An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

Abstract

Source: Kueberuwa, G., et al. A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells. J. Vis. Exp. (2018).

This video demonstrates an in vitro assay to validate chimeric antigen receptor (CAR) T cell activity. B-cell lymphoma cells expressing CD19 antigen and cytoplasmic luciferase are co-cultured with CD19-specific CAR T cells. CAR binding to CD19 on a tumor induces the CAR T cells to release cytokines and toxins that cause tumor cell cytotoxicity. The CAR T cell activity is evaluated by quantifying the released cytokine and decreased luciferase-mediated bioluminescence in the tumor cells.

Protocol

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.

Results

Figure 1. Validation of CAR T-cell activity. αmCD19 CAR T cells were co-cultured with A20 lymphoma cells modified to express luciferase (1 x 10⁴:1 x 10⁴) for 16 h in a U-bottom 96-well plate. After co-culture, cells we...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100X penicillin-streptomycin-glutamine (PSG)	Gibco	10378016
Flow cytometer - LSR Fortessa x20	BD Biosciences	658222R1
Foetal Bovine Serum	Gibco	10270
IL-12 p70 Mouse Uncoated ELISA Kit	Invitrogen	88-7121-76
Ionomycin Calcium Salt	Sigma- Aldrich	I0634
Luminometer - Lumistart Omega	BMG Labtech	415-301
Murine IFN-γ ELISA kit	Diaclone	861.050.010
Phorbol 12-myristate 13-acetate (PMA)	Sigma- Aldrich	P8139
RPMI 1640 Medium	Lonza	BE12-167F
XenoLight D-Luciferin	Perkin Elmer	122799