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. 2020 May 4;17:421–430. doi: 10.1016/j.omto.2020.04.013

Figure 1.

Figure 1

Tumor Cell Lysis Induced by Vγ9Vδ2 T Cells Modified with NKG2D CAR

(A) Schematics of the plasmid constructs used for CAR mRNA production: NKG2D CAR containing the NKG2D extracellular domain (ED) and CD3ζ, and a control CAR replacing NKG2D ED with the membrane binding GFP (mGFP CAR). The DNA templates of the CARs were PCR amplified using a CMV forward primer and reverse primer with 150 Ts. The PCR amplicons were then used for RNA transcription to generate mRNA molecules encoding the CARs for the electroporation of Vγ9Vδ2 T cells (γδ T). (B) Flow cytometric analysis to demonstrate the NKG2D expression on Vγ9Vδ2 T cells. Black lines represent wild-type γδ T cells stained with an isotype control antibody. Red lines represent wild-type γδ T cells stained with an anti-NKG2D antibody to show the expression of endogenous NKG2D receptor. Blue lines represent γδ T cells electroporated with a CAR construct and stained with the anti-NKG2D antibody. Cell samples were collected 24 h post-electroporation for staining. The results of one representative experiment out of three independent experiments with three different donors are shown. (C) Time-lapse analysis of NKG2D CAR expression after electroporation. Vγ9Vδ2 T cell samples were collected at the indicated time points post-electroporation for staining for flow cytometric analysis. Black lines represent wild-type Vγ9Vδ2 T cells stained with an isotype control antibody. Red lines represent wild-type γδ T cells stained with an anti-NKG2D antibody to show the expression of endogenous NKG2D receptor. Blue lines represent γδ T cells electroporated with mGFP CAR and stained with the anti-NKG2D antibody. Yellow lines represent cells electroporated with NKG2D CAR and stained with the anti-NKG2D antibody. The results of one representative experiment out of three independent experiments with three different donors are shown. (D) Delfia EuTDA cytotoxicity assay (3 h EuTDA culturing) used to assess tumor cell lysis efficiency. Human HepG2 hepatocellular carcinoma cell line, MCF7 breast adenocarcinoma cell line, CAOV3 ovarian cancer cell line, Detroit562 pharyngeal carcinoma cell lines that express NKG2D ligands (NKG2DL+), and A549 pulmonary adenocarcinoma cell line that does not express NKG2D ligands (NKKG2DL−) were tested. The results of one representative experiment out of at least two independent experiments with different donors are shown. The differences between NKG2D CAR γδ T and mGFP CAR γδ T are statistically significant (p < 0.001) for all tested tumor cell lines except A549.