| Acute leukemia（AL）is a malignant clonal disease originating from hematopoietic stem cells,which has an acute onset and a high mortality rate.AL can be divided into acute myeloid leukemia（AML）and acute lymphoblastic leukemia（ALL）.Although the remission rate of AL has increased with the continuous improvement of chemotherapy and supportive care in recent years,the long-term survival rate is still low.Severe aplastic anemia（SAA）is a group of diseases in which bone marrow hematopoietic stem cells are severely depleted due to immune abnormalities.The main features of the patient are hematopoietic stem cell damage and peripheral blood pancytopenia.At present,there are many methods for clinical treatment of SAA,but the therapeutic effect is not satisfactory.The occurrence of AL and SAA seriously threatens the life safety of patients.Allogeneic hematopoietic stem cell transplantation（allo-HSCT）is currently an effective treatment for AL and SAA.Graft versus host disease（GVHD）is the most common complication after allo-HSCT,and its occurrence greatly limits the success rate after transplantation and seriously affects the prognosis of patients.According to the time of onset and clinical manifestations of patients,GVHD can be divided into acute GVHD（acute GVHD,a GVHD）and chronic GVHD（chronic GVHD,c GVHD）.At present,the main measures to prevent GVHD include cyclosporine combined with methotrexate,the use of cyclophosphamide after transplantation,and the removal of T cells in the transplant.However,these preventive measures cannot completely prevent the occurrence of GVHD,and are accompanied by Lots of adverse reactions.Studies have shown that the mechanism of a GVHD is mainly due to the activation of donor CD4 T lymphocytes to form cytotoxic T lymphocytes,which attack host tissues and organs,resulting in host pathological damage and multi-organ involvement.The mechanism of c GVHD is more complex.At present,a large number of literatures have reported a GVHD and c GVHD after allo-HSCT in patients with hematological diseases and their target tissues and organs.However,due to the different disease courses of AML,ALL and SAA,the pathological mechanisms of GVHD after allo-HSCT are not exactly the same,so the preventive measures for GVHD for different blood diseases will also be different.At present,there are few studies on the difference of GVHD among AML,ALL and SAA.Clinical studies have shown that immune cells play a key role in the occurrence and development of GVHD,but what are the levels of T cell subsets,B cells,Treg cells and NK cells in peripheral blood of AML,ALL and SAA when a GVHD and c GVHD occur and whether there are differences in changes of immune cells among the three diseases have not been reported in detail,identifying the differences in immune cell changes when GVHD occurs in patients with three hematological diseases is an important basis for preparing targeted drugs for different hematological diseases and realizing individualized and precise treatment of GVHD.At present,it is believed that"cytokine storm"is also one of the important factors causing GVHD.Helper T cells can secrete a large number of cytokines to mediate the immune response of the body,for example:Th1 cells mainly secrete IL-2,IFN-γ,TNF-a,Th2 cells mainly secrete IL-4,IL-5,IL-10,Th17 Specifically secretes IL-17.Studies have shown that cytokines TNF-a,IL-10,and IFN-γare closely related to the occurrence of GVHD.However,the differences in the levels of these cytokines in a GVHD and c GVHD in AML,ALL,and SAA need to be further confirmed.By comparing the changes in the number of T cell subsets,B cells,NK cells,Treg cells and the levels of cytokines in the peripheral blood of AML,ALL and SAA patients who developed a GVHD and c GVHD after allo-HSCT,we elucidate the pathogenesis of different blood diseases.The pathological mechanism of GVHD after allo-HSCT provides an experimental basis for the development of specific GVHD diagnosis and treatment plans for different blood diseases,and guides the effective clinical diagnosis and treatment of GVHD.Objective1.To compare the incidence of GVHD after allo-HSCT in AML,ALL and SAA patients.2.To compare the levels of immune cells and cytokines in peripheral blood of GVHD patients with AML,ALL and SAA after allo-HSCT.3.Provide a basis for clinical formulation of specific GVHD diagnosis and treatment programs.Materials and Methods1.Research subjectsThis study enrolled 73 patients who underwent allo-HSCT in the Department of Hematology,The First Affiliated Hospital of Anhui Medical University（Hefei,China）from December 2020 to December 2021,including AML 38 cases,ALL 20 cases and SAA 15 cases,and normal adults 30 cases were selected as healthy controls.2.Preprocessing schemesAML:haploidentical,busulfan+cyclophosphamide;full congruence,busulfan+cyclophosphamide+antithymocyte immunoglobulin;ALL:haploidentical,busulfan+cyclophosphamide+semustine;full congruence,busulfan+cyclophosphamide+antithymocyte immunoglobulin+semustine;SAA:haploidentical,busulfan+cyclophosphamide+antithymocyte immunoglobulin;fully congruent,cyclophosphamide+antithymocyte immunoglobulin+fludarabine;3.GVHD