The Mechanisms Of Transplant Tolerance Induced By Donor Apoptotic Cells

BackgroundOrgan transplant is one of the most effective treatments for patients with end-stage diseases,while rejections constitute the major obstacle for long-term graft survival. Immunosuppression has been the only applicable clinical method for preventing and treating transplant rejections,although it usually leads to increased opportunistic infections, severe drug toxicity and high medical expense.Theoretically,induction of donor antigen specific tolerance is the ideal way to prevent transplant rejections.Two principles are important for transplant tolerance induction.First,donor antigens should be accessible to the appropriate immune cells residing in the recipient immune system,and second,the immune system should be regulated accordingly.However,the methods successfully established in murine transplant models are hardly transferable to non-human primates and patients,appealing novel tolerance strategies to deal with clinical transplant rejections.In past 10 years,the immune regulatory property of apoptotic cells became a major focus in immunological studies and apoptotic cells were demonstrated to induce tolerance. Rapid phagocytosis of apoptotic cells by antigen presenting cells(APC),more efficient presentation of apoptotic cell-derived antigens to na(i|¨)ve T cells and local immunosuppressive environment initiated by apoptotic cells and their phagocytes all contribute to tolerance induction. The authors also found that apoptotic cells inhibited T cell activation in vitro and intravenous infusion of donor apoptotic cells suppressed donor antigen specific mixed lymphocyte reaction(MLR).More importantly,administration of donor apoptotic cells prolonged heart,skin,and liver graft survival under no immunosuppression,in which phagocytes in the recipients played an important role.Reports in the literature also supported that administration of donor apoptotic cells is an effective strategy to induce transplant tolerance.Many questions remain obscure concerning the mechanisms on tolerance induction by donor apoptotic cells,such as the distribution of infused donor apoptotic cells,their phagocytosis,antigen presentation,and the function of recipient T cells subsets.More importantly,it is critical to understand the theoretical significance of apoptotic cell-induced tolerance in immune recognition and to develop more effective and clinically applicable measures for the treatment of autoimmune diseases,tumors or transplant rejections.The whole project includes four parts:1.The distribution of intravenously infused donor apoptotic cells in recipient organs and its significance in immune regulationPurposes:To investigate the distribution of intravenously infused donor apoptotic cells, and to explore the significance of phagocytotic cells and local cytokines in the induction of local immune regulatory environment.Methods:Inbred mice C57BL(H-2~b) and Balb/C(H-2~d) were chosen to be donors and recipients,respectively.C57BL donor splenocytes were first stained with 2μM CFSE and treated with Dexamethasone(Dex) to induce apoptosis.In some experiments,donor apoptotic cells were enriched with Annexin-V beads(Mitenyl).In vivo,2×10~7 C57BL apoptotic splenocytes were injected intravenously into Balb/C recipients,and liver,spleen and blood samples were collected at different time intervals after injection.The cells were analyzed by fluorescence histochemistry and flow cytometry to examine the in vivo distribution of apoptotic donor cells.Meanwhile,the cytokine concentrations in the tissues of liver and spleen,and in serum were determined with Bioplex system or ELISA. In vitro,CFSE~+ apoptotic C57BL splenocytes were co-cultured with APCs isolated from Balb/C liver and phagocytosis of apoptotic cells by liver APC was analyzed by fluorescence microscopy,immunohistochemistry,and flow cytometry.Results:Apoptotic donor cells were mainly deposited in the liver,but hardly found in the spleen.Thirty to sixty minutes after the injection,donor apoptotic cells mainly accumulated in the liver and were rapidly cleared,usually within 90 minutes.Both donor and recipient apoptotic cells displayed similar distribution,indicating that the deposit of donor apoptotic cells in the recipient liver depends on the nature of apoptosis,not on their allogenicity.In vivo