Evaluation of anti-CAIX BBζ CAR-T cells in vitro
(A) Designs of anti-CAIX BBζ, 28ζ, and 28BBζ CAR-T cells. The same anti-CAIX G36 scFv was used as CAR moiety followed by CD8 hinge, costimulatory domain (s), and activation cassette CD3ζ. ZsGreen was introduced after IRES. (B) Cytotoxicity of anti-CAIX BBζ CAR-T cells with different CD4:CD8 composition ratios. From left to right, the cytotoxicity of CD4, CD4:CD8 = 2:1, CD4:CD8 = 1:1, CD4:CD8 = 1:2, and CD8 anti-CAIX BBζ CAR-T against CAIX-expressing skrc-59 cells is shown. P values are defined by unpaired two-tailed t-tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (C) Heatmap of log2 fold change cytokine and chemokine profiles in supernatant. After 48 h co-culture of skrc-59 tumor cells and CAR-T cells with different CD4:CD8 composition ratios, the media were assessed for cytokine and chemokine release. The fold change was defined by the value of experimental group divided by the one of UNT negative control. All values of fold change are labeled in the heatmap.