Construction Of Lentiviral Vectors Targeting CD80 And CD86 Genes By RNA Interference And Their Effects In Murine Cardiac Allograft Rejection

Organ transplantation has now become an effective method to cure end-stage heart or liver or renal or other organs disease. With the gradual perfection of surgical technique, the progress of organ preservation and tissue culture, and the effective application of immunosuppressive agents, hyperacute rejection and acute rejection prevention have achieved great progress. Chronic graft dysfunction caused by chronic rejection gradually become the bottleneck in successful organ transplantation. Therefore, inducing donor specific immune tolerance is an important subject needed to be solved urgently in current transplant field.Immune recognition, activation, proliferation of recipient T cells aiming at donor antigen is the basis of organ transplant cell immunity, and this also plays an essential role in allograft rejection. Transplantation antigen allorecognition of recipient T cells include direct recognition and indirect recognition. Nowadays more and more scholars consider that indirect recognition pathway may be substantially more important in the occurrence mechanism of chronic graft dysfunction. Therefore, blocking indirect recognition pathway and illustrating its molecular mechanism deserve paying much more attention in transplantation tolerance.Recipient T cells activation demand the co-stimulatory signal which is delivered through interactions between recipient T cells and antigen presenting cells. CD80/CD86-CD28 is the most important co-stimulatory pathway. So we knocked down CD80 and CD86 expression in recipient dentritic cells using lentiviral mediated RNA interference, and tried to find out what happened in murine heart transplantation after blocking indirect recognition pathway in vitro or in vivo, and tried to explore the possible mechanisms.PartⅠConstruction and identification of lentiviral vectors targeting mouse CD80 and CD86 genes by RNA interference Objective To construct lentiviral vectors targeting mouse CD80 and CD86 genes by RNA interference and study their effects on bone marrow-derived dendritic cells in vitro.Methods The effective sequence of siRNA targeting CD80 gene was confirmed in our previous experiment. The complementary DNA containing both sense and antisense oligonucleotides of the targeting sequence was designed, synthesized. After annealed, double-stranded DNA was inserted into the pGCL-GFP vector. The resulting lentiviral vector was named pGCL-GFP-CD80shRNA.293T cells were cotransfected with pGCL-GFP-CD80shRNA,pHelper1.0 and pHelper2.0. The titer of virus was tested according to the expression level of GFP. Lentiviral vector targeting mouse CD86 gene by RNA interference was constructed in the same way. The recombinant lentiviruses infected dendritic cells that were separated from femurs and tibias of mice in vitro. The infection efficiency was assessed by fluorescence microscope. The cell viability of infected dendritic cells was determined by annexin V and propidium iodine staining. The expression of CD80 and CD86 was analyzed by flow cytometry. Results PCR and DNA sequencing demonstrated that LV-GFP-shCD80 and LV-RFP-shCD86 were constructed successfully. The titer of the recombinant lentiviruses was both 2x107TU/ml and the best MOI for lentivirus infecting dendritic cells was 20. Lentiviruses demonstrated a high (85.42%) infection efficiency of dendritic cells without affecting cellular viability. The inhibitory rates of CD80 and CD86 expression were 82.05% and 77.78% respectively. Conclusions Lentiviral vectors targeting mouse CD80 and CD86 genes by RNA interference were constructed successfully. The recombinant lentiviruses show significantly inhibiting effects on CD80 and CD86 expression in dendritic cells. This approach is a potential therapeutic option for allograft rejection.Part IIEffects of blocking co-stimulatory pathway in vitro on alloantigen pulsed recipient dendritic cells pretreated by recombinant lentivirusObjective To measure the phagocytic capacity and the bio-immunological characteristics of recombinant lentivirus pretreated recipient dendritic cells loaded with alloantigen. Methods In the culture process of dendritic cells, donor antigens were added, the validity of dendritic cells loading antigens was investigated by one-way mixed lymphocyte reaction using recipient splenic T cells and recipient dendritic cells. The transcription of CD80/CD86 gene and the expression of CD80/CD86 protein were determined by real-time quantitative reverse transcription-polymerase chain reaction and flow cytometry, respectively. The stimulating capacity to recipient T-cell proliferative response was testified in primary mixed lymphocyte reaction. To