The Study On Effect And Mechanism Of Dendritic Cell NLRP3 Inflammasome And Immune Imbalance In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a hematological malignant disease characterized by clonal proliferation,abnormal differentiation and apoptosis of bone marrow hematopoietic stem and progenitor cells that show massive proliferation and infiltration in peripheral blood,bone marrow and other tissues.Immune system disorders have been found to be related to the occurrence and development of AML,and AML is characterized by an immunosuppressive tumour microenvironment that promotes immune tolerance and disease escape.In recent years,although the treatment strategy of AML has been continuously improved,the long-term prognosis is not optimistic because most patients still suffer from refractory,relapse,and drug resistance.Dendritic cells(DCs)are important antigen-presenting cells that play a key role in the immune response.DCs is an important bridge in connecting innate immunity and adaptive immunity.They have strong antigen-presenting capabilities and control B cells and T cells,and B cells and T cells are important immune cells that play an important role in the protective immune response to pathogens and anti-tumor.Dendritic cells stimulate T cells to produce an immune response and play an important role in the immune response to antitumor.DCs with immunomodulatory properties can overcome the immune tolerance of the bone marrow,enhance the immunity of the bone marrow microenvironment and induce the proliferation of anti-leukemia-specific T cells.Activated T cells can recognize antigens which express on AML cells and subsequently mediate AML cells killing.DCs can be used to stimulate autologous T cells to reduce or eliminate residual leukemia cells,thus preventing AML recurrence.Therefore,DCs-based immunotherapy may be more successful in eradicating residual leukemia cells in AML.The inflammasome is one of the main components involved in innate immunity,and the NLRP3 inflammasome is the most clearly studied in the inflammasome.NLRP3 inflammasome affects the differentiation of Th subsets in infectious diseases and immune diseases,but there are few studies on blood diseases.After activation of NLRP3 inflammasome,it can release IL-1? active cytokines,induce T cells to produce and release IL-2 cytokines,and promote the survival and proliferation of T cells.T cell immunity is an important part of anti-tumor immunity,which eliminates leukemia cells by releasing cytokines and cytotoxic substances.The NLRP3 inflammasome can regulate the proliferation and differentiation of T cells and finally achieve the role of regulating tumors.Although many patients with AML achieve remission with chemotherapy,long-term survival rate is far from ideals.The immunosuppressive bone marrow microenvironment fosters leukemia cells growth and immune tolerance.Therefore,the treatments of immunomodulation may overcome immune tolerance,improve the immunity of the bone marrow microenvironment,enhance the immunogenicity of tumors,and improve the prognosis of AML.We want to explore the effect of DCs NLRP3 inflammasome of AML on bone marrow microenvironmental immunity,and study the relationship between single nucleotide polymorphisms(SNPs)of immune-related genes and AML,and investigate the role and mechanism of bone marrow microenvironmental immunity in the pathogenesis and development of AML,then further explore the methods of anti-leukemia cell immunotherapy in the bone marrow microenvironment,provide strategies for immunotherapy of AML.PART ?The effect and mechanism of dendritic cell NLRP3 inflammasome on Th1 differentiation in bone marrow of acute myeloid leukemiaBackgroundThe abnormal immune function plays an important role in AML development and drug resistance,and the immune-disordered bone marrow microenvironment promotes immune tolerance and facilitates the escape of leukemia cells,and the imbalance of T helper subsets may be related to the pathogenesis of AML.Immune cells mainly include T cells,B cells and NK cells,which are an important part of the bone marrow microenvironmental immunity.Among them,the imbalanced of Th subsets in T cells has attracted great attention from researchers.The ability of T cells to proliferate and produce cytokines in AML bone marrow is significantly reduced.AML cells can inhibit T cells through MDSCs,or prevent T cells from entering the cell cycle or secreting Th1 cytokines.Th1 cells mediate cellular immunity and play an important role in the