Tolerance Induced By Recipient Immature Dendritic Cells Transfected By IKK2dn Gene And Loaded With Donor Antigens

Part I: Generation of Lewis Rat Bone Marrow-derived Dendritic Cells in Vitro and Evaluation of Their Biological Characterization Objective:To establish a suitable method for the generation of Lewis rat immature dendritic cells and evaluate their biological characterization.Methods:Bone marrow cells were developed under the concentration of granulocyte- macrophage colonystimulating factor ranging from 2.5 to 20ng/ml and IL-4(5ng/ml) , and the cell yield and the number of OX62-positive DC was determined at day 7. The phenotypic characterization were analyzed with flowcytometry,the capacity of stimulating T cells was determined by allogeneic mixed leukocyte reaction (MLR),and the levels of IL-12 secreted by DC and interferon-γ(IFN-γ) and IL-12 levels in 5-day mixed leukocyte culture supernatants was detected by ELISA.Results:With the increasing GM-CSF concentrations resulted in a higher number of cell yield(20.3±2.41%,48.7±7.56%,57.5±11.48% and 71.6±9.24%), but at the same time, the percentage of OX62 become lower(88.6±6.27%,84.3±6.29%,59.4±13.07% and 37.8±9.41%)at day 7. Under the condition of GM-CSF(5ng/ml), DC at day 9 expressed an intermediate levels of MHCII and low levels of CD86, revealed a low secretion of IL-12, and they could not stimulate the T cells in MLR effectively. After stimulation of LPS, they showed a higher expression of MHCII and CD86 and a stronger IL-2 and IFN-γproduction and higher stimulatory capacity of allogeneic T cell. Conclusions: GM-CSF at a concentration of 5ng/ml is the best method to generate large number of lewis rat immature dendritic cells, and this make it possible to induce immunological tolerance by DC.Part II: Construction of Adenoviral Vector Encoding IKK2dn Gene and Identification of Their ExpressionObjective: To construct the recombinant adenovirus vector encoding IKK2dn gene for using IKK2dn modified imDC to induce immune tolerance.Methods: IKK2dn cDNA was cloned into adenovirus transfer vector pShuttle- CMV-GFP(-)TEMP, and analyzed by restriction endonuclease KpnI&HindIII digestion. Then, the obtained plasmid, pShuttle-CMV-GFP(-)TEMP-IKK2dn was transferred into pAdxsi vector to construct pAdxsi-GFP-IKK2dn plasmid, and was identified by XhoI digestion. The correct adenoviral recombinant was then cleaved with PacI and transfected into 293 cells to produce and purify viral particles.The virus was then transfected into hela cells,and the infection titer was monitored by green fluorescence protein(GFP)expression, IKK2dn gene was identified by RT-PCR.Results: As confirmed by restriction digestion analysis and RT-PCR, an expectant fragment of 1060bp was observed in proper recombinants, proved that we successfully constructed the recombinant adenovirus vector encoding IKK2dn gene. The viral titer checked by GFP was about 2×1011pfu/ml.Conclusions: The recombinant adenovirus vector encoding IKK2dn gene was constructed successfully, which make it easier to investigate the induction of immune tolerance by imDC transfected by IKK2dn. Part III: Effect of Biological Characteristics of Rat Bone Marrow-derived Dendritic Cells Transfected by IKK2dn in Vitro Objective:To study the effect of biological characteristics of rat bone marrow-derived dendritic cells transfected by IKK2dn gene in vitro.Methods: Recombinant adenovirus expression plasmid IKK2dn was transfected into rat bone marrow-derived dendritic cells to arrest their maturation, construct tolerenence DC. The expression of transfected gene was detected by western blotting analysis, surface molecules of Adv-IKK2dn-DC were detected by FCM. Autologous T cell proliferation stimulated by Adv-IKK2dn-DC was detected by MTT assay, and the level of IFN-γand IL-10 secreted by DC was analyzed by ELISA.Results: Western blotting analysis detected high and stable expression of IKK2dn gene in pAdxsi-GFP-IKK2dn transfected DC, and compared with the untransfected DC, DC transfected by recombinant adenovirus vector encoding IKK2dn didn't up-regulate the expression of CD86(P>0.05). In contrast to untransfected DC, allogeneic T cells proliferation induced by Adv-IKK2dn-DC was obviously lower(P<0.01), and a stronger IL-10 secretion level(P<0.01) and a weaker IFN-γsecretion level(P<0.01) was detected in the IKK2dn transfected DC.Conclusions:IKK2dn gene can be highly and stably expressed in pAdxsi-GFP-IKK2dn transfected DC, and it can restrain their maturation. DC transfected by pAdxsi-GFP-IKK2dn and loaded with BN antigen can lower the allogeneic MLR, which provided an experimental base for immunological tolerance induction by DC.Part IV: Tolerance in Rat Allograft renal transplantation Induced by Dendritic Cells Transfected by IKK2dn GeneObjective: To establish rat allograft renal transplantation model of acute rejective reaction and investigate the effect and mechanisms of recipient-derived imDC transfected by IKK2dn and loaded with donor antigen to induce immune tolerance. To develop a more applicable approach that uses recipient-derived dendritic cells to induce tolerance for clinical renal transplantation.Methods: Renal transplantations were performed from BN or Wistar donors to Lewis recipients. DC were cultured from recipient rats (Lewis) bone marrow with low dose GM-CSF and IL-4. At day 7, they were transfected by IKK2dn to arrest maturation, and at day 9, they were plused with BN splenocyte lysate for another 2 days. Then, this modified recipient bone marrow derived DC were injected into recipient rats 7 days before transplantation. Null treatment, DC-treatment, Adv-0 transfected and plused BN spleen cell lysate, and a third party donor (Wistar) were served as controls. The survival of renal allografts were observed, cytokine levels were analyzed by ELISA. Their allostimulatory activity was assessed in vitro by one-way MLR. Pathological examination was performed to identified the grade of rejection.Results: Compared with Null treatment, DC- treatment, and the third party donor(Wistar) controls, IKK2dn transfected DC plused with BN splenocyte lysate markedly prolonged the survival of renal allografts in an antigen-specific manner(26.8±1.76d, P<0.01). IKK2dn-DC loaded with BN antigen elicited markedly lower proliferative responses and reduced IL-2 and IFN-γproduction. In contrast to other groups, IKK2dn-DC loaded with BN antigen show a lower grade as estimated by pathological examination.Conclusions: Successfully and surely established rat allograft renal transplantation model of acute rejective reaction. Recipient-derived imDC transfected by IKK2dn and loaded with donor splenocyte lysate could induce immune tolerance in a donor-specific manner, it might be associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. Our successful induction of tolerance by DC in a recipient manner provides a more feasible strategy for deceased-donor renal transplantation.