determine immunological capacity of recipient T cells recognizing donor antigens which were presented through indirect or direct recognition pathway, recipient mice were primed with these dendritic cells and secondary mixed lymphocyte reactions were performed. IL-2, IL-4, IL-10 and INF-γlevels in primary mixed lymphocyte reaction culture supernatants were measured using enzyme linked immunosorbent assay.Results Recipient dendritic cells which were cultured with donor antigens could stimulate the proliferation of recipient T cells significantly. The transcription of CD80/CD86 gene and the expression of CD80/CD86 protein enhanced down-regulated when LV-GFP-shCD80 and LV-RFP-shCD86 infected dendritic cells simultaneously, and this inhibition was not reversed by lipopolysaccharide. Recombinant lentivirus pretreated recipient dendritic cells effectively inhibited the proliferation of recipient T cells in primary mixed lymphocyte reaction and induced specific T-cell hyporesponsiveness to donor antigen in secondary mixed lymphocyte reaction. A reduction of Thl cytokines (IL-2 and INF-γ) and an induction of Th2 cytokines (IL-10) were found in primary mixed lymphocyte reaction culture supernatants. Conclusions Recipient dendritic cells could present donor antigens effectively. Recombinant lentivirus could specifically and effectively knock down CD80 and CD86 gene expression. Recombinant lentivirus pretreated recipient dendritic cells markedly suppressed recipient T-cell proliferative response, induced local cytokines prone to Th2, and induced antigen specific T-cell hyporesponsiveness.PartⅢAnti-rejection effect of alloantigen pulsed recipient dendritic cells pretreated by recombinant lentivirus in murine heart transplantationObjective To establish allogeneic heterotopic heart transplantation model in mice. To investigate the mechanism of recipient-derived dendritic cells treated with recombinant lentivirus inducing transplant immune tolerance in vivo. Methods Improved Cuff technique was used in making the model of cervical heterotopic heart transplantation in mice. By using self-made cuffs, the donor pulmonary artery was anastomosed to the recipient right external jugular vein, the donor ascending aorta was anastomosed to the recipient right common carotid artery. Recipient mice were given one injection of recipient dendritic cells treated with lentivirus via the lateral tail vein 7 days prior to heart transplantation, or combined with intraperitoneal injection of a subtherapeutic dose Cyclosporin A. Graft survival was assessed by daily palpation. Rejection was defined by the cessation of heartbeat and further confirmed by histological analysis. The apoptosis of T cells in recipient lymphoid tissues and allografts was determined by Annexin V and CD3 staining. Regulatory T-cell in recipient spleens and allografts was analyzed by flow cytometry. The expression of cytokine genes (IL-2, INF-γ, TNF-α, IL-4, IL-6 and IL-10 mRNA) within cardiac allografts were evaluated by semi-quantity RT-PCR. The expression of apoptosis signal pathway associated genes in splenic T cells were also examined by semi-quantity RT-PCR. Results Murine cervical heterotopic heart transplantations have been performed with a successful rate of 92.5%. The operative time was about 60 minutes, the cold ischemic time for donor heart was smaller than 30 minutes. Recipient-derived dendritic cells treated with recombinant lentivirus significantly prolonged heart allograft survival (Median survival time was 21.8 days). Combined with intraperitoneal injection of Cyclosporin A prolonged the median survival time to 72 days, with 3 grafts surviving beyond 100 days. These dendritic cells down-regulated Thl cytokines (IL-2, INF-y and TNF-a) expression, but enhanced the Th2 cytokines (IL-10) expression within allografts. We found a higher percentage of apoptotic T cells in recipient spleens, mesenteric lymph nodes and grafts. A higher percentage of regulatory T cells were also found in grafts. Splenic T cells expressed high expression of GRP78 and Bax. These changes in endoplasmic reticulum and mitochondrial pathway were enhanced by up-regulation of CHOP and suppression of Bcl-xL expression among these cells. Conclusions Improved Cuff technique successfully established stable heterotopic cardiac allograft model in mice. Recipient dendritic cells treated with lentivirus combined with Cyclosporin A prolonged heart allograft survival, and even induced transplant immune tolerance. Cytokine secretion prone to Th2, regulatory T cells and recipient T-cell apoptosis might be crucial for the equipped dendritic cells prolonging graft survival. Mitochondrial apoptosis pathway and endoplasmic reticulum stress-mediated apoptosis pathway may be involved in allogeneic T-cell apoptosis.