Effects Of Ly49A Transfected Lymphocytes On Experimental Graft Versus Host Disease Post Allogeneic Bone Marrow Transplantation

Graft versus host disease (GVHD) is caused by immunocompetent cells in the allograft, which recognize the hoat's antigens and do harm to the host?tissues. Deletion of the T cells in the allograft and the use of immunosuppressive agents will certainly reduce the morbidity of GVHD, but at the same time, will also contribute to the relapse of leukemia, the failure of transplantation and the infections. No way has been found yet to resolve the problem totally. In recent years the discovery of killer cell inhibitory receptors (KIR) has shed light on the negative regulation of the functions of NK and T cells and also the resolution of GVHD. KIRs express on all NK cells and a portion of T cells. When binding with corresponding MHC I molecules, negative signal is transducted and the cytotoxity of NK and I cells are abrogated. In this research we planed to transfect the Ly49AcDNA into the donor's lymphocytes and transplant them with bone marrow cells in order to find a new way for reducing GVHD but retaining GVL effect after allogeneic bone marrow transplantation. The possibility of the detachment of GVHD and GVL from the aspect of molecular mechanism of the interaction between KIRIMHC I was also discussed. The following methods were adopted in this research: (1) Ly49AcDNA presented by Prof. Raulet was sequenced. The results were compared with the reports in GenBank. After the confirmation of the correction of the ?? sequence, the expression vector pLXSN-Ly49A was constructed. (2) After being packaged by PA3 17 cells, spleen lymphocytes of C57BL/6 mice were transfected. Flowcytometer was used to detect the expression rate of Ly49A receptor; MTT method was applied to assay the killing effects of those lymphocytes cells on fibroblast cells and 4T1 cells of BALB/c origin. (3) The erythroleukemia cells EL9611 H-2d were injected into CB6F1H2d/b mice to establish the FlH2dIb erythroleukemia model, providing the test animal model for detecting GVL effect after halploidentical allogeneic bone marrow transplantation. (4) In the case of the murine model of fully mismatched allogeneic acute GVHD: the donor was C57BL/6H2b, the recipient was BALB/cH2d. After the irradiation (TBI, 60Co 9.OGy) the recipient accepted the transplantation of mixed spleen cells and bone marrow cells to establish the GVHD model. (5) In the case of the murine model of haploidentical allogeneic acute GVHD: the donor was C57BL/6H2b. The recipient was BALB/c×C57BL/6 FlH2d/b (CB6F1). After the irradiation (TBI, 60Co 1O.5Gy) the recipient accepted the transplantation of mixed spleen cells and bone marrow cells to establish the GVHD model. On the basis of these models, the effects of Ly49A transfected spleen cells on GVHD and GVL post fully mismatched allogeneic or haploidentical allogeneic bone marrow transplantation were detected. Results were shown as the followings: (1) The Ly49AcDNA presented by Prof. Raulet was the same as that reported in GenBank. (2) The expression rate of Ly49A receptor was (46.67±0.35)% for lymphocytes transfected with pLXSN-Ly49A; (18.73±O.85)% for lymphocytes transfected with pLXSN and (19.60±0.27)% for untransfected control. The killing effects of Ly49A transfected lymphocytes on 4T1 tumor cells remained almost the same as that of the untransfected control (P>0.05), ?8 ? but the kil...