Experiment Of Anti-Liver Cancer With Dcpulsed With Cancer Testis Antigen OY-TES-1 In Vitro

Objective: Preliminary investigation of the effect of cancer testis antigen OY-TES-1 induced specific cytotoxic T lymphocytes against liver cancer cell lines in vitro.Methods: (1) Target cells, hepatocellular carcinoma cell (HCC) lines HepG2 and Bel-7404 were screened by flow cytometry and OY-TES-1 gene expression in both cell lines were examined by RT-PCR and immunohistochemistry. (2) Peripheral blood mononuclear cells (PBMC) were separated from the peripheral blood of HLA-A2 positive healthy individuals with standard density gradient centrifugation, and cultured with rhGM-CSF,rhIL-4 and rhTNF-αto induce PBMC differentiating into DC. Their morphologies were examined under microscopes and their phenotypes were determined with flow cytometry. DCs were sensitized with OY-TES-1-MBP fusion protein (OY-MBP), o maltose-binding protein (MBP), and enhanced green fluorescent protein (EGFP), respectively. Sensitized DCs were co-cultured with T lymphocytes, and were used to stimulate autologous T lymphocytes proliferation and induce their differentiation into cytotoxic T lymphocyte (CTL). Then the proliferation of T lymphocytes was detected by CCK-8, and IFN-γin against HCC lines was evaluated by the method of LDH releasing assay.Results:(1) Target cell screening: flow cytometry confirmed that HCC cell lines HepG2 and Bel-7404 were HLA-A2+ and HLA-A2-, respectively. RT-PCR and immunocytochemistry showed that both HepG2 and Bel-7404 cell lines were positive for OY-TES-1 gene expression.(2) DC characterization and CTL's cytotoxicity against HCC lines: After induced with cellular factors, PBMC showed typical morphologic characteristics of DCs and had high level expression of HLA-DR, CD86, CD83 and CD80. DCs sensitized with different proteins all could activate T lymphocytes and promote their proliferation, but OY-MBP sensitized DCs induced more significantly proliferation of T lymphcytes than DCs sensitized with other proteins (P<0.05). Also the production of INF-γby stimulated T lymphocytes was much higher for OY-MBP sensitized DCs than for other protein sensitized DCs (P<0.05). CTL induced by OY-MBP sensitized DC could attack and kill target cells HepG2, and the kiling rate was significantly higher (P<0.05) than CTL induced by other protein sensitized DC. CTLs were less capable to attack and kill HLA-ABC antibody blocked target cells.Conclusion: OY-TES-1 fusion protein sensitized DC can induce specific anti-cancer immunological response in vitro, suggesting that OY-TES-1 may be used as a potential immunotherapy target.