Alpha-fetoprotein And IL-18 Gene-modified Dendritic Cells Effectively Specific Induced CTL Response Against Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the commonest malignant disease in China and other parts of Asia. Although encouraging advances have been made in the past few decades, there are still many difficulties in treating advanced-stage patients and preventing recurrence and metastasis. Therefore, novel strategies to prevent proliferation of malignant cells are needed urgently. A promising approach with the dendritic cell vaccination can against the tumor-associated antigen directly.It is promising new tools for the immunotherapy of HCC. AFP is usually expressed with high concentrations in the liver and cellula genitalis cancer. AFP is frequently expressed in HCCs and therefore used as a target for this tumor.In the past research, it was confirmed thatAFP can be a TAA, the target for HCCs immunotherapy and gene therapy, the response of antitumor which were induced by AFP transfected DC vaccine were limited.A number of reports have suggested that DC being tansfected by some cytokine can enhance immune responses, such as IL-18, a well identified Th1-biasing cytokine,can mediates many important biological functions including stimulation of T cell proliferation and induction of CTL: IL-18 gene transfection significantly enhances the antitumor efficacy of gene therapy, and intratumoral IL-18 gene transfer elicits potent antitumor response of antibody-targeted superantigen.therefore. In this study we want to induce antitumor response using DC being cotransfected with IL-18 and AFP gene. To investigate antitumor effect of DC vaccine and evaluate the feasibility of this DC vaccine to enhance the AFP-specific CTL response against the hepatocellular carcinoma cells, thus profoundly potentiate DC-based vaccine efficacy.Part I Construction and examination of a recombinant adenovirus vector expressing human IL-18,AFPObjective To construct a recombinant adenovirus vector expressing IL-18,AFP and examine its efficiency of transfection and to explore the use of IL-18, AFP gene-modified DC in priming antigen specific immune response.Methods For the preparation of recombinant adenoviruses expressing IL-18 (Ad-IL-18), AFP (Ad-AFP) or green fluoresent protein (Ad-GFP), the AdEasy? Adenovirus Vector System was used. These vectors were replication-deficient adenovirus type 5 (Ad 5) deleted for the genes E1 and E3 gene. Transgene were first cloned into the pShuttle-CMV vector. Subsequently transformed the BJ5183, finally, the gene were transfected into 293 cells to product rAd. Finally, the virus particle titre was determined. The expression of AFP and IL-18 by recombinant adenovirus infected 293 and H1299 cells were determined by IFA.Results IL-18,AFP gene in the inserted DNA of adeno-IL-18,adeno- AFP were confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. All the above results indicated that human IL-18,AFP gene had been connected with pAdeno vectors correctly. Virus granule appear after transfected 293 cells. The virus particle titre of Ad-IL-18 and Ad-AFP determined with resulting of 2.65×109 and 4.01×109 PFU/ml。IFA results suggested that they can infect and express relevant genes in the cells respectively.Conclusions The replication-defective recombinant adenovirus which expressed with IL-18,AFP and the control adenoviruses (Ad-GFP) were constructed successfully. The efficiency of transfection of recombinant adenovirus was high and the target antigen was expressed in the infected cells.PartⅡGeneration of DC in vitro and efficiency of Adenoviral vector-mediated transfer of GFP genes to DCObjective To establish a method to generate DC from leukapheresis products (mobilized peripheral blood mononuclear cells)of patients with HCC in vitro and to identify the phenotypes and the biological functions of DC, the efficiency of transfection of Ad-GFP was determined by FCM in DC.Methods MNCs of patients with HCC were isolated by Ficoll-Hypaque density gradient separation. PBMCs were cultured in serum-free X-vivo 15 medium in polystyrene task at 37℃and 5% CO2. On d7 of culture, cells were washed and