Adenovirus Mediated Gene Modified Dendritic Cells In Vitro And Induced CTL Response Against Heptitis B Virus Related Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the most common malignancies in China and Asian countries. Epidemiologic investigation showed that the individuals with HBsAg positive increases the risk of HCC 12 to 300 times compared with that of HBsAg negative population. It has been demonstrated that about 10% of the population are hepatitis B virus (HBV) carriers in Asian countries and about 1 million people die each year from liver cancer associated with HBV infection [1～3]. Recent progress in imaging techniques has enabled the diagnosis of small HCC, and advances in treatment modalities have resulted in the increased survival of patients with HCC. The long-term prognosis, however, is still disappointing because of the high frequency of HCC recurrence; Therefore, novel strategies to prevent proliferation of malignant cells are urgently needed. A promising approach may be the prophylactic vaccination directed against a tumor-associated antigen(TAA)or tumor-special antigen(TSA).The major objective of active specific immunotherapy is to generate tumor- specific cytotoxic T lymphocytes (CTLs) respondes, but to induced CTL respondes depend on the use of professional antigen presenting cells loaded with tumor antigen. In the antigen-presenting cells, dendritic cell( DC) has been considered to be the most potent professional antigen-presenting cell (APC) that can initiates primary immune responses. Its ability to stimulate and regulate T- and B-cell responses makes DC ideally suited to serve as an adjuvant for the purpose of cancer immunotherapy. DC also can be involved in the pathogenesis of graft versus host disease or host versus graft disease after transplantation as well as immunization against viral infections and immunosuppression in autoimmune diseases.In recent years, Abundant experimental evidence indicates that DC primed or loaded with tumor-associated antigens (TAA) or tumor-specific antigens (TSA) stimulate antitumor immune responses, in some studies, such responses have been therapeutic, but immunotherapy approaches targeting to a unique antigen have selectively lost the ability to present the defined antigen effectively. To allow DC to process a broad array of tumor antigens, many method of antigen preparation were used such as irradiated tumor cell coculture with dendritic,RNA extracted from tumor cells tranfected DC, Adenovirus- Mediated multi-tumor related antigen tranfected DC, multi-tumor related antigen pulsed DC, et al. In these methods, the RNA and Adenovirus- Mediated multi-tumor related antigen tranfected DC are most effectively. However, mRNA is not easy to treat because of their low stability. So, in our experiment, we select HBsAg and AFP as targets depend on epidemiologic investigation of our country. To construct recombinant adenovirus vectors containing human HBsAg genes, infected dendritic cell to induce cytotoxic CTL activity against to humen hepatocellular carcinoma cells.We attempt to explore if cotransfected DC could enhance the specific CTL response against the hepatocellular carcinoma cells compared with single antigen transfected DC,and to evaluate the feasibility of adjuvant immunotherapy in patient with HBV related HCC.Part I Construction and examination of a recombinant adenovirus vector expressing HBsAg,AFPObjective To construct a recombinant adenovirus vector expressing HBsAg,AFP and examine its efficiency of transfection and to explore the use of HBsAg,AFP gene-modified DC in priming antigen specific immune response.Methods For the preparation of recombinant adenoviruses expressing HBsAg (Ad-S),AFP (Ad-AFP) or green fluoresent protein (Ad-GFP), the Adeno-XTM Adenovirus Vector System was used. These vectors were replication-deficient adenovirus type 5 (Ad 5) deleted for the genes E1 and E3. Transgenes were first cloned into the pShuttle vector(pShuttle-S, pShuttle-AFP, pShuttle-GFP). Then the gene fragments resulted from the recombinant pShuttle vector digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA to produce recombinant adenovirus (rAd) plasmids, which were linearized with Pac I and subsequently transfected into HEK 293 cells to product rAd. Finally, the virus particle titre was determined. The expression of AFP and HBsAg by recombinant adenovirus infected HEK293 and H1299 cells was determined by IFA.Results HBsAg gene in the inserted DNA of adeno-S was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. All the above results indicated that human HBsAg gene had been connected with pAdeno-X vectors correctly. Virus granule appear after transfected HEK293 cells. The virus particle titre of Ad-S and Ad-AFP determined with resulting of 2.65×109 and 3.16×109 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 HBsAg, AFP and the control adenoviruses (Ad-GFP) were constructed successfully. The efficiency of transfection of recombinant adenovirus we obtained 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 ( 71.45士4.39)%,CD11c ( 89.68士3.16)%,CD86 (91.17士4.43)%,CD80 ( 91.37士4.12)%,HLA-DR ( 94.03士3.01 )%, (DC derived from patients ); CD1a ( 68.45士5.02)%,CD11c ( 91.09士2.01)%,CD86 (92.19士4.36)%,CD80 ( 90.81士3.15)%,HLA-DR ( 93.49士4.18 )%, (DC derived from healthy donors), respectively. 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 nearly 80.62% of the cells were transfected at an MOI of 100 twenty-four hours after infection. As time period of infection prolonged, there were also increasing numbers of infected cells, so that nearly of the cells were 85.25% at an MOI of 100 forty-eight 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 HBV related HCC by dendritic cells infected with recombinant adenovirus vectors containing human HBsAg and AFP genes Objective To investigate the immune functions of DCs transduced with the recombinant adenovirus expressing HBsAg,AFP in vitro, and compare its antiviral and antitumor effect with that of Ad-S DCs and Ad-AFP DCs, and evaluate its feasibility of adenovirus-transduced DCs as an HBV related HCC immunotherapeutic vaccine.Methods The recombinant adenovirus transduced DC cells was determined by IFA, 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-S (S-DC), Ad-AFP (AFP-DC) or Ad-S and Ad-AFP (S/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 S/AFP-DC-L, S-DC-L, AFP-DC-L. Effector cells were harvested and incubated with the target cells HepG2.2.15 for 4 hours, cytotoxicity of S/AFP-DC-L, S-DC-L, AFP-DC-L, DC-L and L were determined by LDH assay.Results IFA 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 S/AFP-DC-L,S-DC-L and AFP-DC-L were higher than those mediated by DC-L and L (P <0.05), and those mediated by S/AFP-DC-L was higher than that mediated by S-DC-L and AFP-DC-L (P <0.05). The difference between S-DC-L and AFP-DC-L was not significant (P﹥0.05). INF-γreleased by S/AFP-DC-L,S-DC-L and AFP-DC-L were higher than those released by DC-L and L (P<0.05), and those released by S/AFP-DC-L was higher than that released by S-DC-L and AFP-DC-L (P<0.05). The difference between S-DC-L and AFP-DC-L was not significant (P﹥0.05)., and the difference between DC-L and L 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 HBsAg genes transfected DC could induce CTL response against HepG2.2.15 higer than single gene transfected DC.