body's normal cellular immunity.Although Th1 is closely related to the occurrence and prognosis of AML,the molecular mechanism is not clear yet.The NLRP3 inflammasome is an intracellular platform that converts the pro-inflammatory cytokines IL-1? and IL-18 to their active forms in response to'danger' signals,which can be either host or pathogen derived,and mediates a form of inflammatory cell death called 'pyroptosis'.The inflammasomes are multi-protein complexes which may be related to the differentiation of Th subsets.NLRP3 inflammasome induces the differentiation of Th1,Th2,Th17 cells and CTL cells in the tumor microenvironment and inhibits tumor immune escape.Immune imbalance and dysfunction of the bone marrow microenvironment lead to the occurrence of leukemia,and may be related to the immune escape of residual leukemia cells after AML treatment.The bone marrow microenvironment with immune dysfunction leads to the relapse and refractory of AML.Th studies have found that the immune imbalance of Th1/Th2 and Th17/Treg in AML bone marrow is involved in the escape of leukemia cells,and plays an important role in the development and drug resistance of AML.However,the mechanism of whether dendritic cells NLRP3 is involved in the differentiation of T cells in AML patients bone marrow is not clear yet.In particular,the mechanism of differentiation of Th1 subsets remains to be studied.ObjectivesThis study aims to investigate whether there is immune imbalance in Th1 subsets in AML bone marrow;Explore the effect and mechanism of dendritic cell NLPR3 inflammasome on the differentiation of Th1 subsets in AML bone marrow;Study the effect of dendritic cell NLRP3 inflammasome on the level of Th1 related cytokine IFN-? and the expression of related transcription factors,and then analyze the key molecules of the influence of NLRP3 inflammasome on the differentiation of bone marrow Th1 subsets.Observe the impact on Th1 subsets through intervention,and clarify the mechanism of NLRP3 inflammasome involved in AML bone marrow immunity,especially the differentiation of Th1 subsets.Methods(1)In this study,bone marrows were collected from 37 AML,AML-CR and 23 refractory recurrent AML patients from Qilu Hospital of Shandong University,and 16 healthy controls were enrolled in the study.BMDCs and CD4+cells were isolated and the plasma was collected.(2)We collected clinical data such as white blood cell count and therapeutic efficacy of AML patients.The percentage of proinflammatory Th1 cells was analyzed by flow cytometry,and then we analyzed the correlation between the percentage of Th1 cells and clinical data.(3)We detected the level of IFN-? cytokine in the bone marrow supernatant by the enzyme-linked immunosorbent assay(ELISA),and then we analyzed the clinical correlation between IFN-? and AML.(4)Bone marrow mononuclear cells(BMMCs)were induced into dendritic cells by using GM-CSF and IL-4,and flow cytometry was employed to detect the expression of CD14,CD11c,CD80,CD83,CD86 molecules in dendritic cells and the proportion of DCs.CD4+T cells in bone marrow BMMCs were sorted by MACS and flow cytometry was used to detect the sorting rate.(5)DCs NLRP3 inflammasome activation and inhibition:The DCs were divided into three groups:?:experimental group(LPS+ATP):BMDCs were pre-treatmented by LPS for 6 hours,and then plus ATP activation for 45 minutes;?Inhibition group:before adding LPS,BMDCs were pre-treated by MCC950 1 hours;?Control group:only sterile PBS was added.The relative expression of NLRP3 inflammasome-related molecules NLRP3,IL-1?,IL-18,ASC,Caspase-1 and NF-?B was detected.(6)The effect of DCs NLRP3 inflammation on the differentiation of CD4+T cells:The above three groups of dendritic cells were co-cultured with CD4+T cells,and the percentage of Th1,Th2,Th17 and Treg cells in CD4+T cells was detected by flow cytometry after 7 days.(7)Cytokine detection:IL-12p70,IL-1?,IL-17A,IL-2,IL-10,IL-4,IL-5,IFN-?,IL-6,IL-7,IL-8 and other cytokine in the supernatant of DCs were detected by multi-cytokine array,and then the expression of key cytokines was detected by the ELISA method.(8)The effect of IL-1? on Thl differentiation:Recombinant IL-1? cytokine was added to BMDCs and CD4+T cell co-culture control group and inhibition group,IL-1? neutralizing antibody was added to the experimental group,and the proportion of Th1 cells was detected by flow cytometry.