1. In vitro Validation of CAR T cell Activity

1. Seed syngeneic target CD19+ tumor cells with or without luciferase expression at a density of 1 x 10⁴ cells in 100 μL complete T cell medium (TCM)/well in a 96-well U-bottom tissue culture plate.
2. Add 1 x 10⁴ CD19 CAR T cells/well in a volume of 100 μL/well to achieve an effector to target (E:T) ratio of 1:1.
   NOTE: E:T ratios should be established for each CAR construct and target cell line.
3. Use T cells alone and tumor cells alone as negative controls and T cells stimulated by phorbol-myristate-acetate (PMA) (50 ng/mL) and ionomycin (1 μg/mL) as positive control for Interferon-gamma (IFNγ) release. Co-culture cells at 37 °C, 5% CO₂ for 16-24 h.
4. Following co-culture, centrifuge the plates at 500 x g for 5 min and collect the supernatant for further IFNγ and IL-12p70 ELISA analysis.
   NOTE: This can be stored at -80 °C.
5. Re-suspend cell pellets in 100 μL of PBS containing luciferin (final concentration of 1.5 mg/mL). Incubate the plates for 10 min at 37 °C. Then measure the luminescence from each well with a suitable luminometer.
   NOTE: Exposure times must be optimized for cell lines and density. Representative results are shown in Figure 1a. Ex-vivo cytotoxicity of CAR T cells can be modified to express luciferin by co-culture with cell lines expressing target antigen. As CAR T cells kill target cells, luciferin is released, therefore a reduction in luminometry signal is correlated with cell kill. Non-transduced cells can often have an effect on target cell viability, particularly over long incubation periods. Measure the concentration of murine IFNγ and IL-12p70 in the supernatant according to the manufacturer's ELISA protocols. Representative results are shown in (Figure 1b and 1c). Ex-vivo activation of CAR T cells by co-culture with cell lines expressing target antigen can be assayed by analyzing supernatant contents using ELISA. The ratio of CAR T cell to target cells and length of co-culture period must be optimized for each CAR construct, target cell line and analyte. PMA and ionomycin treatment can be used as a positive control to confirm quality of T cells and their ability to respond.