| Induction of allograft immune tolerance has been the focus in the field of organ transplantation now. Allograft tolerance induced specific immune tolerance to organ allograft derived from the bone marrow donors which means host immune system appears no or low responsiveness to donor antigens, but normal responsiveness to the third antigens. It is distinguished from induced by immunosuppressive agents and immune defects. A widely variety of host preconditioning regimens have demonstrated the feasibility of the induction of hematopoietic chimerism and donor specific immune tolerance across the histocompatibility barrier in laboratory animals including mice, rats, dogs, and monkeys. However, most of these regimens failed to result in stable chimerism and long-term specific tolerance in human beings.Objective(1)With comparing of varieties of preconditioning regimens, we select a best way which should be low-toxic, suitable to clinical experimental practice .We also demonstrated the powerful theoretical and experimental proofs to the further clinical application of this protocol.(2)We evaluated the roles of anti-donor antigen host mature T cells in acute rejection, engraftment of donor hematopoietic cells and induction of tolerance at the stage of allograft, which would be highly instruction in clinical application. This project includes the following two parts:1.comparing of varieties of preconditioning regimens in induction of long-term specific tolerance to allogeneic heart grafts---a preclinical experimental researchMethods the recipients, BLAB/C mice(H-2Kd),were preconditioned with varieties of combination of TBI,ATS and infusion of bone marrow cells(5.0× 106)from donor C57BL/6 mice(H-2Kb) and divided into 11 groups and then neonatal donor heart allograft were transplanted into the pinna of the ear of recipients without any other immunosuppressive agents. Heart graft were monitored daily for visible contraction under microscope(magnification,×10),and survival was based on the time interval until contraction stopped. Heart grafts stopped beating was defined as rejection. Chimera in peripheral blood of hosts were routinely analyzed by Flow Cytometry on day 28 after BMT.Results:(1) Comparison of heart graft survival time(days) among group:9 of 10 heart graft in hosts preconditionated with combination of ATS+sublethalTBI(450cGy)+BMT survived for more than 100 days, and 1of 10 heart grafts in the group survived for 69 days. heart grafts survival time was non-significantly different between ATS+TBI(450cGy)+BMT group and lethal TBI(800cGy)+BMT group(p>0.05).Heart graft survival time in hosts treated by TBI(450cGy)+BMT was 22.1±9.4 days averagely, which was significantly different from that in the previous two groups(p<0.001).Hosts administered by ATS,ATS+TBI(450cGy), TBI(450cGy) or ATS+BMT rejected their allogeneic heart grafts within 45 days after heart transplantation.(2) Analyses of chimerism in peripheral blood of hosts: All of hosts preconditioned with TBI(450 or 800cGy)+BMT and ATS+TBI(450cGy)+BMT got complete donor-cell chimera in blood on day 28 after BMT, which chimeric indexes were more than 99% and non-significantly different among three groups(p >0.05).But the percentage of donor T cells in group of TBI(800cGy)+BMT and ATS+TBI(450cGy)+BMT was significantly higher than that of group of TBI(450cGy) +BMT(p<0.01),which demonstrated that preconditioned with TBI(800cGy) or ATS+TBI(450cGy) facilitated engraftment of donor T cells. No chimera was detected 28 days after BMT in hosts treated by ATS+BMT(no TBI),which implied that hosts preconditioned with ATS only resulted in loss of chimera at early stage after BMT. In control(no BMT),on day 28 after heart transplantation, percentage of host T cell in the blood of hosts treated by TBI(450cGy) was significantly lower than that of group ATS(no TBI) (p<0.01).TBI is effective in killing host T cells when hosts expose to irradiation. 2.The role of host mature T cells in induction of immune tolerance at the early stage of post transplantationMethods:(1) BALB/C(WT) recipients were divided into three group, and preconditioned respectively with TBI(800cGy)+BMT, TBI(450cGy)+BMT, ATS+TBI(450cGy)+BMT. We examined chimera in peripheral blood,spleen,thymus of hosts on day 7,14,21,28,42 after BMT.(2) Recipients,C57BL/6 TCRα-KO and wild-type C57BL/6,were treated by the same preconditioning regimen TBI(450cGy)+BMT, and then performed heart transplantation from BALB/C donor. We checked chimera in the peripheral blood of recipients on day 28 after BMT.Results:(1) On day 7 after BMT: In TBI(450cGy)+BMT group, some of host T cells still survived in peripheral blood and spleen, and a lot of host cells in thymus; In TBI(800cGy)+BMT and ATS+TBI(450cGy)+BMT groups, host T cells were very few in peripheral blood and spleen, and chimeric index of donor cells was 30% in thymus.(2) All of C57BL/6 TCRα-KO hosts treated by TBI(450cGy)+BNT regimen accepted allogeneic heart graft,and got complete chimera of donor cells. But wild-type C57BL/6 hosts treated by the same regimen rejected allograft which survived for 23.9±13.3 days, and produced unstable chimerism, average chimeric index 18.6%. Both of graft survival time and chimeric index in C57BL/6 TCRα-KO hosts were significantly different from that in wild-type C57BL/6 hosts(p <0.001).Conclusion :(1) Maintenance of donor hematopoietic chimerism was an important factor in induction of long-term, stable donor-host specific immune tolerance. (2)Depletion of anti-donor-antigen host mature T cells at early stage of transplantation not only protected against acute rejection, but also facilitated donor bone marrow cell engraftment and creation of stable chimerism that produced central immunologic cell clone deletion and induced long-term specific immune tolerance. It will be very important for non-myeloablative preconditioning regimens to result in mixed chimerism after BMT and induce immune tolerance in clinical transplantation. (3) Sublethal irradiation therapy is one of the most effective, economical treatments that deplete host mature T cells and facilitate donor hematopoietic engraftment and creation of chimerism. (4) In our experiment, pre-transplant treatment of ATS+sublethalTBI+BMT was the most effective among varieties of non-myeloablative host-preconditioning regimens using different precondition of TBI, ATS and BMT. It can induce long-term, stable. donor-specific immune tolerance in mice and didn't result in any complication, such as GVHD, infection and rejection. The reasonable schedule of treatment time makes it more potential applicability in clinical transplantation.
|
PDF Full Text Request