Fig. 3: Murine and human p32 CAR T cells specifically target p32 expressing glioma cells.
From: P32-specific CAR T cells with dual antitumor and antiangiogenic therapeutic potential in gliomas
a Specific cytotoxicity towards p32-positive and the corresponding p32 KD cells (knockdown; transduced with lentivirus expressing shRNA targeting p32) was measured by LDH activity in culture media. Target cells were co-cultured with either a non-relevant (mSP6) or p32 murine CAR T (mp32) cells for 6 h at the indicated E:T ratios. Data represent mean ± SEM. N = 3. One-way ANOVA, Tukey’s multiple comparisons test. P value shown in graph corresponds to the comparison of the mp32 vs mSP6. P < 0.004 for the remaining comparisons. b CellTracerTM Violet dilution assay of labeled p32 CAR T murine lymphocytes co-cultured with the indicated glioma cells for 3 days (E:T = 3:1). Labeled unstimulated p32 CAR T cells alone were used as control. Representative histogram is shown out of four independent experiments. c, d Transduced murine lymphocytes were incubated with the target cells (E:T = 2:1) for 24 h, and IFN-γ production was measured by FACS intracellular staining (c) and by ELISA (d). N = 3, representative histogram is shown. Dots in graph (d) show average for each independent experiment. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. e Human target glioma cells were co-cultured with control untransduced T cells (hUT) or CAR-p32 T cells (hp32) at the indicated E:T ratios. Eighteen hours later, the cytotoxic action of the human CAR T cells was measured by quantifying luciferase activity in tumor target cells. Data represent mean ± SEM. N = 5 for GBM and N = 4 for U87 independent experiments, two different human donors were used. Unpaired t test with Welch’s correction was used for statistical analysis. Two-tailed P value is shown. f CellTracerTM Violet dilution assay (representative histograms from three independent experiments with two different donors) and g IFN-γ secretion (ELISA; N = 3 independent experiments with two different donors) were also assessed using hUT and hp32 T cells following co-culture with the indicated human glioma cells. Data are shown as mean ± SEM. Unpaired t test was used. Two-tailed P value is shown. All source data are provided as a Source Data file.
