Transplant Immuoregulation Of Recipient Immature Dendritic Cells Loaded With PUVA Treated Donor Spleen Cells

Objectives: 1. To establish allogeneic heterotopic vascularized heart transplant model and orthotopic liver transplant model with two-cuff technique in rat. 2. To establish the method of propagation of rat bone marrow-derived immature dendritic cells (imDC) in vitro, observe maturation and the bioimmunological characteristics of imDC. 3. To establish the method of inducing splenocyte apoptosis by 8-MOP and UVA (PUVA), measure the phagocytic capacity and the bioimmunological changes of imDC loaded with apoptotic splenocytes. To investigate the mechanism of recipient-derived imDC loaded with PUVA treated donor spleen cells inducing transplant immune tolerance and prolonging allograft survival.Methods: 1. Dendritic cells were generated by culturing bone marrow precursors with recombinant rat GM-CSF. Morphological changes of DC were observed using microscopy. The cell-surface expression of co-stimulatory and MHC molecules on DC was analyzed using flow cytometer (FCM). The efect of NF-KB on the maturation and bioimmunological activity of DC was studied. 2. Apoptosis of splenocytes was induced by PUVA and was analyzed using FCM. PUVA treated allogeneic apoptotic splenocytes were co-cultured with imDC over night, then generated immature tolerogeneic DC loaded with PUVA treated allogeneic apoptotic splenocytes (PUVA-SP). The phagocytosis of DC was evaluated by 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 MLRs. 3. Fully allogeneic heterotopic vascularized heart transplantation was performed from normal DA donor rats to size-matched LEW recipient rats. Toassess the effect of recipient-derived DC loaded with PUVA-SP on allograft survival, recipient rat were given one injection of cells intravenously at 4 days before the heart transplantation in the absence of immunosuppression. Animals in this study were divided into 5 groups according to the infusion of various cells:①Control group: only received the pretreatment of PBS (0.2ml) 4 days before heart transplantation; ②DC group: received the donor DC; ③ SP-DC group: received the pretreatment of recipient DC loaded with normal; ? PUVA-SP group: received the infusion of PUVA treated donor apoptotic splenocytes; ⑤ PUVA-SP-DC group: received the pretreatment of recipient DC loaded with PUVA treated donor apoptotic splenocytes (PUVA-SP). 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 serum cytokine (IL-2, IL-10, INF-γ,and, IL-4) and peripheral blood lymphocyte Foxp3 expression were evaluated.Results: 1. We successfully established a stable allogeneic heterotopic vascularized heart transplant model and orthotopic liver transplant model with two-cuff technique in rat. 2. Under the stimulation of GM-CSF and IL-4, a large number of DCs were propagated from bone marrow per rat. 5 day's BM-DC showed immature state in morphology and low expression of co-stimulatory molecules and MHC (CD80, CD86, and OX6) on their surface. 3.PUVA effectively induced splenocyte apoptosis, which shown almost 70% splenocytes in apoptosis analyzed using FCM. 4. After over night co-culturing with apoptotic splenocytes, DC effectively phagocytosed allogeneic apoptotic splenocytes. 5. The maturation of DC was not initiated after phagocytosing allogeneic PUVA-SP. 6. PUVA-SP DC has low ability to stimulate allogeneic T-cell proliferative response in primary MLRs. 8. The mean allograft survival time (MST) in various allograft groups (DA-LEW) is 6.7 days in Control group, 7.8 days in DC group, 5.3 days in PUVA-SP group, 5.5 days in SP-DC group, 12.4 days in PUVA-SP-DC group, respectively. The most profound effects were achieved with infusion of PUVA-SP-DC, compared with any other group. The levels of serum cytokine showed that pretreatment of PUVA-SP-DC partially down-regulated Th1 cytokines (IL-2, IFN-γ) expression, but enhanced the Th2 cytokines (IL-10, IL-4)expression.Conclusions: 1. A great quantity of DC with typical bioimmunological characteristics was propagated from rat bone marrow precursors under GM-CSF and IL-4 stimulation. 3. PUVA is a simple and effective method to induce the apoptosis of splenocytes. DC can phagocytose allogeneic apoptotic splenocytes efectively. PUVA-SP does not stimulate the maturation of DC. DC, which loaded with allogeneic apoptotic cells, induced allogeneic T-cell hyporesponsiveness and up-regulation of Foxp3. 4. The technique of abdominal double vessels anastomose successfully established stable heterotopic cardiac allograft model in rat. Infusion of PUVA-SP-DC interfered with the indirect allorecognition, prolonged cardiac allograft survival, induced cytokine secretion prone to Th2, and increase the number of regulatory T cells.