Fig. 3

TSPO protects GB cells against T cell-mediated cytotoxicity in both allogeneic and autologous co-culture models. a-d Tetrazolium salt-based XTT assay to determine the impact of TSPO expression in GB cells on T cell-mediated killing. Human GB cell lines (a) U-87 MG and (b) U-251 MG were transfected with either control siRNA or a pool of four TSPO-specific non-overlapping siRNAs. After 72 h of transfection, the cells were pulsed with 0.01 μg/ml flu-peptide and cultured with FluTC for 4 h. The primary cell lines BTIC13 (mesenchymal, c) and BTIC129 (proneural, d) were transduced with either non-targeting control or TSPO-specific shRNA (TSPO-proficient/deficient = TSPO ±). Stable cell lines were seeded on a 96-well plate and co-cultured the next day with (c) FluTC after being pulsed with 0.001 μg/ml flu-peptide or (d) pre-activated autologous TIL129 for 4 h. Target-cell lysis was determined as described in the Materials and Methods section. e–g Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze T cell-mediated GB cell killing over 24 h. Incucyte® Caspase-3/7 Green Dye was added as an indicator of apoptosis. The graphs show total apoptotic tumor cell area (green object area) per well (µm²/well). Co-culture of TSPO ± (e) BTIC13, (f) BTIC13 clones and (g) BTIC129 cells with (e, f) FluTC and (g) pre-activated autologous TIL129. a-d Cumulative data of three to five independent experiments, e–g Representative data of at least three independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student`s t-test (* = P < 0.05, ** = P < 0.01, *** = P < 0.005, **** = P < 0.001)