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eISSN: 1643-3750

Programmed Death Receptor 1 (PD1) Knockout and Human Telomerase Reverse Transcriptase (hTERT) Transduction Can Enhance Persistence and Antitumor Efficacy of Cytokine-Induced Killer Cells Against Hepatocellular Carcinoma

Kanghua Huang, Bowen Sun, Nan Luo, Huahu Guo, Jili Hu, Jirun Peng

Department of Surgery, Beijing Shijitan Hospital, Capital Medical University, Beijing, China (mainland)

Med Sci Monit 2018; 24: LBR4573-4582

DOI: 10.12659/MSM.910903

Available online: 2018-07-03

Published: 2018-07-03


#910903

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



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