prevention and treatmentHaploidentical transplant patients:cyclosporine+mycophenolate mofetil+methotrexate+low-dose post-transplant cyclophosphamideConcordant transplant patients:cyclosporine+methotrexate or mycophenolate mofetil4.Analysis of CD3T cells,CD4,CD8T cells,Treg cells,NK cells and B cell subsets in peripheral blood by flow cytometryUsing flow cytometry to analyze CD3T cells,CD4T（CD3+CD4+）cells,CD8T（CD3+CD8+）cells,Treg（CD4+）cells at 1 month,3 months,and 6 months after allogeneic hematopoietic stem cell transplantation in patients with hematological diseases Changes in the content of CD25+FOXP3)cells,NK（CD3-CD56+CD16+）cells and B（CD19+）cells.5.Analysis of the expression levels of cytokines IL10,IL-17,TGF-β1 and IFN-γin peripheral plasma by ELISAThe levels of inflammatory factors IL10,IL-17,TGF-β1 and IFN-γin peripheral plasma of patients with a GVHD and c GVHD after transplantation were detected by enzyme-linked immunosorbent assay（ELISA）.6.The relevant data obtained from the experiment were analyzed using SPSS16.0softwareGraph Pad Prism 5.0 software was used to process experimental data and draw graphs.Differences among three or more groups were compared using one-way analysis of variance（ANOVA）.Results are expressed as mean standard deviation（SD）or mean standard error（SE）.A P value less than or equal to 0.05 was considered statistically significant,and a P value less than 0.01 was considered significantly different.Results1.The occurrence of GVHD in patients after allo-HSCTThis study enrolled 73 patients with hematological malignancies,including 44 males and 29 females,with a median age of 40（14-60）years.Primary diseases included AML38 cases,ALL 20 cases,and SAA 15 cases.Among the 73 patients after allo-HSCT,25patients developed a GVHD,including 11 patients with AML,8 patients with ALL,6patients with SAA,and 10 patients with c GVHD,all of which were AML patients.2.Hematopoietic reconstruction in patients with different hematological diseases after allo-HSCTAfter allo-HSCT,all 73 patients were successfully implanted with hematopoietic stem cells.The median time of peripheral blood neutrophil recovery in AML,ALL,and SAA patients was 11（9-26）days,10（8-17）days,and 13（9-28）days,respectively,and the median time to platelet recovery was 12（9-40）days,12（9-34）days,and 11（8-32）days,respectively.3.Comparison of the degree of GVHD and target organs after allo-HSCT in three diseasesComparing the incidence of GVHD after allo-HSCT in AML,ALL and SAA,it was found that grade II-IV a GVHD was the most common in AML and ALL patients,with an incidence of 72.7%and 75.0%,respectively,while grade II-IV in SAA patients was the most common.The cumulative incidence of a GVHD and grades 0-I a GVHD was equal.Comparing the target organs of GVHD after allo-HSCT in the three diseases,it was found that the most involved organs in AML and ALL with a GVHD were the same,both of which were the gastrointestinal tract,followed by the skin and liver,while the most involved organs in SAA with a GVHD were the skin,followed by the gastrointestinal tract.4.Changes of immune cells in peripheral blood after allo-HSCT in three diseases4.1 Changes in peripheral blood immune cells of a GVHD and c GVHD in AML patientsThe results of flow cytometry showed that compared with 1 month after transplantation,the proportion of CD8 T cells and NK cells in peripheral blood increased significantly when a GVHD occurred in 11 AML patients at 3 months after transplantation;AML patients developed c GVHD at 4 months after transplantation,the proportion of CD8T cells and B cells in peripheral blood increased significantly.4.2 Changes in peripheral blood immune cells of AML patients without GVHDThe results of flow cytometry showed that compared with healthy controls,the proportions of CD3T cells,CD4T cells,Treg cells,NK cells and B cells in peripheral blood of AML patients without GVHD allo-HSCT were significantly decreased at 1month after allo-HSCT.3 months after transplantation,the proportion of CD3T cells,CD8T cells,NK cells and B cells in patients increased significantly,and basically returned to normal levels or even higher than that of healthy controls.CD4 T cells and Treg cells increased slowly,and the proportion of cells was still lower than that of healthy controls6 months after transplantation.4.3 Changes of peripheral blood immune cells in ALL patients with a GVHDThe results of flow cytometry showed that 8 patients with ALL developed a GVHD at3 months after transplantation.The proportion of CD3 T cells and CD8 T cells in patients with a GVHD was significantly higher than that at 1 month after transplantation.At 6months after transplantation,the proportions of CD3 T cells,CD4 T cells,CD8 T cells,Treg cells,NK cells,and B cells in patients with a GVHD were all reduced compared with3 months after transplantation.4.4 Changes of peripheral blood immune cells in ALL patients without GVHDThe results of flow cytometry showed that 12 ALL patients did not develop GVHD,and the proportions of CD3 T cells,CD4 T cells,Treg cells and B cells in patients were significantly decreased at 1 month after allo-HSCT compared