and in vitro experiments revealed that all three kinds of liver APCs, including liver sinusoid endothelial cells(LSEC),Kupffer cells(KC) and dendritic cells (DC),effectively phagocytosed donor apoptotic cells.In the liver,spleen and serum,11 cytokines including IL-1β,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,TNF-α,IFN-γ,GMCS and TGF-β,were detected at different time intervals after the infusion of donor apoptotic cells. Although Th1 response was found both in the liver and spleen,a stronger,more rapid and sustained Th2 response revealed in the liver,but not in the spleen.In the serum,neither Th1 nor Th2 responses were found.Conclusion:The deposition of donor apoptotic cells in the recipient liver and the induction of a liver specific Th2 response might contribute to tolerance inductivity of donor apoptotic cells.2.The effect of donor apoptotic cells on in vivo cytotoxicity against donor cells and on regulatory T cells(Tregs)Purposes:To explore the influence and significance of donor apoptotic cells on the in vivo cytotoxicity against donor cells and on Tregs. Methods:Inbred mice C57BL(H-2~b) and Balb/C(H-2~d) were chosen to be donors and recipients.Donor and recipient splenocytes were stained with different concentrations of CFSE respectively and a mixture of CFSE stained donor and recipient cells were injected intravenously into recipient mice.Then at different time intervals,peripheral blood were harvested and analyzed by flow cytometry to determine the clearance rate of CFSE~+ donor cells in comparison to CFSE~+ recipient cells.In vivo cytotoxicity was calculated according to clearance rates of CFSE~+ donor cells and CFSE~+ recipient cells.The accuracy,specificity and sensitivity of the in vivo cytotoxicity test were verified in mouse skin allograft and immunosuppressive models,and in nude mice.For investigating the influence of donor apoptotic cells on in vivo cytotoxicity and Tregs,2×10~7 apoptotic, necrotic or living C57 splenocytes were intravenously injected into Balb/C mice,and at different time intervals,peripheral blood samples were collected and analyzed by flow cytometry for Tregs.And at the same time,a mixture of CFSE~+ donor and recipient cells was intravenously injected and peripheral blood collected for the evaluation of in vivo cytotoxicity against donor target cells.Results:An injection of CFSE~+ donor and recipient cells with cell number between 1×10~7 and 1×10~8 made those cells easily detected in recipient peripheral blood.In skin allograft sensitized recipients,CFSE~+ donor cells were rapidly eliminated within 1-2 hr, while in those un-sensitized animals the elimination tempo was significantly slower so that they only started to be cleared 4 hrs,significantly eliminated 24hrs and vanished 72 hrs after the injection.In immunosuppressed or nude mice,the elimination rate of test donor cells was significantly decreased.Although CFSE~+ donor cells were rapidly cleared in mice that had been treated with donor necrotic or living cells,they were not eliminated in mice treated with apoptotic cells,indicating that in vivo lymphocyte toxicity were increased in necrotic or living donor cell treated mice,but not increased in apoptotic donor cell treated animals.On the contrary,the proportion of Tregs in the peripheral blood was also increased in mice treated with apoptotic donor splenocytes. Conclusions:In vivo cytotoxicity is an accurate,simple and rapid method for the detection of lymphotoxicity.Donor apoptotic splenocytes,in contrast to necrotic or living donor cells,significantly inhibited lymphocytotoxicity in recipients,during which the increase of proportion of Tregs might play an important role.3.The effect of allogeneic donor apoptotic cells on renal allograft survival and on the proportion of Tregs in monkey recipientsPurpose:To examine the effect of intravenously infused allogeneic donor apoptotic cells on renal allograft survival in a rhesus monkey renal transplant model and to investigate the significance of Treg in donor apoptotic cell-induced immune regulation.Methods:First,we compared three methods to test the human-like blood types in rhesus monkey,including monoclonal antibody test,agglutination inhibition test and reverse blood group test.The results showed that reverse blood group test,i.e,testing A,B-antigen specific antibodies in the serum rather than