Objective To investigate the cytotoxicity of autologous anti-CD3 antibody-induced activated killer cells(CD3AKs) from patients with breast cancer towards MCF-7/adr cells after suppressing PD-1 expression.
Methods Lentivirus-mediated reverse transcription technology was applied to transfer PD-1 recombinant plasmid into CD3AK cells to inhibit PD-1 expression. PD-1 expression on CD3AK and PD-L1 on MCF-7/adr cells surface were detected by flow cytometry (FCM). After co-cultivation of CD3AKs and MCF-7/adr cells, the killing rates of each group towards MCF-7/adr cells were measured by CCK-8 assay. Blank plasmids, PD-1-transfected cells and normal saline(NS) were intravenously injected into BALB/c tumor-bearing nude mice through tail vein. The antitumor effects on tumor-bearing nude mice engrafted with MCF-7/adr cells were observed and evaluated among the three groups through tumor size, weight and tumor inhibition rate.
Results Reverse transcription technology exhibited promising effect on inhibiting PD-1 expression in CD3AKs. On the 14th and 21th days of cultivation, PD-1 expression rates on CD3AKs obtained from breast cancer patients were 44.13% and 60.18%, respectively, however, PD-1 expression rates of PD-1 recombinant plasmid transfection groups were 15.35% and 13.98% (P < 0.01), respectively. RT-PCR results presented that lentiviral vector/PD-1 decreased PD-1 gene expression in CD3AKs. CCK-8 assay demonstrated that on the 14th, 21th days of cultivation, the killing rates of CD3AKs cultivated with blank plasmids and PD-1 recombinant plasmids in MCF-7/adr cells were 42.98% and 62.68%, 47.22% and 66.95% (P < 0.01), respectively. In vivo experiment of nude mice showed that recombinant PD-1 transfected CD3AKs exhibited remarkable inhibition on tumor growth. The anti-tumor rate reached 74.8%.
Conclusion High PD-L1 expression on MCF-7/adr cell surface is demonstrated and the inhibition of PD-1 expression can improve the cytotoxicity of CD3AK cells.
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