Experimental Study Of Nonmyeloablative Bone Marrow Transplantation To Induce Tolerance In Allogeneice Mice

Objective: To explore the effect and mechanisms of immune tolerance induced by"total body irradiation(TBI)+cyclophosphamide(CTX)"regimen combined with intra-bone marrow(IBM) injection of allogeneic bone marrow cells(BMCs).Methods: On day 0 recipient C57BL/6(H-2b,B6) mice received a sublethal dose of TBI(60Coγ-ray) 550cGy followed by IBM bone marrow transplantation(BMT) of 3×107/30μl BMCs from BALB/C(H-2d) mice in 4 hours, and CTX(200mg/kg) was given intraperitoneally(i.p.) 2 days later. The experimental animals were randomly divided into three groups: Blank group(Group A): experimental mice received no treatment; Control group(Group B): experimental mice received TBI+CTX; Experimental group(Group C): experimental mice received TBI+CTX+IBM-BMT. Skin grafting and mixed lymphocyte reactions(MLR) were used to check the status of immune tolerance, and the tolerance mechanisms were investigated by IL-2 reverse test, adoptive transfer assay in vitro and the chimeric analysis. Donor lymphocytes infusion(DLI) was given to mice in group C 150 days after IBM-BMT to investigate it's effect on chimerism.Results: With or without IBM-BMT, none of the mice died during the experiment and no mice in group C suffered from acute and chronic graft-versus-host disease(GVHD).The mean survival time(MST) of skin allografts from BALB/C in group C was significantly longer(>300 days)than that of other groups (P<0.001), but the MST of skin allografts from KM was found no difference among three groups(P>0.05).On day 90 after IBM-BMT, the phenotypic character of the recipient mice(black color) began to convert to that of the donor mice(white color). The MLR demonstrated the immune response of recipient mice was down-regulated and could be reversed by the addition of IL-2. Suppressive activity in the spleen cells of tolerant B6 mice was observed in adoptive transfer assay in vitro, and a high percentage of donor-derived cells was observed in the bone marrow of tolerant B6 mice onday 45,90, but the chimeric degrees of the tolerant mice decreased gradually with the time, and DLI can sustain a high percentage of mixed chimerism.Conclusions: The nonmyeloablative regimen in combination with IBM-BMT can induce a stable, long-term immune tolerance status, no GVHD was found during the experiment, DLI can sustain the degree of chimerism, and multiple mechanisms including clonal anergy, suppressor cells and mixed chimerism may be involved in the development of donor-specific immune tolerance.