BACKGROUND: The weak antitumor efficacy and limited lifespan are the main obstacles that hinder the therapeutic effect of cytokine-induced killer (CIK) cell immunotherapy. In the study, we enhanced the persistence and the antitumor efficacy of CIK cell through PD-1 knockout and hTERT transduction.

MATERIAL AND METHODS: CIK cells were cultured from patients with hepatocellular carcinoma and PD-1 gene was knocked out through the Cas9 ribonucleoproteins (Cas9 RNPs) electroporation. TIDE assay, T7E1 mismatch cleavage assay, and clone Sanger sequencing were used to detect PD-1 knockout efficiency. The immunophenotype was analyzed by flow cytometry. After PD-1 knockout, the hTERT gene was transduced into PD-1 KO/CIK cells with lentiviral transduction. The hTERT expression and persistence of hTERT/PD-1 KO/CIK cells were evaluated by Western blotting and proliferation curve. The antitumor efficacy was detected by ELISPOT and cytotoxicity assay. The telomere length was measured by the Q-FISH and qPCR method. The karyotype assay was used to analyze the chromosome structural stability.

RESULTS: The optimal knockout efficiency of PD-1 gene in CIK cells could reach 41.23±0.52%. PD-1 knockout did not affect the immunophenotype of CIK cells. The hTERT transduction enhanced persistence and increased the telomere length. ELISPOT and cytotoxicity assay showed hTERT/PD-1 KO/CIK cells had an enhanced antitumor efficacy. Meanwhile, PD-1 KO/CIK cells transduced with hTERT showed a normal karyotype.

CONCLUSIONS: PD-1 knockout combined with hTERT transduction could prolong the lifespan and enhance antitumor efficacy of CIK cells against hepatocellular carcinoma cell line.

Keywords: Cytokine-Induced Killer Cells, Programmed Cell Death 1 Receptor, Telomerase