the antigens on erythrocytes,could be used to identify blood types in rhesus monkeys.Rhesus monkey allogeneic renal transplantation was performed by planting donor left kidney in the lower abdomen without nephrectomy of their own kidneys.Usually,donor left kidney was procured and perfused ex vivo with cold HCA solution.For transplantation operation in the recipients,donor renal vein and artery were anastomosed to recipient infra vena cava and abdominal aorta,respectively.Graft ureter was anastomosed directly to the bladder and a double pig-tailed tube placed in its lumen with two ends entending to the renal graft pelvis and to the bladder.High energy X-ray,Co-60-γray,and ultraviolet C(UVC) were tested for the induction of peripheral blood mononuclear cells(PBMC) apoptosis in the rhesus monkeys. Consequently,large number of apoptotic PBMC can be induced by UVC irradiation(dish diameter 6cm,RMPI-1640 supplemented with 10%bovine serum,distance 20 cm and irradiation time 60 minutes). For evaluating the effect of donor apoptotic cells on renal allograft survival,7.2-7.4×10~7 allogeneic donor apoptotic PBMC,or PBS for the control animal,were intravenously injected into recipients one week before allogeneic renal transplantation.The animals were treated with combined immunotherapy,including methylprednisolon(MP), cycloheximide(CHX) and rapamycin(RAPA),in which RAPA was administrated daily for 14 days with a dose approximately about one fifth clinical dose.Ultrasound and CT scan were used to monitor allograft rejection and survival.At the same time,the proportion of Tregs in recipient PBMC was also analyzed weekly after the infusion of donor apoptotic cells.Results:In 43 rhesus monkey tested by reverse blood type method,B type was the most popular,accounting for 58.14%(25 monkeys),followed by AB(34.88%,15 monkeys) and A(7%,3 monkeys),while no O type found.Four monkeys underwent renal transplantation without immunosuppression.In 4 monkeys,the transplantation operation was finished smoothly with immediate urination and blood flow found normal 2 days after operation.One of the monkeys developed swelling of the abdomen 3 days after transplant surgery.Venous embolism was found and the graft removed immediately.Another monkey died 7 days later because of intestinal obstruction and the other two monkeys recovered smoothly although renal graft rejected 5 days after operation.Three monkeys selected for studying the effect of donor apoptotic cells on renal allograft survival were administrated triple immunotherapy, MP+CHX+RAPA.Among them,one week before transplant surgery,two monkeys were injected with 7.2×10~7 or 7.4×10~7 donor apoptotic cells,while the other monkey were infused PBS.In the two monkeys received donor apoptotie cells,renal allograft survival were 14 days and 33 days,while in the monkey infused with PBS,renal allograft survival was 14 days.The proportion of Tregs in the monkeys treated with donor apoptotic cells was higher than that in PBS treated animal.Renal allograft survival was the longest in the animal with highest proportion of Tregs. Conclusion:Reverse blood type test is a reliable and effective method for the detection of rhesus monkey blood types.Donor apoptotic cells may prolong monkey renal allograft survival,possibly by enhancing the proportion of Tregs in the recipients.4.Establishment of a "cell death" immune recognition modelPurpose:To establish an immune recognition model that can better explain various immune phenomena.Method:Theoretical analysis based on the data obtained in our laboratory in the past 10 years and on literature reports.Different immune recognition models,including classic "self nonself" model(clonal selection theory) and contemporary "danger" model and "infectious non-self" model,were compared.Results:"Cell death" immune recognition model mainly comprises four principles in immune recognition and can better explain autoimmunity,tumor immunity,transplant immunity,fetal immunity,liver immunity and the role of Tregs in immune responses.Conclusion:Active immune response or immune tolerance of the immune system to an antigen is neither dependent on whether the antigen is self or non-self,nor on the presence of "danger" or infection,but on the way of cell death during antigen presentation. Apoptotic cells induce tolerance while necrotic cells initiated immune response.