The Preparation For Human B7-2 And DC Vaccines And Their Roles In Anti-tumor Immunity Against Esophageal Cancer In Vitro

BackgroundThe immune exclusion for tumor cells is largely dependent on T cells in vivo. Full activation of T cells requires two kinds of signals: one is the special antigen-presentation signal, the other is the co-stimulatory signal. Lacking of co-stimulatory signal leads to the anergy or apoptosis of T cells. B7 is the most important co-stimulatory molecule, most tumor cells are not able to express B7 or its expression is low, which is one of the reasons leading tumor's immune escape. The immune gene therapy of B7 is a key study direction in tumor therapy. B7-2 molecule is the initial co-stimulatory ligand expressed on APC by its abundant early and easily induced expression pattern, so B7-2 is known as its important role at the initial stage of the immune response. Transfecting B7 gene to tumor cells can enhance their immunogenicity to improve the antitumor immunity. Dendritic cell (DC) is the most powerful professional antigen-presenting cell (APC ), it can activate naive or resting T cells and express MHCⅠ/Ⅱ, CD40 and B7 effectively. The cellular immunity response activated by DC plays a key role in antitumor biology therapy.ObjectiveFirstly, to acquire the B7-2 tumor vaccine, we constructed the expressing vector of human B7-2 fused with enhanced green fluorescent protein; human esophageal cancer cell line EC9706 was transfected by the vector of pEGFP-N3-B7-2 through thetechnique of lipofectamine transfection. Secondly, to acquire the DC-Pr tumor vaccine, DC induced from the mononuclear cell(MNC) of cord blood was loaded with EC9706 cytolysis antigen. Thirdly, human B7-2 and DC vaccines are used together in vitro to study the antitumor immunity effect against esophageal cancer.Methods1. The construction of the eukaryotic expressing vector pEGFP-N3-B7-2we amplified the fragment of human B7-2 cDNA from the thymus tissue of Myasthenia gravis by Reverse Transcription Polymerase Chain Reaction(RT-PCR) and nested PCR. The amplified product was examined by electrophoresis and sequence analysis. This fragment was inserted to pGEM-T plasmid and pEGFP-N3 fluorescent expressing vector.2. The preparation for human B7-2 tumor vaccineHuman esophageal cancer cell line EC9706 was transfected by the vector of pEGFP-N3-B7-2 through the technique of lipofectamine transfection. After 24 hours, we observed the expression of the fused gene under fluorescence inverted microscope. The transfected cells were screened by G418 for 30 days, obtaining the transfected EC9706 cell line consistently expressing fused gene.3. The preparation of human DC vaccine3.1 EC9706 cytolysis antigen was prepared by the method of freezing and melting3.2 The separation of MNC from cord blood and the inducing of DC and T cells MNC was separated from cord blood by density gradient centrifugalism(Ficall-Hypaque). MNC was cultured in complete medium and was allowed to adhere for 2h in 24-well plate, non-adherent cells as T cells were gently removed into a cultured flask and cultured in complete medium supplemented with 5ng/ml rhGM-CSF. The adherent cells as monocyte(Mo) were cultured in complete medium supplemented with 10ng/ml rhGM-CSF and 10ng/ml rhIL-4, on the 3 rd day, EC9706 cytolysis antigen was added into the medium, in which immature DC gradually matured and expressed special tumor antigen, adding to 5μg/ml LPS on the 6th day. On the 7th day, we acquired the mature DC loading the tumor antigen.3.3 DC activated autologous T cell into cytotoxic T lymphocytes(CTL)The DC loading tumor antigen was co-cultured with autologous T cell in the ratioof 1 to 20, DC presented antigen and activated the T cells into tumor specific CTL.4. The effect of killing tumor cells was detected by Methyl thiazolyl tetrazolium (MTT) CTL was used as effector cell, the target cell is EC9706 cell transfectedpEGFP-N3, EC9706 cells transfected pEGFP-N3-B7-2 and EC9706 cells. Theeffector cell was co-cultured with the target cells for 72h in the ratio of 30 to 1. MTTassay was used to detect the inhibition rate of CTL to transfected and untransfectedEC9706.Results1.The amplified fragment of human B7-2cDNA was confirmed correctly by electrophoresis, enzyme digestion and sequence analysis, the base sequence is in accordance with offered by GenBank.2.The recombined plasmid pEGFP-N3-B7-2 was transfected into EC9706. After 24h, observing them under fluorescence inverted microscope, the green fluorescent protein was expressed evenly in EC9706(pEGFP-N3); the fusion gene was located mainly on the membrane of EC9706(pEGFP-N3-B7-2). We successfully detected B7-2 mRNA expression in EC9706(pEGFP-N3-B7-2) with RT-PCR.3.The DC induced from cord blood could load and present tumor antigen, activate autologous T lymphocytes into CTL. MTT assay showed the inhibition ratio of CTL to EC9706(pEGFP-N3-B7-2) is significantly higher than that of control groups (P<0.05).Conclusions1. We have successfully constructed the expressing vector of human B7-2 fused with enhanced green fluorescent protein.2. The result of RT-PCR and the green fluorescent protein marker both show it is efficient to transfect human B7-2 gene into human esophageal cancer cell line EC9706 with lipofectamine.3. The human B7-2 and DC vaccines are used together in vitro can induce significant killing response on human esophageal cancer cell, which provide a new idea about resisting esophageal cancer.