Effects Of Adenovirus Mediated CTLA4Ig Gene Transfer Combining With Donor Specific Cell Transfusion On Induction Of Mice Skin Allograft Tolerance

Graduate student: Zhang Haibin Supervisor: Prof. Xiao XurenTo induce donor-specific tolerance is one of the ultimate solutions to the problem of immune rejection in clinical organ transplantation. Although many protocols have been tested to achieve this goal and some of them have succeeded in animal transplantation models, up to now, few of these protocols are applicable to clinical use. Among all of these protocols, combining blockade of co-stimulatory signals with donor specific bone marrow transplantation to establish allogeneic mixed chimerism is the one that has the best prospect to be used in the clinic. During recent years, protocols of transferring CTLA4Ig gene to block B7/CD28 co-stimulatory signal have proved to be able to induce organ allograft tolerance in many animal models. This project constructed a recombinant adenovirus vector that can express CTLA4Ig, explored the effects of adenovirus mediated CTLA41g gene transfer combined with donor specific cell transfusion on induction of skin allograft tolerance between fully MHC dismatched mice strains. Meanwhile, the mechanisms of this protocol to induce transplantation tolerance are also studied on cellular and molecular level. The conclusions are following:I. The CTLA4Ig cDNA was subcloned into adenovirus shuttle plasmidpCA13. Recombinant adenovirus vectors were obtained by cotransfection of 293 cells with this shuttle plasmid and adenovirus backbone plasmid pJM17. RT-PCR and Western-Blot analyses demonstrated that these vectors are the right recombinant Ad/CTLA4Ig that can express CTLA4Ig.2. To B6 recipient mice that were injected with Ad/CTLA4lg 2X 10SPFU through caudal vein, both RT-PCR and ELISA analysis could detect the in vivo expression of CTLA4Ig gene within 24 hours. 7 days after injection, the expression could be detected in much higher level. The in vivo expression of CTLA4lg could last more than 30 days.3. On the same day of BALB/c to C57BL/6 skin transplant, 2X108PFU Ad/CTLA41g and 1 X 107 donor specific spleen cells were injected into B6 recipient mice through caudal vein. 1 X 107 donor specific bone marrow cells were also injected into recipient mice through caudal vein on day 5. Survival time of skin grafts were recorded. MLR and DTH against donor and third part alloantigens were assayed to test tolerance status. Furthermore, the effect of exogenous IL-2 on MLR of tolerant mice, chimerism analysis and cytokine mRNA detection were performed to explore tolerance mechanisms. The results showed that donor-specific long-lasting skin allograft tolerance was achieved in mice treated with Ad/CTLA4Ig, donor specific spleen cells transfusion (DST) and donor bone marrow transplantation (BMT), the mean survival time of skin grafts was 34.8d. To recipient mice treated with this protocol, the MLR and DTH assays against donor alloantigen were specifically suppressed. Exogenous IL-2 could partly reverse the hyporesponsiveness of MLR of tolerant mice. Peripheral mixed chimerism could be detected for more than 30 days in the tolerant mice. Difference of Thl/Th2 cytokine profile between the tolerant mice and the blank control mice was not found.4. When the protocol of Ad/CTLA4Ig+DST+BMT was joined with intraperitoneally injection of 25 mg/kg Busulfan (BU) to recipient mice on day 4, the effects of this protocol in inducing donor specific skin graft tolerance and establishing allogeneic mixed chimerism were significantly enhanced. The mean survival time of donor skin grafts was extended to 69.8 d. Peripheral chimerism level was elevated and the chimerism state lasted more than 60 days. Intra-thymus chimerism could also be detected in recipient mice of this group.