(9)The way in which dendritic cells NLRP3 inflammasome promote Th1 differentiation:CD4+T cells and dendritic cells came into contact or got separated with transwell.The percentage of Th1 cells was detected by flow cytometry in CD4+T cells.ELISA was used to detect IFN-? and IL-1? cytokine levels in the supernatant.(10)The mechanism of IL-1? affecting Th1 differentiation:The freshly isolated CD4+T cells were divided into three groups:first group Add IL-12 alone;second group IL-12 combined with IL-1?;third group Add sterile PBS,RT-PCR was used to detect the expression of IFN-?,IFN-? receptor and STAT1 in CD4+T cells in each group.(11)The effect of DCs NLRP3 inflammasome on the differentiation of Th1 cells in vivo:WT or Nlrp3-/-C57BL/6J mice dendritic cells were cultured in vitro,then WT mice DCs were divide into 3 groups:?Experimental group:BMDCs were treated by LPS and ATP;?Inhibition group:before adding LPS,BMDCs were pretreated by MCC950 for 1 hours;?Control group:add sterile PBS.The DCs of Nlrp3-/-mice were divided into experimental and control group.Then the DCs of each group were injected into WT mice through the tail vein to observe the effect of DCs NLRP3 inflammasome on the differentiation of Th1 subset in the mice.The pertentage of Th1 cells in mice bone marrow and spleen was detected by flow cytometry.(12)The effect of IL-1? on the differentiation Th1 cells:IL-1? neutralizing antibody was injected into the experimental group of mice to observe the effect of IL-1? on the differentiation of Th1 subsets in mice.Flow cytometry was used to detect the percentage of Th1 cells in the bone marrow and spleen in mice.Results(1)The percentage of Th1 cells was significantly lower in the bone marrow of newly diagnosed patients with AML than that in the control group.After chemotherapy treatment,the percentage of Th1 cells in AML-CR patients was significantly increased;The percentage of Th1 cells was negatively correlated with the peripheral blood white blood cell count of newly diagnosed patients with AML,and peripheral blood white blood cell count was significantly high in the AML patients whose Th1 cells ?10%rather than Th1 cells>10%.(2)The level of IFN-y cytokine in the bone marrow supernatant of newly diagnosed patients with AML was significantly lower than control group,and the level of IFN-y increased after the AML patients chemotherapy reaches CR.(3)Dendritic cell culture and CD4+T cells:The dendritic cells expressed CD11c,not CD80 and CD 14,and slightly expressed CD83 and CD86.They were detected by flow cytometry detects.DCs induced conversion rate is over 90%.The separation purity of CD4+T cells in bone marrow using CD4+magnetic beads is>90%.(4)Activated and inhibited NLRP3 inflammasome in dendritic cells:The mRNA expression levels of caspase-1 and IL-1? in BMDCs were increased after NLRP3 activation and reversed by MCC950.IL-1? concentration in the supernatant of LA-activated BMDCs was also elevated,while MCC950 inhibited LA-induced IL-1? secretion significantly.Western blot result showed that IL-1?(p17),the activated form of IL-1?,was increased after NLRP3 activation.(5)The influence of DCs NLRP3 inflammation on the differentiation of CD4+T cells:NLRP3-activated BMDCs enhanced the percentage of Th1 cells in CD4+T cells,while MCC950-treated BMDCs decreased the frequency of Th1 cells.There was no significant change in the percentage of Th2,Th17 and Treg cells.(6)Cytokine detection:IL-1? in BMDCs supernatant was significantly elevated after LA stimulation in the experimental group and then recovered after adding MCC950 in inhibition group.We further confirmed the elevation and recovery of IL-1? level in another 12 AML patients by ELISA.(7)The effect of IL-1? on Th1 cell differentiation:CD4+T cells co-cultured with BMDCs differentiated into markedly higher Th1 cells in the presence of IL-1?cytokine in control group and inhibited group.The promotion of CD4+T cells differentiation into Th1 by LA-treated BMDCs was inhibited by the addition of IL-1? neutralized antibody,while the suppressive effect by MCC950 could be reversed after adding IL-1?.(8)The way in which dendritic cells NLRP3 inflammasome promotes Th1 differentiation:CD4+T cells in contact with NLRP3-activated BMDCs were differentiated into Th1 cells at the same manner as untouched Transwell assay.As for the concentration of cytokine IFN-? and IL-1? in culture supernatant,and no significant difference was found between the direct contact and Transwell cultured manner.(9)The mechanism of IL-1? affecting Th1 differentiation:IL-12 combined with IL-1? promoted higher expression of IFN-?,IFN-? receptor and STAT1 compared with IL-12 alone in CD4+T cells.