with healthy controls.CD3T cells and CD8T cells were significantly increased at 3 months after transplantation,and the proportion of cells returned to normal levels or even higher than that of healthy controls.B cells basically returned to normal levels 6 months after transplantation,CD4T cells and Treg cells increased slowly,and the proportion of cells was still lower than that of the healthy control group 6 months after transplantation.4.5 Changes of immune cells in peripheral blood of a GVHD patients with SAAThe results of flow cytometry showed that 6 SAA patients developed a GVHD at 3months after transplantation,and the proportion of CD8 T cells in SAA patients with a GVHD was significantly increased compared with 1 month after transplantation.The proportions of CD3 T cells,CD4 T cells,Treg cells,NK cells,and B cells in patients with a GVHD at six months after transplantation were all reduced compared with healthy controls.4.6 Changes of peripheral blood immune cells in patients with SAA without GVHDThe results of flow cytometry showed that 9 patients with SAA did not develop GVHD after allo-HSCT.Compared with the healthy control group,the proportions of CD3 T cells,CD4 T cells,Treg cells,NK cells and B cells in patients were decreased at1 month after transplantation.The proportions of CD3 T cells,CD8 T cells,and NK cells in patients at 3 months after transplantation were significantly increased compared with1 month after transplantation.CD4 T cells,Treg cells and B cells increased slowly,and the proportion of cells was still lower than that of healthy controls 6 months after transplantation.5.Changes of cytokine levels in peripheral blood after allo-HSCT in patients with different blood diseases5.1 Expression of a GVHD cytokines in AML patients after allo-HSCTBy collecting peripheral blood samples of AML patients after allo-HSCT,ELISA was used to detect the changes of cytokines IL-10,TGF-β1,IL-17 and IFN-γin peripheral blood serum of AML patients with and without GVHD after allo-HSCT.The results showed that the serum levels of cytokines IL-10 and TGF-β1 in AML patients who developed a GVHD after allo-HSCT were significantly lower than those in the healthy control group and the non-GVHD group,and the levels of IFN-γwere significantly higher than those in the non-GVHD group and the healthy group.level did not change significantly.5.2 Expression of c GVHD cytokines in AML patients after allo-HSCTELISA was used to detect the levels of cytokines IL-10,TGF-β1,IL-17 and IFN-γin serum of AML patients with c GVHD and AML patients without c GVHD after allo-HSCT.The results showed that c GVHD patients with AML had significantly lower levels of cytokines IL-10 and TGF-β1 than healthy controls and non-GVHD groups,while no significant changes in cytokines IL-17 and IFN-γwere observed in the c GVHD group.5.3 Expression of a GVHD cytokines in ALL patients after allo-HSCTBy collecting the peripheral blood samples from ALL patients at 1,3,6 months after allo-HSCT,ELISA was used to detect the changes of cytokines IL-10,TGF-β1,IL-17 and IFN-γin serum of ALL patients with and without GVHD after allo-HSCT.The results showed that the levels of IL-10 and TGF-β1 in a GVHD with ALL were significantly lower than those of the healthy control group and the non-GVHD group,and the level of IL-17 was significantly higher than that of the healthy control group and the non-GVHD group.However,the levels of IFN-γwere not significantly changed.5.4 Expression of a GVHD cytokines in SAA patients after allo-HSCTThe changes of cytokines IL-10,TGF-β1,IL-17 and IFN-γin serum of SAA patients with and without GVHD after allo-HSCT were detected by ELISA.The results showed that the levels of IL-10 and TGF-β1 in the serum of SAA patients with a GVHD were significantly lower than those of the healthy control group and the non-GVHD group,and the levels of IL-17 and IFN-γwere significantly higher than those of the healthy control group and the non-GVHD group.Conclusions1.AML patients are more likely to develop a GVHD and c GVHD in patients with AML,ALL,and SAA after allo-HSCT.For this,preventive measures can be strengthened before transplantation to reduce the incidence of GVHD.2.In AML and ALL,the occurrence of a GVHD is more common in gastrointestinal tract,followed by skin type and liver;in SAA,the occurrence of a GVHD is more common in skin,followed by gastrointestinal tract.3.The occurrence of a GVHD in AML patients after allo-HSCT may be related to the levels of CD8 T cells and NK cells in peripheral blood,while the occurrence of a GVHD in ALL and SAA patients may be related to the levels of CD8 T cells.4.The occurrence of a GVHD in patients with AML,ALL and SAA after allo-HSCT may affect the reconstitution of the patient’s immune cells,thereby affecting the prognosis of the patient.5.The occurrence of a GVHD in AML patients after allo-HSCT may be related to IL-10,TGF-β1 and IFN-γ,the occurrence of a GVHD in ALL patients may be related to IL-10,TGF-β1 and IL-17,and the occurrence of a GVHD in SAA patients May be related to the above four cytokines. |
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