The Study Of Prolongation Of Murine Cardiac Allograft Survival Mediated By Adenoviral Vector Encoding 4-1BBIg Gene Therapy

Transplantation has been adopted as the therapy of choice for end-stage organ failure. Recently, with developing of high efficency gene transfer vector and the knowledge about role of immune regulatory molecule, it improves the further study of gene therapy in allograft transplantation. Organ transplantation has supplied a unique application area for gene therapy. The aim of gene therapy in allograft transplantation is to prevent and alleviate the allograft rejection, prolong the survival of allograft. The essential of allograft rejection is the cell immune response mediated by alloreactive T cell after recognised alloantigen, just like against other antigens. It has a complicated mechnism. In allograft rejection, dendritic cell derived of donor and recipient can migrate to the secondary lymphoid tissures of recipient, where DC differentiation and maturation. Mature DC can interact with alloreactive T cell homing there, and activate it through direct and indirect mechnism, then due to the occurrence of alloresponse. Thus, it becomes the highlight in the study of transplantation immunology to interfere the DC and T cells interaction through gene therapy, and has recently created considerable excitement. This approach is based on disrupting costimulation of DCs and T cells in the immune response. If deficiency of the costimulatory signal, antigen-primed T cells can lead to anergy or apoptosis, so it can lose immune function.Many receptors have now been described to be costimulatory. These can be divided into two main groups: the immunoglobulin superfamily (IgSF) and the tumour necrosis factor receptor (TNFR) superfamily. Engagement of IgSF costimulatory molecules such as CD28 with B7-1 (CD80) and B7-2 (CD86) alone does not avtivate T cell for long time, and not developed memory T cells, there also has other cell-cell interaction systems, such as TNFR superfamily, e.g. CD40/CD154 (CD40L), HVEM/LIGHT have been shown to enhance T cell activation.Recently, a new receptor/ligand, 4-1BB/4-1BB ligand system has been identified. Such 4-1BB, a member of the TNFR superfamily, is a type I surface glycoprotein expressed on activated T cells, interacts with 4-1BBL expressed on activated antigen-presenting cells (APC), including B cells, macrophages and dendritic cells (DC) and delivers a costimulatory signal for T cell activation and growth. Recent studies have shown that 4-1BB expression is not restricted to subpopulations of lymphoid cells but is distributed across a variety of cells. For example, myeloid cells, including monocyt.es, neutrophils and DCs, express 4-1BB constitutively. The ligand of 4-1BB (4-1BBL) is expressed on activated APCs, such as DCs, B cells and macrophages. This expression pattern raises the possibility that 4-1BB and 4-1BBL could be involved in multiple steps in various innate and adaptive immune responses. Of special note is the fact that, like CD40, 4-1BB is one of the DC-activating molecules, in that signaling via 4-1BB results in cytokine production (e.g. IL-6 and IL12) and upregulation of costimulatory molecules B7-1 and B7-2. However, unlike CD40, which are triggered by their respective ligands on activated T cells, 4-1BB probably receives its stimulus from adjacent APCs (including the DCs themselves), rather than from T cells. Recent studies strongly support 4-1BB regulation of both clonal expansion and survival of CD8~+ T cells in vitro and in vivo. These findings and the observation that naive T cells initially activated through CD28 signals become refractory to repeated CD28 stimulation suggest the need for costimulatory signaling via 4-1BB to sustain the response. 4-1BB has a crucial role in the generation of effector and memory cytotoxic T cells (CTLs) by increasing the numbers of CTLs and their survival and in the development of some effector functions, when on absent of CD28 signals.These above studies have demonstrated that 4-1BB may be an important molecule for preventing the activation and proliferation of alloreactive T cells. In the present study, we'll construct a recombinant E1/E3-deleted adenovirus vector encoding murine 4-1BB extracellular domain and human lgG1 Fc (Ad4-1BBIg), and develop the murine cardiac graft model to test that 4-1BBIg gene therapy whether inhibiting allogeneic T cells proliferation and function. These studies may provide a new route for the therapy of transplantation reject.1. The construction and identification of the recombinant eukaryotic expressing plasmid pcDNA3.1-4-1BB-IgG Fc.In this study, the total RNA is extracted from the murine spleen and human PBMC, respectively; the cDNA encoding the mouse 4-1BB extracellular domain and human IgG Fc are acquired by RT-PCR technique and then inserted them into the eukaryotic expressing plasmid pcDNA3.1. The recombinant plasmid pcDNA3.1-4-1BB-IgG Fc is identified to be correct by restriction endoniclease digestion and sequence analysis. 