GFP Reporter Cell Line - NALM6

Using a range of methods including single-cell imaging, dynamic imaging of fluorescent reporters, and scRNA-seq of patient-infused products, the researchers investigated the cytotoxic mechanisms of tumor cell killing by individual CAR T cells. The GFP reporter cell line NALM6 was used to track cytotoxic activity in tumor cells. The study found that although overexpression of the GZMB inhibitor PI9 inhibited the molecular activity of GZMB, this did not alter the cytotoxicity of CD19-specific CAR T cells against NALM6 or SkOV3-CD19 cells. By designing and validating reporter genes, the researchers directly measured the activity of T cell-delivered GZMB. They confirmed that CAR T cell killing requires combined inhibition of multiple tumor cell-specific pathways. B cell lines such as NALM6 were sensitive to combined inhibition of GZMB and GZMA, while solid tumor targets such as SkOV3-CD19 were sensitive to combined inhibition of GZMB and Fas ligand. Through scRNA-seq analysis, the researchers demonstrated the expression correlation of GZMB and GZMA at the single-cell level, highlighting the redundancy of CAR T cell killing mechanisms and their importance for effective T cells.

Figure 1. The researchers used the GFP reporter gene cell line NALM6 to verify the effect of PI9 overexpression on cell-killing activity through 19-41BB CAR T cell-killing cytotoxicity analysis. The CAR structure used included a single-chain variable segment (scFv) against CD19, a human CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ activation domain, and a T2A peptide chain to enhance GFP for cell sorting. (Montalvo MJ, et al., 2024)