Study On Antitumor Activity Of Interleukin-27 Gene-modified Dendritic Cell Vaccines

Objective: Breast cancer is one of the most common tumors in women, which is a life-threatening disease that is seriously affecting the physical and mental health of the female patients. In recent years, tumor biotherapy has gained more attention from the people all over the world as a new mode and method for carcinoma treatment, in which cytokine gene therapy has always been the hot spots in this area. The routine theory is that cytokine genes which are transduced into tumor cells or other immune effector cells can secrete cytokines, which could induce tumor cell apoptosis or accelerate tumor regression by enhancing the immune activities in the body. Interleukin-27 (IL-27) is a novel IL-6/IL-12 family member[1], reported by Pflanz in 2002. In natural and adaptive immune systems, IL-27 plays a broad role in the immunological regulation of various kinds of cells. Besides the important function to the Th1 reaction, IL-27 has various biological activities[2], including anti-infection, anti-tumor and inflammation-inducing reaction, et al. Dendritic cells (DC) are the most powerful professional antigen presenting cells (APC) that can intake, process and present antigen effectively, and possess a high potency of migration and obvious activation to naive T cells. Thus, DC is the core in the process of initiating, regulating and maintaining of primary immune responses[3-4]. In this study, we set up the IL-27/DC vaccine by transfecting IL-27 gene into human dendritic cells (DC), and investigated its activities on tumor-killing. We are anticipating that our research could provide a new vision and experimental guide for the clinical immunotherapy on breast cancer in the future.Methods: 1 Establishment of IL-27/DC vaccine Preparation of dendritic cells (DC) from human peripheral blood mononuclear cells (hPBMCs), Observe morphous of mature DC by light microscope, and analyze the expression of CD80, CD86 and MHCⅠ, MHCⅡmolecul on the surface of DC and the mature condition of DC by flow cytometry. Transfect IL-27 gene carried by retrovirus vector into mature human DC and assess; Observe morphologic changes of DC and IL-27/DC cells by light microscope; IL-27 gene expression was confirmed by RT-PCR and the secretion of IL-27 in the supernatant of IL-27/DC cells was detected by ELISA.2 Analysis on IFN-γinduced by IL-27 ELISA was used to detect the secretion of IFN-γand IL-4 in cell supernatant.3 Lymphocyte proliferation analysis Differentiation and cultivation of T lymphocyte, MTT was used to detect the proliferation potency of T lymphocyte stimulated respectively by DC and IL-27/DC in vitro, and draw the growth curve.4 The assessment of the lethal effects of cytotoxic T lymphocyte (CTL) MTT was used to measure the lethal effect to 4T1 cells in vitro of activited cytotoxic lymphocyte (CD8+T) (CTL) stimulated by IL-27/DC and DC cells respectively.5 Establishment of animal model and analysis on antitumor activity of IL-27/DC in vivo Divide mice to three groups: IL-27/DC group, DC group and control group. Mice were immunized respectively with DC and IL-27/DC cells administered subcutaneously in the right flank, and challenged in the left flank after the last immunization with 4T1 cells. The tumor volume and the survival time were observed. ELISA was used to detect the quantity of IFN-γsecreted by splenocytes.Results: 1 There was an obviously higher level of expression of CD80, CD86 molecul in mature DC than immature. The IL-27/DC vaccine constantly expressing IL-27 was obtained by transfection. Positive expression of IL-27p28 and IL-27EBI3 gene in IL-27/DC cell were showed by RT-PCR. There was no significant difference except for some decrease of the quantity of cells in the morphous of DC and IL-27/DC observed by light microscope. ELISA result indicated that the supernatant of IL-27/DC cell could produce higher level of IL-27 compared to DC cells (P<0.01), suggesting that IL-27 gene was transfected successfully into dendritic cell lines in gene and protein level.2 ELISA results indicated that there was a higher level of IFN-γsecretion in the supernatant of IL-27/DC cell compared to DC cells (P<0.01). However , there was no significant difference in the two groups on the results of IL-4 production(P>0.05).3 MTT results showed that IL-27/DC could notably stimulate the proliferation of T lymphocyts in vitro, which had made a statistic significance compared to other groups (P<0.05).4 Under different effect-target ratios (E/T), the group of IL-27/DC always showed a higher level of performance of killing-activity to 4T1 cells compared to other groups (P<0.05), whose potency was demonstrated to be an obvious enhancing trend as the increasing of the effector cells.5 The growth of tumor in mice immunized with IL-27/DC vaccine was markedly retarded compared to the DC and control group (P<0.05), and the survival time of the mice immunized with IL-27/DC vaccine was longer than the other groups (P<0.01). The quantitation of IFN-γproduction in vitro from splenocytes of IL-27/DC vaccine group demonstrated a higher level compared to the other groups (P<0.01).Conclusions: Successfully establish IL-27/DC vaccine by transfecting IL-27 gene into human dendritic cells (DC). IL-27/DC vaccine could notably stimulate the proliferation of T lymphocyts in vitro. IL-27/DC vaccine had a higher killing-activity to 4T1 cells. The obvious antitumor activities of the vaccine in vivo was speculated to be associated with its function to stimulate IFN-γproduction in mice splenocytes.