(10)The effect of DCs NLRP3 inflammasome on the differentiation of Th1 cells in vivo:NLRP3-activated WT BMDCs promoted the differentiation of Th1 cells in WT murine BM or spleen,and these effects were reversed by MCC950.However,LA-activated Nlrp3-/-BMDCs failed to promote Thl differentiation in WT mice.(11)The effect of IL-1? on the differentiation Th1 cells:Th1 frequency in murine spleen was up-regulated after injection of LA-activated BMDCs and rescued by anti-IL-1?.Conclusions1.The percentage of Th1 cells and the level of IFN-? in the bone marrow of newly diagnosed AML patients are reduced,and can be recovered by chemotherapy.The reduction of the percentage of Th1 cells is negatively correlated with the white blood cell count,which indicates that there is an immune imbalance in the bone marrow microenvironment of AML.2.The differentiation of Th1 cells promoted by NLRP3-activated BMDCs requires a functional inflammasome.This result provides a new research direction for correcting the immune imbalance of the AML bone marrow microenvironment.3.NLRP3-activated BMDCs promotes Th1 cell differentiation through IL-1?secretion independent of cell contact.These data identify IL-1? is the key factor that favors the NLRP3-dependent differentiation of CD4+T cells into Th1 cells,suggesting that IL-1? can be used to improve the immunity of the bone marrow microenvironment.PART ?Study on the anti-leukemia effect and mechanism of dendritic cell NLRP3 inflammasome in acute myeloid leukemiaBackgroundAcute myeloid leukaemia(AML)is a lethal haematological malignancy and the most common acute leukemia in adults,accounting for 20%of childhood leukemia.Standard treatment for AML consists of induction chemotherapy with remission achieved in 50%to 75%of cases,but standard therapy is often inadequate in sustaining durable remissions due to the emergence of chemotherapy-resistant clones.AML with unfavorable cytogenetics has a poor outcome,even when treated with aggressive chemotherapy especially.Unfortunately,most patients will relapse and die from their disease,as 5 years survival is roughly 25%.By contrast,the prognosis of patients aged 65 years or older has not improved considerably over time,the latest reported five-year survival rate being<10%,therefore,other treatment options are urgently needed.It is within this context that immunotherapy has stand out in recent years.In recent years,immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers.Immunotherapy is based on the premise that tumors suppress the immune system.Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML.There is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival.AML/dendritic cell fusion vaccine can cause the expansion of leukemia-specific T cells and prevent disease recurrence.Therefore,a new type of personalized dendritic cell vaccine is a new treatment method that produces immunity to prevent disease recurrence.T cells produce an immune response though stimulation by DCs and play an important role in the immune response against tumor.Activated T cells can recognize antigens expressed on AML cells and then mediated the effect on anti-leulemia cells.In recent years,DCs have attracted great interest as a tool for AML immunotherapy.Stimulation of autologous T-cells by immunization with DCs is an innovative strategy to combat relapse of AML,which acts via the reduction or eradication of residual leukemia cells.Therefore,DCs-based immunotherapy may be more successful in eradicating residual leukemia cells in AML patients.Non-specific and antigen-specific immunological effects have been obtained in a considerable number of DC-treated AML patients.NLRP3 inflammasome has a controversial role in cancer development and progression.Many studies considered that NLRP3 inflammasome and its components contributed to the promotion of tumor growth,proliferation,invasion and metastasis in different malignancies such as melanoma,lung cancer,breast cancer,and multiple myeloma.However,colon cancer induced by AOM/DSS in NLRP3 knockout animal models had shown that the NLRP3 inflammasome had a protective effect on cancer.The function of NLRP3 inflammasome in different tumors emphasizes the therapeutic potential of inflammasome.Our previous research results showed that the percentage of Th1 cells in the bone marrow of AML patients is reduced,and there is immunosuppression of the bone marrow microenvironment.The activation of dendritic cell inflammasome can promote the differentiation of CD4+T cells into