Protective Effects Of PD-L1 Gene Transfer Mediated By Adeno-associated Virus On Liver Allograft Of Rat And Its Mechanisms

BackgroundLiver transplantation is an effective or even only approach of treatment to some end stage liver diseases, but acute rejection remains to be the primary clinical problem and challenge of transplantation. The outcome of allogeneic organ transplants is, in part, dependent on the balance between co-stimulatory and modulatory signals that accompany TCR-mediated recognition by recipient's T cells. The local expression of a gene encoding an immunosuppressive protein within a graft can generate local immunosuppression, which enhanced co-inhibitory signals in recipients, making gene therapy a viable approach for facilitating transplantation.Programmed death ligand-1 (PD-L1), a newer member of the CD28 family, has been implicated as a negative regulator of T cell activation. In previous research, engagement of programmed death-1 (PD-1) by PD-L1 inhibited proliferation and cytokine production by activated T cells. Conversely, blockade of PD-L1 using specific monoclonal antibodies stimulated T cell proliferation and cytokine production. Meanwhile, recent studies showed that interfering with the PD-L1/PD-1 co-stimulatory pathway prolonged allograft survival in various organ transplantation models, suggesting that they might play an important role in maternal acceptance of liver allograft. Thus, the recombinant PD-L1 gene was chosen as the immunomodulator.This study focused on establishing acute rejection model of orthotopic liver transplantation in inbred rats. The effective way of transferring the target gene into the liver allograft with PD-L1-AAV2-RFP was also explored. Furthermore, we investigated the protective effects and mechanisms of modification of liver allograft with target gene on the survival in vivo. The main results and conclusions were as follows: Part 1 Establishment of acute rejection model of orthotopic liver transplantation in inbred ratsObjective To establish acute rejection model of orthotopic liver transplantation in inbred rats.Methods We established LEW-BN rat orthotopic liver transplantation model using"the innovated two-cuff technique". Experimental groups included:①G1: Isogenic (ISO) group.②G2: Allogeneic (ALLO) group.③G3: CsA group injected CsA (2.5mg/kg.d) intraperitoneally daily from day 0 to day 7 postoperatively. Recipients were sacrificed on POD 3, 7, 14, and 21 respectively. Liver tissues and blood samples were collected. The general states, median survival time (MST), and histopathological characteristic of recipients were observed. Serum levels of ALT and TBIL were measured with automatic biochemical analyser.Results In G2, typical mild, moderate and severe acute rejection occurred on POD 3, 7 and 14 respectively, and the delayed occurrence of graft rejection were observed in G3. The MST of G3 (28.7±0.98)d was prolonged significantly compared with that of G2 (18.3±1.15)d (P<0.05). Serum ALT and TBIL levels of G2 increased obviously on POD 3 and has been booming ever since. Serum ALT and TBIL levels of G2 were significantly higher than those of G1 and G3. Typical histological change occurred on POD 14 in G2 and the light rejection in G3. There was no significant difference in rejection activity index (RAI) scores between G2 and G3 on POD 3. Whereas on d7, d14 and d21 after operation, RAI scores of G2 were marked higher than that of G3 and G1 (P<0.05).Conclusions1. Rat orthotopic liver transplantation of LEW to BN strain is an ideal acute rejection model.2. The model has stable and typical allograft rejection. It can be used in the study of liver transplantation immunity.Part 2 The security and effectiveness of recombinant adeno-associated virus transfecting PD-L1 gene into livers of donorObjective To find the effective way of transfecting PD-L1 gene into livers of donor with recombinant adeno-associated virus. Methods The PD-L1-AAV2-RFP was identified by PCR. The transfection efficiency of PD-L1-AAV2-RFP was determined by observing the expression of RFP in BHK cell with fluorescence microscope. We established the PD-L1-AAV2-RFP gene transfer model and recipients were randomly divided into three groups:①G1: ISO group.②G2: AAV group introduced through back-table portal vein perfusion with AAV2-RFP for 2 hours, and the graft was flushed with Ringer's lactate solution to remove any free virus before implantation.