Figure 3.

Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (A) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. (B) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. (C) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab’)2 domain IgG (H+L) and CD107a. Each dot represents an experiment. Data represent means ± SEM from three independent experiments. (D) Representative 4-hour standard Cr⁵¹ release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK-92MI and NK-92MI. Experimental groups were performed in triplicates. Error bars represent means ± SD. The experiment was repeated at least two times. Non-parametric test was used for panels (C). ns p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001.