The Dendritic Cells Silenced By RelB ShRNA Can Induce Immune Tolerance

BackgroundSolid organ transplantation is the most effective treatment for end-stage organ failure.With the development of immunosuppressive drugs,5-year survival rate for kidney transplantation have been improved significantly, and strides long-term curative effect of liver transplantation. The immunization inhibitors'side effects still exist, including the occurrence of infectious diseases, posttransplantation diabetes mellitus, hypertension, high blood cholesterol, and so on.All these factors hamper recipients and long-term graft survival. Immune tolerance after organ transplantation is the ideal way to solve these problem. Allograft-specific tolerance can be induced and anti-rejection drugs could be reduced to avoid side effects, and prolong long-term survival of grafts. Currently the dendritice cell is the ideal choice,which is the most potential cell applied to induce immune tolerance.DC is the most poweful antigen present cell in human.In recent years, it was found that DC also can induced T cell low response, this kind of DC is named for regulatory DC. As the number of DC was low in human peripheral blood, which most of are reactive DCs.How to induce immune tolerance by expansion of DC in vitro and in vivo is the hot spot in the field of transplantation immune tolerance. In the1990's, the DC can be cultured in culture medium with the cytokines GM-CSF and IL-4in vitro.It provided a basis with the help of DC infusion to induce immune tolerance.ObjectiveLentiviral-mediated shRNA against RelB was used to produce tolerogenic dendritic cells from murine bone marrow dendritic cells (BMDCs). The tolerogenic dendritic cells were produced by silencing the RelB expression and was applied in the prevention of rejection in cardiac transplantation.Methods1. The RelB lentivirus was produced according to the lentivirus system. The experimentwas divided into four groups.In the Group1(imDC group), the BMDCs were isolated from C57BL/6mice on the1st day and maintained in the absence of LPS stimulation.In the Group2(mDC group), the BMDCs were isolated fromC57BL/6mice on the1st day, maintained and treated with LPS on the7th day.In the Group3(LucR siRNA-DC group), the BMDCs were isolated first from C57BL/6mice on the1st day and then infected with LucR-siRNA expressing lentivirus as control lentivirus on the5th day and stimulated with LPS on the7th day.In the Group4(RelB shRNA-DC group), the DCs were firstly isolated from C57BL/6mice on the1st day and then infected with RelB-shRNA expressing lentivirus on the5th day and stimulated with LPS on the7th day.The DCs were infected by lentivirus at a multiplicity of infection (MOI) of50. At MOI of50, the transfection ratio can reach above92%detected by fluoroscopy and flow cytometry. The DCs were stimulated with LPS at50ng/ml. RelB expression in the BMDCs was silenced by Lentivirus carrying RelB shRNA. The apoptosis rate and surface markers of DCs were assessed by flow cytometry. IL-12,IL-10,TGF-?1secreted by DCs were detected by ELISA and the DNA binding capacity of NF-kB subunits in the nucleus were measured respectively by ELISA.2. The toleranic dendritic cells were acquired by transfection the DCs with Lentivirus-RelB shRNA as described above.The1st Mixed Lymphocyte Reaction (MLR) was performed by RelB shRNA-DCs with allogenic antigen-specific T cells.The T cells acquired from the1st MLR was reacted with the same specific antigen and the third party sources of DC for the second MLR respectively.Secretion of cytokines IL-2, IFN-?, IL-10and IL-4were detected by ELISA after1st MLR.The T cell apoptosis rate and the ratio of T cell differentiation into CD4+CD25+Foxp3+regulatory T cell was detected by flow cytometry after the1st MLR.3. With the murine model of cardiac transplantation, every group had10mouses.The experiment were divided into four groups. Male C57BL/6(H-2b) and BALB/c (H-2) mice aged8-12weeks were used as donors and recipients, respectively. RelB shRNA-DCs, LucR siRNA-DCs, imDCs (5*106cells) and PBS were independently injected to BALB/c recipient mice via tail vein. Sevendays later, treated and untreated BALB/c mice were subjected to allogeneic cardiac transplantation using organs from C57BL/6donors.Heterotopic heart transplantation was performed according to Niimi' procedures and some modifications were made according to our experiences. The survival time was observed after infusion of RelB shRNA-DCs before transplantation. HE staining was used to detect rejection degree in the specimen of allograft.Results1. RelB expression was significantly inhibited in DCs following lentiviral mediated delivery of RelB specific shRNA. The RelB shRNA-DC produced lower IL-12and higher IL-10than mature dendritic cells (mDCs) and silencing control DCs. There were no difference in the apoptosis rate between shRNA RelB-DCs and mDCs. The expression levels of co-stimulatory molecules (CD80, CD86and CD83) and MHC-II class molecule were lower in the RelB shRNA-DCs than in the mDCs and silencing control DCs. In addition, RelB shRNA also inhibited the RelB DNA binding capacity but had no effect on other NF-kB subunits.2. The shRNA RelB-DCs can significantly inhibit mixed lymphocyte reaction (MLR). Mature DCs and LucR siRNA DCs had more strong ability in the stimulation of the proliferation of T cells, than immature DCs and RelB shRNA DCs. The RelB shRNA DCs can induce allogeneic T cell hyporesponsiveness, but had no specific reactivity on the third kind T cells.Compared to other three groups,the RelB shRNA DCs group secrete lower level of IL-2, IFN-y and higher level of IL-10and IL-4after MLR.RelB shRNA-DCs induced T cell apoptosis and prompt T cells differentation into CD4+CD25+Foxp3+regulatory T cells.3. In the mouse cardiac transplantation model, infusion of donor origin of the Lenti-RelB shRNA-DC can prolong graft survival time and inhibited rejection occurrence.Conclusion1. Compared with other three groups,the RelB expression in the RelB shRNA-DC groups was inhibited.The DC silenced by RelB shRNA acquire the tolerogenic features,including down-expression of surface molecules(MHC-2,CD80,CD86, CD83), with higher secretion of inhibitory cytokines, and the same survival.2. The DC transfected with RelB shRNA Lentiviral induce the specificity T cells no responses, The main mechanism includes that it induced T cell apoptosis, prompt the T cell differentiation into regulatory T cell, regulated the secretion of cytokine shift from Thl to Th2. 3. Within heart transplantation mouse model, the infusions RelB shRNA-DCs before transplant can prevent acute graft rejection, down regulate the degree of rejection, and prolong the survival in mice.