2. The recombinant adenovirus encoding 4-1BBIg was obtained by homologous recombination in bacteria BJ5183 using AdEasy system.The two sequences were subcloned into the adenovirus shuttle vector pAdTrack-CMV. The resultant plasmid was linearized by digesting with restriction endonuclease, and subsequently cotransformed into E. coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1 by electroporation. The recombinant plasmid DNA was linearized by restriction endonuclease and transfected into HEK293 cells. The adenoviruses were typically generated within 7 to 10 days. The formation of recombinant adenovirus was confirmed by monitoring GFP expression with fluorescence microscope.3. Characterization of recombinant adenovirus and expression of 4-1BBIg in 293 cells.To determine whether the Ad4-1BBIg really worked, expression of 4-1BBIg (48 h after Ad4-1BBIg infection) was assayed by western blot. The culture supernatants from normal and infected 293 cells, those infected with Ad4-1BBIg or infected with the control AdEGFP virus were analyzed with anti-4-1BB Ab. The analyses showed the cells infected with Ad4-1BBIg to express high levels of 4-1BBIg fusion protein. In these experiments, we investigated whether the soluble molecule 4-1BBIg could bind the 4-1BB ligand expressing on the surface of DC cell line DC2.4. As expected, the DC2.4 cell line was found to bind comparable highly levels of 4-1BBIg. The percentage of 4-1BBIg stained positive cells was 84.81%. To assess the inhibitory effect of 4-1BBIg on proliferation of allogeneic T cells, we examined its effect against allogeneic T cells in mixed lymphocyte reaction by CFSE labeling assays. When human IgG was added to the culture liquid, there was no obvious difference on proliferation of T cells. In contrast, when 4-1BBIg fusion protein (0.1, 0.2 and 0.4μg/ml) was added to the MLR assay system, significant dose-dependent of inhibitory effect against the proliferation of allogeneic T cells was observed. To determine the effect of 4-1BBIg fusion protein on cytokine production, we analyzed IL-2 and IFN-γ production by allogeneic T cells by ELISA. When T cells were cultured with stimulating cells, high levels of IL-2 and IFN-γ were produced. Compared with these results, coculture of T cells and stimulating cells with 4-1BBIg fusion protein resulted in a statistically significant marked reduce of IL-2 and IFN- γ.4. Gene therapy using adenoviral vector encoding 4-1BBIg gene significantly prolonged murine cardiac allograft survival.To screen for the efficacy of adenovirus mediated production of 4-1BBIg in transfected mice, we analyzed the concentration of serum 4-1BBIg using a specific ELISA in AdEGFP treated mice and Ad4-1BBIg transfected mice. There was no detectable 4-1BBIg signal in the non-transfected mice (control), nor was any 4-1BBIg detected in AdEGFP group. However, the serum from the Ad4-1BBIg transfected mice showed a large and significant increase in the 4-1BBIg concentration (348.5 ± 23.5 ng/ml) at 72 h after 4-1BBIg gene transfection but decreased rapidly at one week after treatment and transplantation. All isografts survived long-term. No significant prolongation of allograft survival was obtained in the recipients transfected with AdEGFP, comparing to the no-treated allogeneic group. The median grafts survive time (MGST) was 7.67±0.27 and 7.75±0.21 days, respectively. In contrast, the transfection with Ad4-1BBIg prolonged the survival of heart allografts (13.25±0.34 days) compared with the control. To determine whether Ad4-1BBIg affected leukocyte infiltration, we analyzed the histopathology of grafted hearts at day 7 after transplantation. The results revealed a moderate decrease in total infiltrating leukocytes in Ad4-1BBIg-treated hearts compared with AdEGFP or untreated grafts. By contrast, allografts in the control mice showed extensive leukocyte infiltration. We then used RT-PCR to analyze cytokine gene expression patterns. Results shows a time-course study in which grafts infected with Ad4-1BBIg or AdEGFP were screened at days 3, 5 and 7 for mRNA of cytokine coding for IFN-γ and IL-2. PCR products derived from IFN- γ and IL-2 mRNA were not detected in Ad4-1BBIg group at day 3 after transplantation. In contrast, the expression of IFN-γ and IL-2 in untreated and AdEGFP-treated grafts remained consistently and significantly increased throughout from day 3 to day 7, compared with the Ad4-1BBIg group. However, Ad4-1BBIg resulted in a significant and progressive delay in gene transcript levels for IFN-γ and IL-2. We investigated the effect of Ad4-1BBIg on the production of IFN-γ , which was associated with the function of effector T cells. There were several differences noted for intracellular T-cell cytokine production between Ad4-1BBIg and control groups. As expected, the population of CD8~+ and CD4~+ T-cells producing IFN- γ was significantly reduced from the group treated with Ad4-1BBIg compared to controls. The reducing in the population of T cells expressing IFN-γ was more marked for CD8+ T cells than for CD4~+ T cells in Ad4-BBIg group. However, the population of IFN-γ producing cells was very high in the untreatment control and AdEGFP treated group. The percentage of CD8~+ and CD4~+ IFN-γ producing cells in normal spleen was 9.7 ± 0.97 % and 1.78 ± 0.31%, respectively.In conclusion, our studies show that blockade of 4-1BB/4-1BBL interactions by 4-1BBIg gene transfer can prolong murine cardiac allograft survival. Our findings suggest that 4-1BB may be a good target for immunotherapy particularly at the CD8~+ T cell subset.