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 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. 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 C…

Representative 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 were pelleted, and supernatant was collected. A) Cells were re-suspended in PBS and luminometry was used to assess the viability of the tar…

Materials

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