Recipient Tolerogenic Dendritic Cells Loaded With Donor Apoptotic Splenocytes Prolong Concordant Xenogeneic Pancreatic Islet Grafts Survival

The introduction of the Edmonton protocol has demonstrated that islet transplantation is a safe and viable route to achieve insulin independence in a population of patients with type1diabetes, but this protocol requires islets from two or more donors to achieve euglycemia in a single recipient, which is a serious drawback given the prevalence of diabetes and the limited cadaveric organ donor pool. Shortage of donor becomes the chief obstacle for islet transplantation. So it is necessary to find alternative islet sources, such as xeno-islets. However, the rejection for xenotransplantation is severe, the usual way of preventing rejection lies in using high dose of immunosuppressivedrugs, which gives rise to serious side effects such as infection and tumour. The ideal strategy is to induce antigen specific tolerance in transplantation. In general, xeno-islets are mainly rejected by T lymphocytes activated via the indirect pathway of antigen recognition. Dendritic cells(DC) are specialized antigen-presenting cells that have a dominant role in initiating T-cell responses, playing a pivotal role in the induction of both immunity and tolerance. The ability of DC to initiate immune responses or induce immune tolerance is strictly dependent on their maturation state. In addition, DC can acquire antigen from apoptotic cells and turn to maturation. It has been recently demonstrated that suppressor of cytokine signaling 1(SOCS1) is essential for suppressing DC activation, so overexpression of SOCS1 in DC by gene transfer may arrest DC in an immature state, prolonging their window of tolerogenic opportunity. In this study, we use SOCS1 gene modified recipient immature DC to load with donor-derived apoptotic splenocytes, and use these DCs to treat recipient at 7 days prior to islet transplantation. Then, we investigate whether the SOCS1 gene modified recipient immature DCs loaded with donor-derived apoptotic splenocytes could induce T-celll cross-tolerance and prolong xeno-islets graft survival. The whole study consists of six parts as follow:PartⅠConstruction and Identification of Recombinant Adenovirus Vector Containing Mouse SOCS1 GeneObjective: To construct recombinant adenovirus vector containing mouse suppressor of cytokine signaling-1gene (Ad5F35-SOCS1), and to investigate the expression of SOCS1 in mouse dendritic cells transfected by Ad5F35-SOCS1. Methods: Full length SOCS1 cDNA was obtained from pEF-FLAG-1/mSOCS1 plasmid by PCR amplification. The PCR product was digested by restriction endonucleases AgeI and NheI, and then inserted orientationally into plasmid pDC316-LacZa digested by AgeI and NheI. Once constructed, the recombinant pDC316-SOCS1 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then cotransfected with rescue plasmid pBHGF35 into 293 cells by liposome reagent. Recombinant adenovirus vector containing mouse SOCS1 gene (Ad5F35-SOCS1) was generated by site-specific recombination and confirmed by PCR, and then Ad5F35-SOCS1 was propagated in 293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay. Results: Construction of pDC316-SOCS1 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5F35-SOCS1 was confirmed to be correct by PCR. The infective titer of Ad5F35-SOCS1 was 1.4×109 IU/ml. Conclusions: Recombinant adenovirus vector containing mouse SOCS1 gene (Ad5F35-SOCS1) was successfully constructed, and it laid the foundation for producing tolerogenic dendritic cells via gene transfection. PartⅡIn Vitro Culture and Identification of Dendritic Cells from Mouse Bone MarrowObjective: To establish a simple and economic method of propagation and culture of mouse bone marrow derived dendritic cells (BM-DC) in vitro, and identify them with morphological, immunological phenotype determination, and functional examination. Methods: DC progenitors were isolated from bone marrow of mice, and propagated in culture medium with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF). The culture system was divided into 4d group and 8d group. The suspension cells, which were DCs, were collected on day 4. The morphology were observed with light inverted microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM) , and cell phenotype was analysed with flow cytometry. The biological function was studied with antigen phagoeytosis test and mixed lymphocyte