Immunological Study On Murine Bone Marrow-derived Dendritic Cells Infected With Human Livin α Recombinant Adenvoiral Vectors Against Lewis Lung Carcinoma

PartⅠConstruction of a recombinant adenoviral vector encoding functional human livinαgene and its identification[Background]Livin gene was recently discovered a antiapoptotic gene, which has two two splicing variants (livinαand livinβ). Because antiapoptotic characteristics of livin and it is overexpressed in a variety of human tumors, but unexpressed or poorly expressed in most normal adult differentiated tissues, it may been a good target site for immunotherapy of tumor. Hence it is a potential therapeutic method using dendritic cells (DCs)-based tumor vaccine which target livin antigen. Recently with development of method of culturing DCs in vitro, it has been a concerned focus of antitumor immunotherapy using tumor vaccine which was prepared by transferring tumor antigen into DCs. Therefore developing efficient method of transduction gene is a key for DCs-based antitumor immunotherapy.To date, transduction with recombinant, replication-defective adenoviral (rAd) vectors encoding a targert gene is an one of the most efficient methods for gene transfer into DCs. This study was aimed to construct and identify a recombinant adenovirus vector carrying human livinαgene and enhanced green fluorescent protein gene (rAd-hlivinα) by using AdMax system.[Objective]To aimed to construct and identify a recombinant adenovirus vector carrying human livin a gene and enhanced green fluorescent protein gene (rAd-hlivinα) by using AdMax system.[Methods]The full-length cDNA of human livin a was amplificated from the plasmid pIRES2-EGFP-hlivinα, and was subcloned into the adenovirus shuttle plasmid pDC316-EGFP-cmv. The newly constructed adenovirus shuttle plasmid pDC316-EGFP-cmv-hlivin a was identified using enzyme digestion and sequencing of inserted livinα. Subsequently, the pDC316-EGFP-cmv-hlivin a was transfected into HEK293 cells, together with the helper plasmid pBHGlox(delta)E1,3Cre. Based on the homologous recombination of the two plasmids within HEK293 cells, rAd-hlivin a was generated. The rAd-hlivinαwas identified using PCR and proliferated using HEK 293 cells. Enhanced green fluorescentprotein (EGFP) expression were detected after rAd-hlivin a infected HEK 293 cells. The titer of the virus was determined by the method of tissue culture infectious dose 50 (TCID50).[Results]The results showed that the bands of recombinant plasmid pDC316-EGFP-cmv-hlivin a by enzyme digestion were in the right range corresponding with expectation. Sequencing of inserted human livin a was correspond to the report in Genebank. EGFP expression could be observed in rAd-hlivinα-infected HEK 293 cells and PCR amplification of the rAd-hlivin a specifically produced the bands with expected sizes. The titer of virus was 2.7×1010 TCID50/ml.[Conclusions]Our study had constructed successfully the recombinant adenovirus vector encoding human livin a gene as well as a EGFP gene using AdMax system.This is the first report showing the construction of a recombinant adenovirus vector encoding human livin a gene as well as a EGFP using AdMax system, which will provide material basis for researches of DCs-based tumor immunotherapy. PartⅡConstruction and identification of vaccine of dendritic cells infected with human livinαrecombinant adenoviral vectors[Background]Dendritic cells (DCs) are professional antigen-presenting cells (APC) which are able to initiate innate and adaptive immune responses. DCs play a central role in the immune response and interact with many cells of immune system. DCs are able to act as natural adjuvants in the induction of antitumor-specific cytotoxic T lymphocytes that can directly kill target cells. Currently tumor vaccination strategies utilizing DCs have made many progress in animal studies and clinical research and have been a hot spot of tumor therapy. It has been a importment approach to induce antitumor immune response using DCs vaccine which was prepared by transferring tumor antigen into DCs. Furthermore transduction with recombinant, replication-defective adenovirus vector encoding a transgene is an efficient method for gene transfer in to murine DCs or human DCs and recombinant adenovirus vector (rAd) is widely used in basic research vector. To date, rAd medicated gene therapy is one of the most promising treatment. This study is to prepare DCs vaccine by using recombinant adenovirus vector encoding human livinα(rAd-hlivinα) constructed in the previous part to infect murine DCs, which will provide experimental basis for next researches of DCs-based lung cancer immunotherapy.