Induction Of Allograft Tolerance By NF-κB Oligodeoxyribonucleotide Decoy-Pretreated Donor And Recipient Dendritic Cells Loaded With Donor-Derived Apoptotic Cells

Objectives: 1. To establish the method of propagation of mouse bone marrow-derived dendritic cells (BM-DC) in vitro, observe the effect of NF-KB oligodeoxyribonucleotide decoy (NF-KB ODN Decoy) on the maturation and the bioimmunological characteristics of BM-DC, induce immature tolerogeneic DC, and investigate the function of NF-KB on the maturation of DC. 2. To establish the method of inducing splenocyte apoptosis using ultraviolet B (UVB) irradiation, measure the phagocytic capacity and the functional changes of NF-KB ODN Decoy-pretreated DC loaded with apoptotic splenocytes, and study the mechanism of cross-presenting and cross-priming, further explore the method of inducing DC cross-tolerance. 3. To establish allogencic heterotopic vascularized heart transplantation model in mice. To investigate the mechanism of donor and recipient-derived DC treated with NF-KB ODN Decoy inducing transplant immune tolerance and prolonging allograft survival.Methods: 1. Dendritic cells were generated by culturing bone marrow precursors with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF). The activity of NF-KB in dendritic cells was blocked by ODN Decoy containing two special binding sites for NF-KB, which was proved by electrophoretic mobility shift assay (EMSA). Morphological changes of DC were observed using microscopy. The cell-surface expression of co-stimulatory molecules and MHC class I/II on DC was analyzed using flow cytometer (FCM). The capacity of stimulating allogeneic T lymphocyte proliferative reponse was determined in mixed leukocyte reaction (MLR). The effect of NF-KB on the maturation and bioimmunological activity of DC was studied. 2. Apoptosis of splenocytes was induced by UVB irradiation (200mJ/cm2) and was analyzed using FCM. UVB-irradiated allogeneic apoptotic splenocytes were co-cultured with NF-KB ODN Decoy-pretreated BM-DC for 48 hours, then generated immature tolerogeneic DC loaded with allogeneic apoptotic cells (Decoy Apo-SCs DC). The phagocytosis of DC was evaluated byFCM and fluorescence microscopy. The expression of co-stimulatory molecules was analyzed using FCM before and after DC phagocytosing allogeneic apoptotic cells. The stimulating capacity to allogeneic T-cell proliferative response was testified in primary MLRs. To determine the cross-presenting and cross-tolerance capacity of DC loaded with allogeneic apoptotic cells, C57BL/6 mice were primed with Decoy Apo-SCs DC and secondary MLRs were performed. 3. Fully allogeneic heterotopic vascularized heart transplantation was performed from normal BALB/c or C3H/HeJ (third party) donors to size-matched C57BL/6 recipient mice. To assess the effect of donor- or recipient-derived DC on allograft survival, recipient mice were given one injection of 210 cells via the portal vein at 7 days before the heart transplantation in the absence of immunosuppression. Animals in this study were divided into 7 groups according to the pretreatment of various DC: (DControl group: only received the pretreatment of PBS (0.2ml) via the portal vein 7 days before heart transplantation; 〥C group: received the pretreatment of donor BM-DC untreated with NF-KB ODN Decoy; 〥ecoy DC group: received the pretreatment of donor BM-DC pretreated with NF-KB ODN Decoy; 瓵po-SCs DC group: received the pretreatment of recipient DC loaded with donor-derived apoptotic splenocytes (Apo-SCs) and untreated with NF-KB ODN Decoy; 〥ecoy Apo-SCs DC group: received the pretreatment of NF-KB ODN Decoy-pretreated recipient DC loaded with donor apoptotic splenocytes (Apo-SCs); ︰nited infusion group: combined pretreatment of donor Decoy DC and recipient Decoy Apo-SCs DC; ㏕hird party donor group(3rd party group): C3H/HeJ mice used as donor, recipients (C57BL/6) were pretreated as the sixth group as above. Graft survival was assessed daily by transabdominal palpation of the transplant heart. Rejection was defined by the cessation of heartbeat and further confirmed by histological analysis. The expression of intragraft cytokine genes (IL-2, IL-10, and INF-y mRNA...