Study On Treg Immune Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation And Mechanism Of ZFP36L2 In Regulating Acute Graft-versus-host Disease

Objective: The high incidence of acute graft-versus-host disease(aGVHD)after allogeneic hematopoietic stem cell transplantation(allo-HSCT)and the damage to the target organs seriously affect the patients’ prognosis.Regulatory T cells(Treg)are a group of cells that regulate immune homeostasis and maintain immune tolerance,the immune reconstruction of Treg plays an important role in controlling aGVHD.We explored the mechanism of Treg regulating aGVHD from three aspects: clinical study,single-cell RNA sequencing and in vivo experiment of a mouse aGVHD model,aiming to analyze the mechanism of immunosuppressive function of Treg cells in aGVHD and find a target gene for regulation.Methods: Firstly,283 retrospective and 63 prospective cases of allo-HSCT in our center were included.The prospective cases were randomly divided into the training set and the validation set.The dynamic changes of lymphocyte subsets and Treg subsets within 3months after transplantation were analyzed.Logistic regression models were constructed with the retrospective cases and the training set,and their predictive effects on aGVHD were compared with the validation set.Then,the peripheral blood collected from allo-HSCT patients were divided into the aGVHD group and the control group.CD3+T cells were sorted from the peripheral blood for single-cell RNA sequencing analysis.The functional changes of Treg cells in aGVHD were emphasized,and the target gene of Treg cells regulating aGVHD were screened.Finally,a mouse model of aGVHD was constructed,and the target gene was verified in the target organs of aGVHD through experiments in vivo.Also,we explored the mechanism of the target gene in regulating aGVHD,and provided experimental basis for its clinical application.Results: The peak period of aGVHD was 2 weeks to 1 month after HSCT.The proportion of lymphocytes,CD3+T cells and suppressive T cells were significantly increased in aGVHD group,and the degree of elevation were correlated with the severity of aGVHD.During 2 weeks to 1 month after HSCT,the proportion of CD4+Treg decreased,turning from resting type to active type to exert immunosuppressive function;while CD8+Treg was amplified by aGVHD induction.The comparison of area under under the ROC curve(AUC)suggested that the clinical risk prediction model containing Treg had more advantages over the traditional clinical risk prediction model in predicting the occurrence of aGVHD(AUC=0.85 vs.0.56,P<0.001).In the analysis of aGVHD single-cell RNA sequencing,T cells were divided into 9 subgroups,and the proportion of Treg cells in aGVHD group was significantly higher than that in non-aGVHD group.T cell subsets in aGVHD were mainly differentiated from proliferating T cells,forming 2 differentiation paths(path I and II),and Treg cells were mainly differentiated from the later stage of path I.Pathway enrichment analysis of Treg intergroup differentially expressed genes showed that lymphocyte proliferation activation,T cell activation and chemokine-related pathways were downregulated,and protein localization mucosal pathway and ribosome signaling pathway were up-regulated.The interaction of Treg cells with CD8+ effector T cells,CD8+ effector memory T cells,natural killer cells and proliferating T cells were enhanced in aGVHD group than that in control group.The Treg cell gene set from the single-cell RNA sequencing in this study was intersected with 2 aGVHD-related gene sets from GEO database,and finally ZFP36L2 was selected as the key target gene of Treg regulating aGVHD.In the mouse aGVHD models,down-regulated expression of ZFP36L2 reflected aGVHD damages to target organs in skin,liver,spleen,small intestine,and colon tissues.The expression level of ZFP36L2 in aGVHD target organs could be increased by Rapamycin and the relative expression level of p-AKT/AKT decreased.Conclusions: The immune reconstitution of Treg cells after allo-HSCT is closely related to the occurrence and development of aGVHD.Incorporating the proportion of Treg cells into the aGVHD clinical risk prediction model could make it better to predict the occurrence of aGVHD.The immunosuppressive effect of Treg cells in aGVHD was further illuminated with the aid of single cell RNA sequencing.ZFP36L2 could be used as the target gene for Treg cells regulating aGVHD.The down-regulation of ZFP36L2 expression level is related to damages on aGVHD target organs,and its mechanism may be related to protein phosphorylation modification,which could be reversed by rapamycin to a certain extent.