The Study Of Engraftment Of Haplo-HSCT Enhanced By Transfusion Of Donor Cells Before BMT

Background Hematopoietic stem cell transplantation (HSCT) is one ofthe most effective therapy for patients who are suffering from hematologicalmalignant. However, transplantation of allogeneic graft produces two-wayrejections, namely, graft versus host and host versus graft reaction, due to thedispatrty of MHC or HLA between individuals and biological species. Becauseof the significant disparity of HLA between donor and recipient inhaploidentical transplantation, large dose of chemotherapy beforetransplantation is needed to overcome the severe reject reaction, exceptdepletion of T cells in grafts and megadose of G-CSF mobilized hematopoieticstem cells. It is reported that nonmyeloablative hematopoietic stem celltransplantation (NST) reduces transplantation-relanted toxicity. So this treatmentconsists with modern therapeutic thinking. Nevertheless, NST or mismatched-HSCT reduced the intensity of conditioning combining withdisparity of HLA. They always induced graft rejection and transplantationfailure.As to be known, T cells and natural killer cells are the main factorsmediating allo-rejection. It will be important to eliminate them or induceimmune anergy for alloengraftment. It has been shown that depletingdonor-alloreactive T lymphocytes can indeed reduce the incidence of GVHD,indicating that eliminating of alloreactive cells definitely reduces allogeneicrejection. So we presume that selectively depleting host alloreactive Tlymphocytes in vivo maybe enhance engraftment. We scheme a novel regimenof transfusing donor cells before BMT followed by chemotherapy to eliminatehost alloreactive T cells and induce immune tolerance. Cytosine rabinoside(Ara-c) is a kind of antimetabolites of miazines and mainly affects the cell cycleof S stage. Alloreactive cells are sensitive to it. It has been shown that cytotoxitywill reach peak when T cells are exposed to alloantigen. We intensionly inducedhost alloreactive T cells directely against donor cell antigen by transfusion ofdonor cell before BMT. Then host alloreactive T cells are eliminated beforecellular immunity reaches peak by interperitoneal injecting Ara-c. So when itwas exposed to the same alloantigen within short time, host will be tolerant as aresult of the immunity directly against the antigen, which has been relativelyexhausted.SectionⅠ The model establishment of haploidentical bonemarrow transplantation using nonmyeloablative conditioning Objects Establish mice model of haploidentical BMT usingnonmyeloablative conditioning, to provide a platform for the study of immunetolerance induced by transfusion of donor cells before HSCT.Methods Mice were devided into two groups, experimental and control.All female CB6F1recipients were exposed to a dose of450cGy total bodyirradiation (TBI), followed by intervenously injection5×107nucleated bonemarrow cells (BMC) from male C57BL/6mice or RPMI1640medium. Thenwe monitored the recoverment of hemogarm and engraftment by detection ofsex determining gene (SRY) and donor cell chimerism especially in the CD3+cells. At the same time we monitored the signs of GVHD.Results No mice showed significant signs of GVHD or dead. The valueof white blood cells can recover to normal level within thirty days aftertransplantation. SRY can be observed in recipient peripheral blood bypolymerase chain reaction (PCR). Donor cells chimerism in lymphocytes,monocytes and granulocytes at days14,30and60after BMT were23.8%±4.6%,36.9%%±13.7%,19.4%±7.5%vs49.9%±3.6%,53.2%±7.6%,54.4%±4.8%vs67.6%±3.1%,51.6%±4.3%,56.9%±2.9%, respectively. In CD3+cells, chimerism rates were4.4%±4.6%,21.2%±6.4%,54.4%±4.1%,respectively.Conclusion The nonmyeloablative conditioning of single dose of450cGy TBI can induce immune tolerance and donor bone marrow cellsengraftment with a lower level chimerism.Section Ⅱ The study of engraftment of haplo-HSCT enhancedby transfusion of donor cells before BMT Objects To observe whether transfusion of donor cells before BMT caninduce immune tolerance and enhance donor bone marrow cells engraftment.Methods Mice were devided into three groups, pretransplanting spleencell, pretransplanting BMC and control. Three days before BMT, spleen or bonemarrow cells from male C57BL/6mice were transfused intravenously intofemale CB6F1recipient followed by a dose of Ara-c0.75g/kg at24h and48h.The next day, recipients were exposed to a dose of450cGy TBI. On the day ofBMT recipients were injected intervenously5×107nucleated bone marrow cellsfrom C57BL/6mice. Then we monitored the recoverment of hemogarm, donorcell chimerism of T cells and signs of GVHD.Results Each group had two mice dead and all of them show disparitysigns of graft-versus-host disease. Rapid white blood cell engraftment wereobserved in group of pretransplanting SC and group of control at day17(from12d to37d), while the group of pretransplanting BMC was slightly slower at day22. The chimerism of T cells from group of pretransplanting SC was as high asthat of control. At days30after BMT, the chimerism of pretransplanting SC was93.5%±4.8%, significantly higher than that of pretransplantating BMC (P<0.05).At days60, the chimerism of pretransplanting BMC was rised to87.2%±7.4%,as high as that of control groups (94.1%±2.5%, P>0.05).Conclusion It seems that giving donor lymphocytes cells before BMTcan enhance haploidentical bone marrow cells engraftment.