[Objective]This study is aim to transfer livinαgnen into murine DCs using recombinant adenovirus vector encoding human livinα(rAd-hlivinα) and investigate expression of human livinαprotein in murine DCs[Methods]1. C57BL/6 murine bone marrow-derived mononuclear cells were isolated using lymphocyte separation medium in vitro. DCs were harvested after incubating the mononuclear cells with complete RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; 20 ng/ml) and recombinant murine interleukin-4 (IL-4; 20 ng/ml).2. On day 7, the bone marrow-derived DCs were infected with either rAd-hlivin a or rAd-control, at a multiplicity of infection (MOI) of 200. After that time, complete medium was added and cells were cultured for an additional 48h.3. On day 9, rAd-control infected DCs (rAd-c DCs) or rAd-hlivin a infected DCs (rAd-hlivina DCs) were harvested. Efficiency of transduction of DCs were evaluated using flow cytometry and cell viability was assessed by trypan blue dye-exclusion.4. On day 9, non-infected DCs, rAd-c DCs or rAd-hlivina DCs were collected and resuspended in cold FACS buffer. Phenotype of cells were analysed using flow cytometry.5. rAd-c DCs or rAd-hlivin a DCs were used to assess human livin a protein expression by western blot.[Results]1. Murine bone marrow-derived mononuclear cells were successfully isolated and the characteristic dendritic profile was observed using microscope after mononuclear cells were induced culture.2. DCs were transduced with rAd-hlivin a to analyze the efficiency of transduction. About 75% of DCs were positive for enhanced green fluorescentprotein (EGFP) expression by flow cytometry and cell viability was>90%. Therefore MOI 200 was considered optimal for gene transduction.3. By analyzing the cell surface phenotype of rAd-hlivin a DCs, rAd-c DCs or non-infected DCs, expression of CD80, CD86 and MHC class II on rAd-hlivin a DCs did not differ from that on rAd-c DCs. rAd-infected DCs (rAd-hlivin a infected DCs or rAd-control infected DCs) enhanced the CD80, CD86, and MHC class II expression levels compared with the non-infected DCs (P<0.05).4. Endogenous livin a protein expression was assessed in rAd-hlivin a DCs and rAd-c DCs. Human livin a protein was expressed in rAd-hlivin a DCs, but not in rAd-c DCs.[Conclusions]In this study, human livin a gene was successfully transfer into murine DCs using recombinant adenovirus vector encoding human livin a gene and sustained, high levels of livin a protein expression in infected DCs. The CD80, CD86, and MHC classⅡexpression levels of cell surface molecules were upregulated after DCs were infected by rAd which suggest that infection of recombinant adenovirus enhances maturation of DCs. This will provide experimental basis for next researches of DCs-based lung cancer immunotherapy. PartⅢAntitumor effects of murine bone marrow-derived dendritic cells infected with human livin a recombinant adenoviral vectors against Lewis lung carcinoma[Background]Livin is one tumor antigen (TA) that is a novel member of the inhibitor of apoptosis protein (IAP) family. Livin is overexpressed in most tumor, but poorly expressed in most normal adult differentiated tissues. Although expression profiles of livin seem to be very similar to those of Survivin, its antiapoptotic activity is more robust than that of survivin. It has been reported that livin protein was overexpressed in certain lung cancer cell lines and primary lung cancers. A substantial fraction of patients with lung cancer develop autoantibodies, aswell as a cellular immune response toward livin. Taken together, these reports suggest that livin could play a role in the development and progression of lung cancer. Hence, livin is a good candidate for lung cancer immunotherapy.Self proteins like livin may not stimulate potent antitumor immuneresponses due to central immunologic tolerance.However, small variations in protein sequence that may exist between homologous proteins of different species can break tolerance to the native antigen. To study the immunogenicity of a xenogeneic livin protein, we explored an immunization strategy using H-2b positive mouse bone marrow dendritic cells (DCs) transduced with adenoviral vectors containing the human livin a gene (rAd-hlivinαDCs) to induce resistance of C57BL/6 mice to subcutaneous challenge with Lewis lung cancer (LLC) cells.