Evaluating CAR T cell Cytotoxicity

The luciferase-based cytotoxicity assay was carried out as previously described [38 (link)]. On day 7 post-transduction, effector CAR T cells were co-cultured with target MCF-7 (Her2⁺) tumour cells at a 10:1 ratio of effector to target (E: T) cells in triplicate using Firefly Luc One-Step Glow assay (Thermo Fisher #16197). To evaluate the role of TNF-α in CAR T cells killing breast cancer cell line, 5.0 × 10⁶ of each CAR T cell constructs were treated with 100 μg/mL infliximab (IFX) and incubated for 24 h at 37 °C, 5% CO₂ prior to cytotoxicity assay. For IL-2 and IFN-γ cytokine release assay, CAR T cells were stimulated with Her2⁺ target cells at 2:1 ratio. The concentration of cytokines secreted in cell supernatant was measured 24 h post-incubation using sandwich ELISA according to manufacturer’s protocol (BD Biosciences, San Jose, CA, USA). Plates were read on a Varioskan Lux multimode microplate reader (Thermo Fisher, USA). To measure intracellular cytokines, perforin and granzyme, CAR T cells were co-incubated with target cells at 2:1 ratio for 6 h and brefeldin A for 4 h (Biolegend, San Diego, CA, USA, #420601) prior to intracellular labelling.