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. 2021 Sep 10;11:664236. doi: 10.3389/fonc.2021.664236

Figure 2.

Figure 2

In vitro characterization of 806 EGFR CAR T cells. Antigen-specific cytolytic activity of 806 and 2173 CAR T cells against cell lines expressing EGFR and its variants. (A) U87MG-EGFR and GSC5077-EGFR cell lines expressing EGFRvIII, EGFRR108K/G, EGFRA289D/T/V, and EGFRG598V mutant variants were stably transduced with Click Beetle Green (CBG) and cocultured with CAR T cells at indicated effector-to-target ratios for 24 h. One representative experiment from three normal donors is shown. Samples were performed in triplicates in three replicative experiments. C225-BB-ζ CAR, and CD19-BB-ζ CAR were used as positive and negative controls, respectively. (B) Antigen-specific cytolytic activity of 806 and 2173 CAR T cells in EGFR and its variants expressed in K562 cells in a 4-h chromium release assay at indicated effector-to-target ratios. (C) K562 cells expressing wtEGFR, EGFRvIII, or EGFR-mutants were cocultured with 806 CART cells for 48 h. IFN-γ, TNF-α, and IL2 secretion was measured in the supernatant by ELISA. Bar charts represent results from a single experiment, and values represent the average ± SD of triplicates. (D) CD107a upregulation on CAR T cells stimulated with K562 cells expressing wtEGFR, EGFRvIII, or EGFR-mutants for 4 h. The percentage of CD107a expression was quantified on CD3 cells (values represent the average of ± SD of two repeated experiments).