Figure 8.

Anti-CD33 CAR-NK cells bearing the anti-HLA-DR iCAR preferentially target HLA-DR^neg AML cells

(A) Flow cytometric analysis of NK cells, and CD33-targeted single and dual CAR-NK cells. Cells were stained for flow cytometry with a PE-labeled anti-HA antibody and an APC-labeled anti-FLAG antibody to assess for CD33 CAR and HLA-DR iCAR expression, respectively. (B) IFN-γ production of CD33-targeted single and dual CAR-NK cells against HL-60 (CD33⁺HLA-DR^neg) and KG-1 (CD33⁺HLA-DR⁺) cells. CAR-NK cells were incubated with either HL-60 or KG-1 cells for 4 h. The supernatant was collected to assess for the IFN-γ level by ELISA. (C) CD107a degranulation assays of single and dual CAR-NK cells cocultured with different target cells. CAR-NK cells were incubated with HL-60 or KG-1 for 1 h at 37°C with a PE-conjugated anti-CD107a antibody. Monensin (GolgiStop) was added to the cell culture. After incubation for 4 h, cells were stained with an anti-CD56 antibody. The CD107a⁺ population in CD56⁺ cells was determined by flow cytometry. Data are shown as mean ± SEM of two independent experiments. Statistical significance is calculated by unpaired two-tailed Student's t test. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001; n.s., not significant.