[Objective]To investigate antitumor effects of murine bone marrow-derived dendritic cells infected with human livin a recombinant adenoviral vectors against Lewis lung carcinoma.[Methods] 1. Endogenous livin expression was assessed by western blot and immunohistochemical method in LLC cells.2. LLC tumor models were made using LLC cells (H-2b) tissues of tumor were stained by hematoxylin and eosin (H&E) staining method.3. For the prophylactic study, female C57BL/6 mice (H-2b) were injected subcutaneously with DCs, rAd-c DCs or rAd-hlivin a DCs at the lift flanks 3 times with 7-day interval. Seven days after the last immunization mice were inoculated subcutaneously into the right flank with LLC cells and mice were then observed for 8 weeks. Survival of mice was assessed using standard methodology.4. For the therapeutic study, mice were injected subcutaneously with LLC cells at the right flanks. Eight days after having been injected subcutaneously with LLC cells the treatment mice were treated subcutaneously with DCs, rAd-c DCs or rAd-hlivin a DCs at the lift flanks three times. As an indication of antitumor effect the size of each tumor was assessed using calipers every 2 days, and was recorded as the tumor volume (lengthxwidth2x0.52). After mice were inoculated subcutaneously into the right flank with LLC cells, survival of mice were observed for 7 weeks. Survival of mice was assessed using standard methodology.5. To examine the ability of the rAd-hlivin a DCs treatment to induce tumor-specific CTL, C57BL/6 mice were immunized subcutaneously two times with 7-day interval with rAd-hlivin a DCs, rAd-c DCs or DCs. On day 7 after the last immunization, spleen lymphocytes were obtained from immunized mice.These spleen lymphocytes, as effector cells(E), were incubated with LLC cells, target cells (T) at different E:T ratios of 20:1,40:1 and 60:1. A nonradioactive cytotoxicity assay-lactate dehydrogenase (LDH) release was used to measure cytotoxic T lymphocyte (CTL) activity.[Results]1. Murine livin protein was detected by western blot and immunohistochemical method in LLC cells.2. Eight to ten days after mice were inoculated subcutaneously into the right flank LLC cells (per mouse 5×105) tumor models were finished. different sized, disorganized and pathological karyokinetic tumor cells were observed using H&E staining of tissues of tumor by microscope.3. Effector cells from mice vaccinated with rAd-hlivin a DCs induced remarkable killing responses by CTL against LLC target cells, but control effector cells of mice vaccinated with rAd-c DCs or non-transduced DCs exhibited minimal lysis. As increase of E/T ratio, CTL activity of effector cells from mice vaccinated with rAd-hlivin a DCs were also gradually increased.4. In prophylactic study, immunization with rAd-hlivin a. DCs significantly induced antitumor effects resulting in complete survival of mice compared to that seen in the mice immunizated with the rAd-c DCs or the DCs alone during the 8-week observation period after challenge with LLC cells (Kaplan-Meier analysis, P< 0.001).5. In the therapeutic study, immunization with rAd-hlivin a DCs significantly attenuated growth of tumor as compared to that seen in the mice immunizated with rAd-c DCs or DCs during the 24-day observation period (days 12, P<0.01; days 14-24, P< 0.001). Tumor size in treated groups (immunization with rAd-hlivinαDCs) was at least 2-fold smaller than in control groups (immunization with rAd-c DCs or DCs) (days 16-24). Immunization with rAdhlivin a DCs leaded to complete survival in 5 of 10 mice during the 48-day observation period; whereas, rAd-c DCs or DCs alone provided no protective effect (Kaplan-Meier analysis, P<0.001). There was no difference of survival in the two control groups.[Conclusions]The results indicated that rAd-hlivin a DCs vaccine generated potent mouse livin-specific immune responses and anti-LLC effect. Therefore, xenogeneic differences between human and murine sequences might be exploited to develop more immunogenic tumor vaccines. This approach may offer a useful strategy for DCs-based clinical immunotherapy.