③G3: PD-L1 group introduced with PD-L1-AAV2-RFP. Liver allografts harvested on POD 3, 7, 14, and 21 were fixed in 10% buffered formalin and embedded in paraffin for histological study by hematoxylin-eosin staining. Part of the grafts were frozen for RFP expression analysis. PD-L1 mRNA and protein expression in grafts were detected by using Realtime RT-PCR and IHC respectively. The function of liver was also measured. Results The fragment was verified by PCR analysis to make sure that the cDNA is just the one we expected. Evident red fluorescence was observed and the transfection rate increased with the prolongation of time, suggesting that the donor liver might be transfected efficiently by PD-L1-AAV2-RFP. Serum levels of ALT and TBIL increased obviously on POD 3 and 7 respectively, then gradually returned back to normal with time. Pathological changes of the specimens showed only mild ischemia-reperfusion injury on POD 7, with no statistical difference among three groups (P>0.05). Red fluorescence was not observed in sections of heart, lung, kidney, and spleen after transplantation. However, evident red fluorescence was observed in sections of livers after operation except day 3 and increased in the later. The expressions of PD-L1mRNA and protein were both upregulated in the liver of donor at 7th day after transfection, and reached its peak at 21st day in G3, but still lower in G1 and G2.Conclusions1. PD-L1-AAV2-RFP is effective and safe for gene delivery in rat liver transplantation.2. Back-table perfusion through the portal vein with PD-L1-AAV2-RFP (1×1011v.g./animal) and preservation of grafts for 2 hours is the optimal protocol to induce sufficient protein expression.Part 3 The protective effects of PD-L1 gene transfer mediated by AAV-2 on liver allograft of rat and its mechanisms Objective To evaluate the protective effects and mechanisms of the liver allograft transfected with PD-L1 gene in vivo.Methods Recipients were randomized into six groups:①G1: ISO group.②G2: ALLO group.③G3: AAV group introduced with AAV2-RFP.④G4: CsA group injected CsA (2.5mg/kg.d) intraperitoneally daily from day 0 to day 7 postoperatively.⑤G5: PD-L1 group introduced with PD-L1-AAV2-RFP.⑥G6: Combination therapy group introduced with PD-L1-AAV2-RFP plus a subtherapeutic regimen of CsA. We established ROLT model using"the innovated two-cuff technique". Gene delivery to donor liver was done by intracoronary instillation of virus containing solution (1×1011v.g./animal) through the portal vein. On POD 3, 7, 14 and 21, grafts were taken for IHC to assess the expression of PD-L1.Ig, CD4+, CD8+ and CD25+ lymphocytes, for ELISA to analysis the concentration of IFN-γ, IL-17, IL-10, and TGF-β1 respectively. The MST, liver function and histopathological changes were also observed.Results In G1, no acute rejection was found, biochemical datas showed that the levels of ALT and TBIL increased during the first weeks after operation and decreased to normal with time. Fewer PD-L1-positive cells in the graft were detected. The expression of IFN-γ, CD4+, CD8+, and CD25+ lymphocytes were not observed in various period of time. Otherwise, IL-17, IL-10, and TGF-β1 were all low-expressed. The MST was (106.7±0.24)d. In G2 and G3, typical mild, moderate and severe acute rejection occurred on POD 3, 7 and 14 respectively. The function of liver were markedly higher than those of other groups (P<0.05). Fewer PD-L1-positive cells in the graft were detected. A lot of CD4+, CD8+ and CD25+ lymphocytes infiltrated in portal area of grafts, especially CD8+ T cells (P<0.05). On POD 3, the concentration of IFN-γand IL-17 in grafts increased continuously and even decreased on POD 21, while the expression of IL-10 and TGF-β1 were down-regulated gradually. The MST were (18.3±1.15)d and (18.3±0.72)d. In G4, G5 and G6, acute rejection still occurred after operation with light rejection sign in histopathological change. The function of gene transfected liver was inferior to that of G2 and G3 significantly (P<0.05). Although fewer PD-L1-positive cells were detected in G4, a certain number of PD-L1-positive cells were found in G5 and G6. Compared with G2 and G3, those allografts treated with PD-L1 or CsA had a significantly reduced cellular infiltrate, especially CD8+ cell. The concentration of IFN-γand IL-17 in grafts were greatly inhibited, accompanying with greatly increased secretion of IL-10 and TGF-β1. Allografts transduced with the PD-L1 survived for longer periods of time (P<0.05). PD-L1 gene transfer combined with a subtherapeutic regimen of CsA was superior to CsA alone: (34.0±1.49)d vs. (28.7±0.98)d.Conclusions1. Adeno-associated virus mediated PD-L1 gene delivery significantly prolongs liver allograft survival in a rat model.2. Enhanced expression of PD-L1 protein in donor liver attenuates acute allograft rejection. The effect is additive to that of a subtherapeutic regimen of CsA.3. PD-L1 gene may take the effect in liver transplantation via reducing intragraft infiltrates of T lymphocytes, suppression of INF-γand IL-17 production as well as stimulation of IL-10 and TGF-β1 production.