Caspase Activation in CAR-T Cells

The following monoclonal antibodies conjugated with fluorochromes were used: Coxackie-adenovirus receptor, GD2, CD95 (Fas), CD80, CD86, CD40L, OX40L, CD25, CD69, IFN, CD3, granzyme B (BD Biosciences), TRAIL, TRAIL-R1 and TRAIL-R2 (Biolegend). Expression of GD2.CAR by T cells was detected using a specific anti-idiotype, 1A7 (TriGem, Titan), followed by staining with secondary antibody RAM-IgG1 (BD Biosciences). FACS data were collected with a FACSCalibur (Becton Dickinson) and analyzed using FloJo software version 9.3 (Tree Star). For the caspase assay, CHLA-255 cells were seeded in 24-well plates (1 × 10⁵/well), infected with Ad5Δ24 or mock (100 vp/cell), and cultured for 4 days. Control and GD2.CAR-T cells (2.5 × 10⁵/well) were then added to the tumor cells. Active caspases in CHLA-255 cells were measured at 0, 2 and 4 hrs by FACS. The apoptotic cells were stained using Vybrant FAM Poly Caspases Assay Kit (Molecular Probes) according to manufacture’s instructions. The frequency of early apoptotic cells was determined as percentage of FAM⁺ (carboxyfluorescein group as a reporter) cells excluding CD3⁺ and PI⁺ cells from the analysis(32 (link)).