time Cell Analyzer System (RTCA) (ACEA Biosciences, San Diego, USA). Eighteen h after irradiation, tumor cells were seeded into an E-Plate 96 (ACEA Biosciences) at a density of 7.2 × 10³ cells/well and rested overnight at 37 °C. Next day, CTLs were added at an effector/tumor cell ratio of 2.5:1. PD-L1-specific antibody (20 µg/ml) (Bio X Cell, Inc., West Lebanon, USA) was added 3 h prior to CTL addition and at the time point the co-culture was started, resulting in a final concentration of 10 μg/ml αPD-L1 antibody. The cell index (CI), representing the relative impedance as a measure for the number of adherent cells was determined every 5 min for at least 24 h. CI values were normalized to the time point of CTL addition using the RTCA Software 2.0 (ACEA Biosciences). Percentage cytolysis was calculated according to the formula: Cytolysis [%] = ((CI_wo.CTLs − CI_w.CTLs)/CI_wo.CTLs) × 100. Standard deviation (SD) of mean CI values was calculated using error propagation formulas established by the Biostatistics Department of the DKFZ. Specificity of the CTL line was controlled using parental PDA30364 cells in comparison to PDA30364/OVA cells as described previously^{25 (link)}. “Kill-Time-50” (KT50) was defined as time span between CTL addition and eradication of 50% of PDA30364/OVA cells.