Establishment Of A Mixed Donor Chimeric Mouse Model Of Allogenic Bone Marrow Tranplantation And Its Realated Reseaarch

Objectives:To construct a mouse mixed donor chimerism(MDC)model of nonmyeloablative allogeneic bone marrow transplantation(Allo-BMT),explore its influencing factors and build a relative mathematical modeltoexplore the mechanism of donor immune cells augmenting hematopoietic stem cell(HSC)engraftment.Methods:The MDC model was constructed by nonmyeloablative Allo-BMT followed by high-dose posttransplant cyclophosphamide(PTCY).The retrospective study was performed on 123 mice during the construction process.The univariate and multivariate logistic regression analysis was performed on the related factors affecting the chimerism.A multivariate linear regression was constructed by R project to obtain a mathematical model for predicting the chimeric level with relevant influencing factors.Single-cell sequencing analysis was used to compare the MDC and the full donor chimerism(FDC)after DLI and describe the changes in cytokines,thedifferences in bone marrow mononuclear cells,and CD3+T cell subsets in recipient mice.Results:The model presented mixed chimerism on day 14 post-transplantation,which was characterized by a donor lymphocyte infusion(DLI)infusion on day 15 that significantly promoted donor engraftment,whereas PBS control group failed.There were 47[38.21%]and 76[61.79%]of MDC and non-MDC in 123 mice,respectively;univariate analysis showed that the baseline weight of mice(p=0.001,17.87[17.10-18.40]and 18.41[17.88-18.96]),total body irradiation(TBI,p=0.048)and the use of cyclophosphamide(p=0.16)affected the chimeric state of the mice,while the number of cells transplanted and the time of detection had no significant effect.Multivariate regression analysis showed that under certain conditions,mice weight in day0 was an independent factor affecting chimeric levels(OR=0.493,95%CI 0.307-0.791,p=0.003).Through R project multiple linear regression showed that chimerism=6.09-12×weight(g)+80.03×TBI(Gy)-4.4×cell-counts(×10~7)+0.38×CTX(mg/kg),R~2=0.5841,p<2.2e-16.Compared with MDC,single-cell analysis showed that CD8+T cell and DC werethe dominant cell subsets in the bone marrow of the FDC recipient mouse;multiple cytokine expression levels were increased in peripheral serum,including CCL5,IL-27 and IFN-γ.The cell that express IL-27 most were DC cells in FDC mouse,and its ligand IL-27Ra were mainly expressed on the surface of CD8+T cells;DC cells that express IL-27 were also highly expressed T cell co-stimulatory factors ICAM1 and CD83.Conclusions:This study established a method for constructing a mixed chimeric mouse model and built its mathematical model with relevant influencing factors.And the mixed chimerism followed by DLI or not could presented different outcomes,which effectively simulated the clinical DLI intervention process when it comes to a reduction of chimerism after allo-BMT.Further analysis by single-cell technology suggests that DC may participate in the process of T cellspromoting donor engraftment by secreting IL-27.