The Immunotherapeutic Effect Of Dendritic Cells Vaccine Modified With MouseIL-15 Gene And Cell Lysate On Mice With Prostate Cancer

Objective:To construct the replication-deficient adenovirus vector encoding mouse IL-15 gene (Ad-GFP-mIL-15) and prepare a lot. To establish the methods of isolation, purification and propagation of mouse bone marrow-derived dendritic cells(DC), investigate the changes of morphological structure and bio-immunological characteristics of DC during its development, differentiation and maturation, study the related factors involved in proliferation and polarization of DC. To construct the DC vaccine modified with mouse IL-15 gene and cell lysate on mice with prostate cancer. To detect and analyze the specific antitumor activity and immunotherapeutic effect of DC vaccine(IL-15/lysate-DC)both in vitro and in vivo.Methods:Mouse kidney cDNA as a template, by using pfx DNA ploymerase amplified mIL-15 gene. Amplified target gene fragment was cloned into pGEM-T Easy Vector. mIL-15 gene recevied from the mIL-15-T cloning vector EcoRI digestion and connected to the pShuttle-GFP-CMV vector.Ad-GFP-mIL-15 virus plasmid was received by pShuttle-GFP-mIL-15 transfer to pAdxsi vector. The correct adenovirus plasmid was substantially amplified and purified. The mouse bone marrow progenitors were isolated andcultured with cytokines of mrGM-CSF plus mrIL-4, and a large amount of BM-DC were harvested after selection and culture for 5 or 7days. The bio-immunological features and functions of DC in different stages were studied by using methods of light- microscopy, mixed leucocyte reaction(MLR), flow cytometry(FCM), and so on. Mouse bone marrow DC were transfected with the recombinant adenovirus vector encoding mIL-15 gene and pulsed with cell lysate from RM-1 mouse prostate cancer cell line. The ability of stimulating syngeneic T cells, T-lymphocyte subsets analysis, IFN-γ, IL-10 level of lymphocyte culture supernatant and the effect of inducing specific kill activity of IL-15/lysate-DC vaccine were detected. The immunotherapeutic effect IL-15/lysate-DC vaccine and IFN-γ, IL-10 level of serum on mice with prostate cancer was assessed.Results:1.By digestion, electrophoresis, sequencing and RT-PCR identification, we successfully constructed the recombinant adenovirus vector encoding mIL-15 gene.2.About 5～10×106 DC were havested from bone marrow progenitors per mouse followed culturing the cells with rmGM-CSF plus rmIL-4 for 5 days. In early stage, DC expressed very low level of costimulatory molecules, adhesion molecules and MHC molecules and failed to stimulate allogeneic T cell proliferation. Cultured DC transfected with Ad-GFP-IL-15 on 5 days, they expressed very high level of costimulatory molecules, adhesion molecules and MHC molecules and stronger activity of stimulating T cell proliferation on 7 days. IL-15/lysate-DC vaccine was contructed by transfected with the recombinant adenovirus vector encoding mIL-15 gene and pulsed with cell lysate from RM-1 mouse prostate cancer cell line.3.The ability of stimulating T cells proliferation, CD8+T cells proportion in T-lymphocyte subsets and IFN-γlevel of lymphocyte culture supernatant were significantly increased by IL-15/lysate-DC stimulating with the allogeneic of mouse spleen T lymphocyte proliferation reaction. The mice immunized with DC pulsed with RM-1 cell lysate (Lysate-DC) exhibited a specific CTL response, but the highest CTL activity against RM-1 cells was induced by immunization with IL-15/lysate-DC(P<0.05).4.In the mice model with pre-established subcutaneous RM-1 prostate cancer cell, vaccination with Lysate-DC and GFP/lysate-DC vaccine could inhibit tumor growth, but the immunization of IL-15/lysate-DC vaccine inhibit the tumor growth most significantly when compared with Lysate-DC and GFP/lysate-DC(P < 0.05). The survival time of the mice treated with IL-15/lysate-DC Vaccine was also greatly extended(P<0.05). 5.Histological examination showed that the most obvious tumor necrosis and infiltration of inflammatory cells was present inside and around the tumor of the tumor-bearing mice immunized with IL-15/lysate-DC Vaccine when compared with Lysate-DC and GFP/lysate-DC(P<0.05). Immunotherapy of tumor-bearing mice with IL-15/lysate-DC vaccine could increase the the production of IFN-γby serum test when compared with Lysate-DC and GFP/lysate-DC(P<0.05).Couclusions:1.The recombinant adenovirus vector encoding mIL-15 gene was constructed successfully.2. A sufficient number of functional DC were havested from bone marrow progenitors mouse followed culturing the cells with rmGM-CSF plus rmIL-4. After DC were transfected with Ad-GFP-IL-15, they expressed very high level of costimulatory molecules, adhesion molecules and MHC molecules and stronger activity of stimulating T cell proliferation.3.The ability of stimulating T cells proliferationand and IFN-γlevel of lymphocyte culture supernatant were significantly increased by IL-15/lysate-DC stimulating with the allogeneic of mouse spleen T lymphocyte proliferation reaction. The highest CTL activity against RM-1 cells was induced by immunization with IL-15/lysate-DC.4.IL-15/lysate-DC might elicit significant antitumor effects through efficient induction of systemic and local immune responses against prostate cancer.