suspended in medium containing TNF-αto generate mature DC, which was identified by morphological features, surface antigens expression(analyzed by FCM ) and the ability to stimulate alto-T cells. After 7 days in culture, cells were infected with Ad-GFP at various multiplicity of infection (MOI). The phenotypes were analyzed by flow cytometry using a FACS can analytical flow cytometer. And the efficiency of transfection of Ad-GFP was determined by FCM in DCs.Results After induction,the cultured cells displayed typical morphology with elongated dendritic processes viewed by inverted microscope as well as electron microscope. DCs obtained from patients were identical to those from healthy donors in terms of phenotypes and the abilities to stimulate proliferation of allo-T cells. Both DCs expressed high level surface antigens including CD1a,CD11c,CD86,CD80,HLA-DR (DC derived from patients and DC derived from healthy donors). The results of mixed lymphocyte reaction (MLR) showed that both DCs had the ability to high proliferation of alto-T cells. Ad-GFP infected DCs in a dose-dependant and time-dependant manner. As MOI(multiplicity of infection) increased, there were increasing numbers of infected cells, so that more than 70% of the cells were transfected at an MOI of 200 twenty-four hours after infection. And greater degree of maturation was observed after transfection with Ad. All these proved that adenoviral vector can transfer GFP gene into DCs in culture efficiently and the viability of DCs was not affected following infection with an adenoviral vector.Conclusions DC could be generated from enriched MNC of patients with HCC,which presents the feasibility for further clinical application in the immunotherapy of cancer. An adenoviral vector could be used to efficiently direct the expression of a foreign gene in DCs derived form MNC.PartⅢIn vitro induction of anti-tumor CTL effect against HCC by dendritic cells infected with recombinant adenovirus vectors containing human IL-18 and AFP genesObjective To investigate the immune functions of DCs transduced with the recombinant adenovirus expressing IL-18,AFP in vitro, compare its antiviral and antitumor effect with that of Ad-IL-18/ Ad-AFP DCs and Ad-AFP DCs, and evaluate its feasibility of adenovirus-transduced DCs as an HCC immunotherapeutic vaccine.Methods The recombinant adenovirus transduced DC cells was determined by western blot and ELISA, the phenotypes were analyzed by flow cytometry. Then non-adherent PBMCs were resuspended in complete medium supplemented with IL-2 and cultured at 37℃and 5% CO2. Culture medium was refreshed every 4 days. DCs transfected with Ad-IL-18 (IL-18-DC), Ad-AFP (AFP-DC) or Ad-IL-18 and Ad-AFP (IL-18/AFP-DC) were plated with the effector T cells and cultured for 72h at 37℃and 5% CO2. Then the effector T cells were harvested, washed, counted, and restimulated. The obtained cells were named as IL-18/AFP-DC-T, IL-18-DC-T and AFP-DC-T. Effector cells were harvested and incubated with the target cells HepG2 for 4 hours, cytotoxicity of IL-18/AFP-DC-T, IL-18-DC-T, AFP-DC-T. DC-T and T were determined by LDH assay.Results Western blot and ELISA results suggested that they can infect and express relevant genes in the dendritic cells respectively. The expression of CD11c, CD1a, CD80,CD86 and HLA-DR on adenovirus transduced DCs were up-regulated, and Ad-tranduced DCs can expressed mature markers. The cytotoxicity mediated by IL-18/AFP-DC-T,IL-18-DC-T and AFP-DC-T were higher than those mediated by DC-T and T(P <0.05), and those mediated by IL-18/AFP-DC-T was higher than that mediated by IL-18-DC-T and AFP-DC-T (P <0.05). INF-γreleased by IL-18/AFP-DC-T,IL-18-DC-T and AFP-DC-T were higher than those released by DC-T and T (P<0.05), and those released by IL-18/AFP-DC-T was higher than that released by IL-18-DC-T and AFP-DC-T (P<0.05). The difference between DC-T and T was not significant (P﹥0.05).Conclusions DCs co-infected with these vectors can induce antitumor CTL response in vitro. And adenovirus vectors containing AFP and IL-18 genes transfected DC could induce CTL response against HepG2 which higer than AFP gene transfected DC.