Inhibition Of Chronic Allograft Rejection By Blockade Of Indirect Recognition

Objectives: 1. To establish the method of culturing and propagation of the recipient mouse bone marrow-derived dendritic cells(BM-DC) in vitro and verify its validity of donor soluble antigen presentation. Also to investigate the effect of the inhibition of the one-way mixed lymphocyte reaction (MLR) by B7 antisense peptide(B7AP) and find the most suitable B7AP concentration of marked inhibition to one-way MLR. 2. To investigate donor-specific immune hyporesponsiveness induced by the B7AP-pretreated donor antigen-loaded recipient derived DC using the the response system of the recipient-donor indirect pathway in vitro, then to observe the validity in vivo and analyze the mechanism initially. 3. To investigate the effect and its regional mechanism of chronic rejection of the transplanted artery via the blockade of indirect recognition after establishing the allogeneic murine model of carotid artery orthotopic transplantation.Methods: 1. The recipient C3H BM-DC were generated by culturing bone marrow precursors with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and tumor necrosis factor-alpha (TNF-α). BM-DC were identified by the cell morphous,function and the expression of the molecules on the cell surface. In the culturing process, donor C57BL/6 soluble antigens were added, then the validity of the recipient DC loading the donor antigen was investigated by the one-way MLR using the spleen T cells of the recipient C3H and the cultured recipient DC. Also the MLRs of the cultured recipient DC and the T cells of the recipient spleen were performed after the cultured recipient DC were incubated with B7APs of different concentrations, to display the curve of the logarithm of the B7AP concentrations and the CPMs of the MLRs and to find the most suitable concentration of the B7APs associated with marked inhibition on the one-way MLR. 2. The response system in vitro of the donor antigen indirect presentation to the recipient was established. The primary one-way MLR of the B7AP-pretreated donor antigen-loaded recipient DC and the recipient spleen T cells were taken , then the secondary MLR of the collected recipient T cells after the primary MLR and the donor-antigen or the third antigen loaded recipient DC or donor DC to investigate its immunological competence. Also the recipient spleen T cells were isolated after the recipient was pretreated by the B7AP-pretreated donor antigen-loaded recipient DC and the MLR was taken with the donor antigen or the third party antigen loaded recipient DC or donor DC to identify the vadility of the B7AP-pretreated donor antigen-loaded recipient DC in vivo. Meanwhile the cytokine genes(IL-2, IL-10, and INF-γmRNA) and the lymphocyte apoptosis of the recipient spleen were analyzed to find the other possible mechanism of the immune tolerance initially after stimulated by the donor antigen in the earlier period or 2 months later. 3. The reciepient was pretreated by B7AP-blocked donor antigen-loaded recipient DCs, then the allogeneic murine model of carotid artery orthotopic transplantation was established (C57BL/6→C3H) . The transplanted artery was harvested to be analyzed pathologically after 2 months. Also the TGF-β1 immunohistochemistry analysis of the transplanted artery were performed to find the regional mechanism of the immune tolerance.Results: 1. Under the stimulation of the rmGM-CSF and TNF-α, large number of the mature BM-DC were propagated from the mouse bone marrow. They match with the DC morphologically, highly express the CD80,CD86n MHC- II on their surface and stimulate the proliferation of the other strain's T cells intensively. 2. The recipient DC which were cultured by adding the donor antigen could stimulate the proliferation of the recipient T cells significantly. 3. B7AP-pretreated donor antigen-loaded recipient DC could effectively inhibit the proliferation of the recipient T cells and it was sure that the B7 molecular could be fully blocked when the concentration of B7AP was 10mg/L or 20mg/L incubated with DC. 4. The result of the secondary MLR showed B7AP-pretreated donor antigen-loaded recipient DC could induce the immune hyporeponsiveness to the donor antigen, but immune responsiveness to the third party antigen. 5. The experiment in vivo also showed the recipient spleen T cells would hyporesponse to the donor antigen after the recipient was pretreated by the B7AP-blocked donor antigen-loaded recipient DC. Meanwhile after stimulated by donor antigen, the Th2 cytokines (IL-10) mRNA of the recipient spleen were up-regulated and the Th1 cytokine (IL-2,IFN-γ) mRNA were down-regulated, which was maintained in some time, but the apoptosis of the recipient spleen lymphocytes were not changed. 6. The recipient T cells pretreated by the blockade of indirect recognition were hyperresponsive to the donor antigen presented via direct recognition. 7. We successfully established the allogeneic murine model of carotid artery orthotopic transplantation. 8. The transplanted artery was harvested in 2 month and the intimal proliferative lesion was observed pathologically. The result showed the relative thickness of the intima pretreated was 0.071±0.034, but 0.179±0.056 of the control group. The difference was statistically significant. The TGF-β1 in the artery wall pretreated was low-expressed by the immunohistochemical analysis, which was matched with the levels of intimal proliferative lesion.Conclusions: 1. A large quantity of mature DC was propagated from mouse bone marrow precursors under rmGM-CSF and TNF-αstimulation. 2. The recipient DC could present the donor antigen effectively. 3. The donor-loaded recipient DC pretreated by the B7AP could inhibit the one-way MLR with the recipient T cells. 4. The B7AP-pretreated donor antigen-loaded recipient DC could induce the specific hyporesponsiveness to the donor antigen, which may be enhanced by the immune deviation (Th1→Th2). 5. The specific hyporesponsiveness of the recipient T cells to the donor antigen via the blockade of the indirect recognition would not take effect on its response to the donor antigen via direct recognition. 6. The B7AP-pretreated donor antigen-loaded recipient DC could effectively inhibit the intimal proliferative lesion of the transplanted artery in the allogeneic murine model of carotid artery orthotopic transplantation, and the TGF-β1 may play a regionnal role. 7. Blockade of indirect recognition by B7AP can effectively inhibit chronic allograft rejection.