Thl cells.It is well known that Thl cell effector cytokine is IFN-?,and IFN-? has anti-tumor immunity.However,whether dendritic cell NLRP3 inflammasome activation promotes AML bone marrow Th1 cell differentiation has a killing effect on leukemia cells and its mechanism remains to be studied.ObjectivesThis study aims to explore whether the activation of dendritic cells NLRP3 inflammasome to promote the differentiation of Th1 cells in AML bone marrow has an effect on the proliferation and apoptosis of leukemia cells.The key molecules affecting the proliferation and apoptosis of AML cells were studied,and the mechanism of their influence on the proliferation and apoptosis of AML cells was analyzed through intervention of key molecules,to clarify the mechanism of NLRP3 inflammatome participating in AML bone marrow immunity,especially in promoting Th1 subsets differentiation and anti-leukemiaMethods(1)The effect of BMDCs NLRP3 inflammatome on the proliferation and apoptosis of leukemia cells:The AML BMDCs were divided into three groups:?Experimental group(LPS+ATP):BMDCs were pre-treatmented by LPS for 6 hours,and then plus ATP activation for 45 minutes;?Inhibition group:before adding LPS,MCC950 was added and pre-treated for 1 hours;?Control group:only add sterile PBS.CD4+T cells co-culture with three groups of BMDCs in transwell,and then co-culture the CD4+T cells with THP-1 and U937 AML leukemia cell,and detect cell apoptosis of THP-1 and U937 leukemia by flow cytometry,and use CCK-8 kit to detect the proliferation of leukemia cells.(2)Mechanisms affecting the proliferation and apoptosis of leukemia cells:The level of Th1 cell main effector cytokine IFN-? in the supernatant of co-cultured cells was detected by ELISA.EFN-? neutralizing antibody was added to the co-culture system of the experimental group,the apoptosis of THP-1 and U937 leukemia cells was detected by flow cytometry,and the CCK-8 kit was used to detect the proliferation of leukemia cells,then to explore the effect and mechanism of IFN-?on apoptosis and proliferation of leukemia cells.(3)The effect of dendritic cell NLRP3 inflammasome on AML leukemia mice:WT or Nlrp3-/-C57BL/6J mice dendritic cells were cultured in vitro,then WT mice DCs were divide into 3 groups:?Experimental group:BMDCs were treatmented by LPS and ATP;?Inhibition group:before adding LPS,BMDCs were pre-treated by MCC950 for 1 hours;?Control group:add sterile PBS.The DCs of Nlrp3-/-mice were divided into experimental and control groups.First,WT mice were injected with MLL-AF9-GFP leukemia cells through the tail vein.After 3 days,the BMDCs of each group were injected into the mice through the tail vein.The spleen and liver volume or weight of each group of leukemia mice was measured 7 day later,and the percentage of MLL-AF9-GFP leukemia cells in spleen,liver and bone marrow was detected by flow cytometry in mice.(4)The effect of IFN-? on leukemia cells in leukemia mice:MLL-AF9-GFP leukemia cells were injected through the tail vein of WT mice.Three days later,the control and experimental groups WT/BMDCs were injected into the mice through the tail vein,and IFN-? neutralizing antibodies were injected into experimental group of mice on the 4th,6th and 8th day.On the 10th day,the volume or weight of the spleen and liver of each group were measured in leukemia mice,and the MLL-AF9-GFP leukemia cell ratio in the spleen,liver and bone marrow of mice were measured by flow cytometry.(5)The effect of IL-1? on leukemia cells in leukemia mice:MLL-AF9-GFP leukemia cells were injected through the tail vein of WT mice.Three days later,WT/BMDCs of the control and experimental groups were injected into the mice through the tail vein.IL-1? neutralizing antibody was injected into experimental group of mice for consecutive 4 days.On the 10th day,the volume or weight of the spleen and liver of each group of leukemia mice were measured,and the MLL-AF9-GFP leukemia cell ratio in the mouse spleen,liver and bone marrow was measured by flow cytometry.Results(1)The effect of BMDCs NLRP3 inflammatome cells on the proliferation and apoptosis of leukemia cells:Compared with the control group,the experimental group CD4+T cells co-cultured with THP-1 or U937 leukemia cells were significantly increased leukemia cell apoptosis,while the inhibition group MCC950 would reduce the pro-apoptotic effect.Compared with the control group or the inhibition group,the CD4+T cells of the experimental group reduced the proliferation of THP-1 or U937 leukemia cells significantly.