reaction (MLR). Results: These collected cells exhibited typical DC morphology. These cells of 4d group,which had low expression of MHCⅡ,CD40 and CD86, could phagocytize antigen. Those cells of 8d group, which had high expression MHCⅡ, CD40 and CD86, could stimulate allogenic mixed lymphocyte proliferation. Conclusion: DC can be generated from mouse bone marrow cells through rmGM-CSF induction in vitro.Part III Effect of SOCS1 Gene Transfection on the Biological Characteristic of Mouse Dendritic CellsObjective: To investigate the effects of SOCS1 gene transfection on the biological characteristic of dendritic cells (DCs) in mice. Methods: Recombinant adenovirus containing mouse SOCS1 gene was employed to transfect mouse DCs, and the expression of SOCS1 was detected by immunohistochemistry and Western blot. The phenotype changes of DCs were analyzed by FCM. The influence of the transfected DCs on the proliferation of T cells was tested by mixed lymphocyte reaction (MLR). The secretion of cytokines were assayed by ELISA. There were control group, Ad-EGFP group and Ad-SOCS1 group. Results: The expression of SOCS1 was obvious in Ad-SOCS1 DCs, and the expression of MHCⅡand costimulatory molecules in Ad-SOCS1 DCs were much lower than those in control DCs and Ad-EGFP DCs. There were no diferences in the IL-12 secretion between Ad-SOCS1 DCs and control DCs, but when stimulated by LPS, IL-12 secretion in Ad-SOCS1 DCs was much lower than that in control DCs. The proliferation of T lymphocyte cells was inhibited by Ad-SOCS1 DCs, but control DCs and Ad-EGFP DCs stimulated the proliferation of T lymphocyte obviously. In MLR, the secretion of IL-2 and IFN-γin Ad-SOCS1 group decreased obviously (P<0.05), while the secretion of IL-10 increased obviously(P<0.05) . Conclusion: SOCS1 gene transfection could suppress DCs maturation and induce Th2 type differentiation, and it was a effective strategy to prepare tolerogenic DCs.Part IV Immunological Characteristic of SOCS1 Gene Modified Mouse Dendritic Cells Loaded with Apoptotic Rat SplenocytesObjective: To investigate the immunological characteristic of SOCS1 gene transfected mouse dendritic cells loaded with apoptotic rat splenocytes. Methods: Apoptotic rat splenocytes was induced by ultraviolet B irradiation (200mJ/cm2) and analyzed by using FCM. Rat apoptotic splenocytes were co-cultured with SOCS1 gene transfected mouse DCs (SOCS1-DC) for 48h to make SOCS1-DCs load with xenogenic apoptotic splenocytes (SOCS1-Apo-SCs DC). The expression of co-stimulatory molecules and MHCⅡwere analyzed using FCM before and after DC phagocytosing xenogenic apoptotic splenocytes. The capacity to stimulate T-cell proliferative response was detected by primary MLR. To investigate whether SOCS1-Apo-SCs DC could induce antigen-specific T cell hyporesponsiveness, C57BL/6 mice were primed with SOCS1-Apo-SCs DC and secondary MLR was performed. Results: Mouse DCs could efficiently phagocytize rat apoptotic splenocytes(Apo-SCs DC). After phagocytizing apoptotic splenocytes, the expression of MHCⅡand costimulatory molecules was conspicuous high in Apo-SCs DC and relatively low in SOCS1-Apo-SCs DC. Apo-SCs DC could stimulate T cell proliferate obviously while SOCS1-Apo-SCs DC could not activate T cell. In second MLR, T cell primed by SOCS1-Apo-SCs DC is antigen-specific hypo-responsive. Conclusion: SOCS1 gene modified DCs loaded with xenogenic apoptotic splenocytes could induce antigen-specific T cell hyporesponsiveness.Part V Isolation, Purification and Identification of Rat Pancreatic IsletsObjective: To optimize conditions for rat islets isolation and purification, and lay foundation for islet transplantation experiment. Methods: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase P digestion, and purified by discontinuous Ficoll density gradient centrifugation. The protocol was carried out to investigate the influence of collagenase concentration and digestion time on the result of islet isolation. Rat islets were identified by Dithizon staining, and islets viability was assessed by Acridine orange /Propidium iodide staining. Glucose-stimulated insulin secretion was detected using rat insulin ELISA kits and used to assess the function of rat islets. Results: The concentration of collagenase and digestion time were important factors for efficacious isolation of islets. Under the condition of digestion by 1mg/ml collagenase P for 45min, the effect of islet isolation was optimal (P<0.05). The purity of islets isolated under optimizing conditions was above 90%, and the viability was nearly 90%. The insulin secretion upon 2.8mmol/L and 16.7mmol/L glucose simulation was (0.67±0.14)μg/L/10islet and (5.44±0.09)μg/L/10islet, respectively (P<0.05). The simulation index was 8.25±1.87. Conclusions: The result of islet isolation was concerned with collagenase concentration and digestion time. Optimizing conditions for islets isolation can improve the result of rat islet isolation .Part VI Establishment of Xeno-islet Transplantation Model in Diabetic Mice and Observation of Its Curative Effect on Diabetes MellitusObjective: To establish the model of xeno-islet transplantation in diabetic mice and to observe its curative effect on diabetes mellitus. Methods: Diabetic mice was induced by a single intraperitoneal injection of 200mg/kg streptozotocin. The mice was considered diabetic after two consecutive blood glucose of 16.7mmol/L or higher. 600 rat islets were transplanted into the renal subcapsular space of diabetic mice. The time of graft failure was determined as the day of diabetes recurrence, defined as the first of 2 consecutive days when blood glucose was above 11.2mmol/L. The morphological change of transplanted xeno-islets was evaluated by histopathology. Results: Xeno-islet transplantation could restore normoglycaemia in diabetic mice, but the majority of xeno-islets were rejected within 7 days after transplantation without any administration of immunosuppressive treatment. The survival time of xeno-islets was 6.50±1.05d. The rejection of xeno-islets was confirmed by histopathology. Conclusions: The model of xeno-islet transplantation in diabetic mice was successfully established, and it laid the foundation for investigating the rejection or tolerance of xeno-islet transplantation. Part VII Prolongation of Concordant Xeno-Islet Grafts Survival by SOCS1 Gene Modified Recipient Dendritic Cells Loaded with Donor-Derived Apoptotic SplenocytesObjective: To investigate the effect of recipient tolerogenic dendritic cells (Tol-DCs) loaded with donor-derived apoptotic cells on inducing tolerance in concordant xeno-islet transplantation. Methods: Diabetic mice was induced by a single intraperitoneal injection of 200mg/kg streptozotocin. The mice was considered diabetic after two consecutive blood glucose of 16.7mmol/L or higher. Xenogenic islet transplantation model in mice renal subcapsular space was performed from Lewis or SD donor rat (third party) to diabetic C57BL/6 recipient mice. Recipient mice were given one injection of 2×106 DCs via the tail vein at 7 days before islet transplantation in the absence of immunosuppression drugs. Animals in this study were divided into 6 groups according to the pretreatment of various DC: (1) Control group: only inject 0.2ml PBS; (2) DC group: recipient DC; (3) SOCS1-DC group: SOCS1 gene transfected recipient DC; (4) Apo-SCs DC group: recipient DC loaded with donor apoptotic splenocytes; (5) SOCS1-Apo-SCs DC group: SOCS1 gene transfected recipient DC loaded with donor apoptotic splenocytes (Apo-SCs); (6) Third party donor group: SD rats were used as donor, recipients (C57BL/6) were pretreated as the fifth group as above. Graft survival was assessed daily by nonfasting blood glucose concentration. The time of graft failure was determined as the day of diabetes recurrence, defined as the first of 2 consecutive days when blood glucose was above 11.2mmol/L. Histopathology and immunohistochemistry were performed at day7 after transplantation, Results: The xeno-islets survival time of Control group, DC group, SOCS1-DC group, Apo-SCs DC, SOCS1-Apo-SCs DC group, and Third party donor(SD) group was 6.50±1.05d, 6.14±1.57d, 6.67±1.03d, 4.86±1.35d, 17.29±4.54d, and 6.17±1.17d, respectively. SOCS1-Apo-SCs DC group survival time was much longer than other groups(P<0.01). Histopathology and immunohistochemistry staining confirmed the rejection of SOCS1-Apo-SCs DC group is slight at 7 day posttransplantation. Conclusion: SOCS1 gene modified recipient DC loaded with donor apoptotic splenocytes could prolong concordant xenogeneic islet grafts survival.SummarySOCS1 gene transfection could suppress DCs maturation and induce Th2 type differentiation. SOCS1 gene modified recipient DCs loaded with donor apoptotic splenocytes could induce antigen-specific recipient T cell hyporesponsiveness, and they could be used to prolong concordant xenogeneic islet grafts survival.