Establishment Of A Mouse Model Of Skin-dominant Chronic Graft Versus Host Disease And Characteristic Analysis Of T Cell Subsets

BackgroundHematopoietic stem cell transplantation(HSCT)is currently an important means to treat malignant hematologic diseases,but the occurrence of graft versus host disease(GVHD)after transplantation seriously affects the survival and quality of life.In particular,the incidence of chronic GVHD(c GVHD)is as high as 30%to 70%,of which the skin is the most common target organ.Due to the complex pathogenesis and lack of targeted treatment,it is difficult to achieve ideal therapeutic effect.c GVHD mouse models are often constructed for basic research,but it is very critical to select appropriate models for different research purposes.Currently constructed c GVHD mouse models are often accompanied by acute graft versus host disease(a GVHD)and the involvement of multiple target organs in c GVHD,which may affect the research results of skin c GVHD.ObjectiveThe purpose of this study is to construct the mouse model of skin-dominant c GVHD,and explore the characteristics and analyze T cell subsets in this model.MethodsFor the construction of c GVHD mouse model,lethal dose irradiation was used to simulate myeloablative condition and bone marrow cells from donor mice were transplanted to simulate the process of stem cell transplantation(BM group).Meanwhile,spleen cells were transplanted to simulate the occurrence of c GVHD(c GVHD group).Because of the same major histocompatibility complex(MHC)and different minor histocompatibility antigens(mi HAs)between donor and host mice,the constructed mouse model developed slowly and was similar to human c GVHD.In this study,B10.D2 mice were used as donors and BALB/c mice as hosts to construct the skin-dominant c GVHD mouse model.The occurrence of c GVHD was evaluated by observing the skin appearance and the thymus function.The basic characteristics of this model were evaluated by survival,arched back,diarrhea and body weight.The effects of c GVHD on skin,liver,lung and intestine were evaluated by tissue sections.The cell subsets of skin,lung and liver were analyzed by flow cytometry.In order to avoid the limitation of this single model,other c GVHD mouse models were constructed to verify the conclusions.Due to the differences between mice and humans,the humanized GVHD model was also constructed,and the changes of human cells in GVHD were compared.ResultsIn this study,the skin-dominant c GVHD mouse model was successfully constructed by transplanting bone marrow cells(10×10~6)and spleen cells(18×10~6)from B10.D2 mice to lethal dose irradiated BALB/c mice.Mice in BM group could gradually return to normal after transplantation,excluding the influence of experimental factors on mice in c GVHD group.Mice in c GVHD group mainly showed skin lesions,including hair depilation,scar and skin shrinkage,which began to appear 20 days after transplantation and gradually became worse over time.Compared with the normal state of BM group,the survival rate and body weight of c GVHD group decreased gradually,and the arched back and body weight scores increased simultaneously,but there was no significant difference in diarrhea scores,suggesting no intestinal c GVHD.The tissue sections of skin,liver,lung and colon showed that only skin fibrosis and collagen deposition were obvious in c GVHD group,and the pathological scores of skin were significantly increased.Therefore,skin was the main target organ in this model and showed typical c GVHD manifestations.The results of flow cytometry showed that Th1,CD4~+IL-13~+T cells and Tc1 increased significantly,which was closely relevant to the pathogenesis of c GVHD.Further analysis revealed that CD4~+IFN-γ~+IL-13~+T cells were present in CD4~+T cells.Single cell transcriptional analysis of CD4~+T cells showed that the cells in BM group tended to normal state,while in c GVHD group proliferated and differentiated in entirely different directions.It was also found that there were two directions of differentiation in CD4~+IFN-γ~+IL-13~+T cells.Therefore,it was speculated that CD4~+IFN-γ~+IL-13~+T cells had heterogeneity,and the cells only existing in c GVHD group might be closely related to the occurrence of c GVHD.After the knockout of IL-13,the expression of IL-4 was significantly increased in CD4~+T cells and IL-4 was co-expressed with IFN-γ.The humanized GVHD mouse model was constructed and CD4~+INF-γ~+IL-4~+T cells were found,reflecting the difference between human and mouse.ConclusionIn this study,the skin-dominant c GVHD mouse model was successfully constructed.The skin c GVHD was the main manifestation.Th1,CD4~+IL-13~+T cells and Tc1 were closely related to the pathogenesis,and CD4~+IFN-γ~+IL-13~+T cells were observed.However,CD4~+IFN-γ~+IL-4~+T cells appeared in the humanized GVHD mouse model,reflecting the difference between mice and humans.