Effect Of Anti-CD3Monoclonal Antibody In Combination With Busulfan And Fludarabine Conditioning Regimen On Preventing GVHD In Mouse Haploidentical Hematopoietic Stem Cell Transplantation

Objective：To investigate the effect and mechanism of anti-CD3monoclonalantibody in combination with busulfan and fludarabine conditioning regimenpreventing GVHD in mouse haploidentical hematopoietic stem cell transplantation.Methods：A parents�'F1haplo-HSCT mouse model was set up by using the CB6F1mice(the generation of BALB/C and C57BL/6)as the recipient and C57BL/6mice asthe donor.CB6F1mice were assigned to three groups:mice in anti-CD3group wereconditioned with Anti-CD3,busulfan and fludarabine, and tranplanted withsplenocytes and TCD bone marrow cells; mice in SPL group were preconditionedwith busulfan and fludarabine and transplanted with splenocytes and TCD bonemarrow cells; mice in BM group were preconditioned with busulfan and fludarabineand only injected with TCD bone marrow cells. Flow cytometry was used to confirmthe engraftment of donor-derived lymphocytes and bone marrow cells. GVHD wasmonitored by the loss in total body weight、clinical scores and the survival of eachgroup. Histology of the skin, small intestine, liver and lung were used to analysis theseverity of GVHD. Also, the percentage of donor T cells in GVHD target organswas determined by flow cytometry.Results：①Optimized preconditioning regimen:We found busulfan15ug/g andfludarabine10ug/g twice a day for4days were good for Haplo-HSCT model. A doseof Anti-CD35ug/g twice (at thirteen day and eight day before transplantation) can beused to condition the recipient.②Establishment of Haplo-HSCT aGVHD mousemodel: we preconditioned the mice as previously described, donor lymphocytes and TCD bone marrow could induce lethal GVHD, including body weight lost, hunchback, diarrhea, and hair lost. Engraftment was confirmed by flow cytometry,measuring donor-derived CD4-Positive lymphocytes、CD8-positive lymphocytes andB cells. In the Haplo-HSCT aGVHD model, we observed donor T cells seriousinfiltration and tissue damage in GVHD target tissue.③The effect of anti-CD3precondition in preventing GVHD:the survival in Anti-CD3group was higher thanSPL group, as well as lower clinical score. Furthermore, we observed that anti-CD3preconditioning markedly reduced infiltration and tissue damage in GVHD organs.④the mechanism of anti-CD3preconditioning preventing GVHD: we observed thatanti-CD3preconditioning markedly reduced infiltration of donor T cells in peripherallymphoid tissue. Also, a lower expression of in donor T cells was found in themesenteric lymph node..Conclusion：1. Preconditioning with anti-CD3monoclonal antibody can prevent GVHDeffectively in a haplo-HSCT model.2. Anti-CD3monoclonal antibody prevent GVHD is associated with the inhibition ofdonor T cells infiltration to the target tissues of GVHD.