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. 2021 Nov 29;24:14–25. doi: 10.1016/j.omto.2021.11.018

Figure 2.

Figure 2

CAIX-CAR-T exerted selective cytotoxicity to CAIX-positive renal cancer cells

(A) Schematic representation of CAIX-CAR. (B) Different cell lines were stained with anti-CAIX to calculate the levels of CAIX expression. (C) CAIX-CAR-T or the Control Ctrl-T cells were cocultured with ACHN, 786O, and OSRC-2 cells at the designed E:T. Viable cells were calculated as “cell index” using the xCELLigence RTCA system. (D) The killing ability of CAIX-CAR-T or Control Ctrl-T cells was also detected by flow cytometry. ACHN-GFP and OSRC-2-GFP cells were cocultured with CAR-T cells for 1 or 2 days. CAR-T cells were labeled with anti-CD3-APC. (E) Supernatants were obtained 786O or OSRC-2 after 48 h coculturing (E:T = 2:1) with CAIX-CAR-T, and the concentration of IFN-γ was analyzed by ELISA assay. (F) CAIX-CAR-T cells were pre-stained with CFSE before coculture with 50 MOI OAV-DEC and collected for flow cytometry at serial day 1, 2, 3 or 4 to monitor the results of division. (G) OSRC-2 and ACHN cells were treated with OAV-DEC (MOI = 5) following by adding Ctrl-CAR-T or Ctrl-CAR-T (E:T = 1:1). Viable cells were calculated as “cell index” using the xCELLigence RTCA system. Right panels were representative values for OSRC-2 or ACHN cells at the end of RTCA analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and “ns” means not significant.