Fig. 4

Cytotoxicity of cryopreserved pNK cells against ovarian cancer cell lines. a. Cytotoxicity of cryopreserved pNK cells against various ovarian cancer cell lines was analyzed using CFSE/7-AAD assay with the indicated E:T ratio. b-c. Expression of HLA-class I, ULBP-1, ULBP-2, MIC-A/B, CD112, and CD155 in three different ovarian cancer cell lines was analyzed using flow cytometry. Representative histograms of the expression of receptors in each cell lines (b). Grey histograms represent isotype controls. Expression intensity of MICA, ULBP1, ULBP4, B7-H6, and HLA-ABC in three different ovarian cancer cell lines. Mean fluorescence intensity determined using flow cytometry (c). Data represented as mean ± SD. Statistical analysis was performed using an unpaired t-test (*P < 0.05 and **P < 0.01). d. Expression of HLA-C1 and HLA-C2 mRNA in A2780 and A2780cis cells. Data are represented as mean ± SD. e-f. Efficiency of siRNA treatment of HLA-C on A2780 cells. Gene knockdown efficiencies were obtained using quantitative RT-PCR (e) and FACS (f). Data are represented as mean ± SD. Statistical analysis was performed using an unpaired t-test (**P < 0.01 and ****P < 0.0001). g. Cytotoxicity of cryopreserved pNK cells against A2780 cells with and without HLA-C knockdown was analyzed using CFSE/7-AAD assay with the indicated E:T ratio. h. Cryopreserved pNK cells were pre-incubated with blocking antibodies against KIR2DL1, KIR2DL3, and KIR3DL1, and cytotoxicity was analyzed against A2780 using CFSE/7-AAD assay in triplicate. E:T ratio was 10:1. Each group was compared to the isotype control. Statistical analysis was performed using an unpaired t-test (**P < 0.01). i. Cryopreserved pNK cells were pre-incubated with blocking antibodies against NKG2D, NKp30, NKp44, and DNAM-1, and cytotoxicity was analyzed against A2780 or A2780cis cells using CFSE/7-AAD assay in triplicate. E:T ratio was 10:1. Percentage inhibition of cytotoxicity was calculated as the percentage of inhibition via the isotype control antibody. Data are represented as mean ± SD. Each group was compared to the isotype control. Statistical analysis was performed using an unpaired t-test (*P < 0.05 and **P < 0.01)