(2)The mechanism that affects the proliferation and apoptosis of leukemia cells:The IFN-? level in the supernatant of the co-cultured BMDCs and CD4+T cells was significantly higher in the experimental group than that in the control group,while the level of IFN-y was significantly reduced in the inhibitory group.Adding IFN-y neutralizing antibody to the co-culture system of the experimental group significantly reduced the apoptosis of THP-1 or U937 leukemia cells.In addition,the proliferation of THP-1 or U937 leukemia cells were increased in the experimental group which added IFN-? neutralizing antibody.(3)The effect of dendritic cell NLRP3 inflammasome on tumors in leukemia mice:Compared with the control,the spleen and liver volume or weight of leukemia mice in the experimental group injected with NLRP3-activated WT/BMDCs were significantly reduced.And the load of MLL-AF9-GFP leukemia cells in the bone marrow,spleen and liver was lower than that in the control group,and MCC950 can reverse the effect of NLRP3-activated WT/BMDCs inhibiting the proliferation of leukemia.However,the NLRP3-activated BMDCs of Nlrp3-/-were injected into leukemia mice has no inhibitory effect on leukemia cells.(4)The effect of IFN-y on leukemia cells in leukemia mice:Compared with mice in the experimental group without IFN-? neutralizing antibody,the spleen volume or liver weight and volume of mice was significant increased in the experimental group injected with IFN-? neutralizing antibody.The load of MLL-AF9-GFP leukemia cells in the bone marrow,spleen or liver of the mice increased significantly.(5)The effect of IL-1? on leukemia cells in leukemia mice:The experimental group was injected with IL-1? neutralizing antibody compared with the experimental group without injected with IL-1? neutralizing antibody.The volume or weight of spleen and liver of leukemia mice was increased significantly,and the load of MLL-AF9-GFP leukemia cells in the bone marrow,spleen,and liver increased significantly.Conclusions1.NLRP3-activated BMDC promotes Th1 cell differentiation and has anti-leukemia effect,which is providing a new research direction for AML immunotherapy2.IFN-? is an important effector molecule of NLRP3-activated BMDCs against leukemia.3.NLRP3 inflammasome are involved in anti-leukemic action through the IL-1?/Thl/IFN-?,regulating dendritic cells NLRP3 inflammasome and IL-1? are expected to be a new target for AML treatment.PART ?Immunerelated Gene Polymorphisms associated with Acute Myeloid LeukemiaBackgroundAcute myeloid leukemia(AML)is characterized by the proliferation of myeloid blasts in the bone marrow,resulting in impaired normal hematopoietic function,and is characterized by recurrent cytogenetic abnormalities.It is the hematological malignant tumor with the highest incidence in adults.The presence of immune imbalance in the occurrence of malignant tumors and the presence of immune system damage in AML,among which T-cells,the most important component of the immune system,which are deficient in number and function.T cells immune,important for anti-tumor immunity,eliminates AML cells through releasing cytokines and cytotoxic substances.AML cells influence T cells differentiation and proliferation,and play an immunosuppressive role by releasing inhibitory cytokines or other kinds of mechanisms.T helper(Th)cells play a pivotal status in T cell immune system network.Th1/Th2 imbalanced involved in the pathogenesis of solid tumors as well as hematological malignancies.However,researches on Th helper cell in AML bone marrow(BM)microenvironment are limited.Single nucleotide polymorphisms(SNPs)is the most common type of genetic variation,and its function has gradually been discovered in many fields of biology,especially in human diseases.The role of inflammation-related SNPs in AML patients has been studied and SNPs associated with chemotherapy-sensitive,disease prognosis and survival time have been found.For example,IL-1?(rs 16944)GA genotype contributed to the cytogenetics favorable-risk and the GT genotype of IL18(rs 1946518)statistically led to a poorer AML-specific survival.G single mutant and GG mutation homozygote of IL17F were related with AML development.Nursal et al.suggested that functional variants of the TNF-?,IL10,and TGF-?1 may have a significant association with the etiopathogenesis of AML.In our previous study,we found that Th subsets imbalance and related cytokine secretion disorder in AML bone marrow,and the unbalanced Th subsets and the disordered cytokines may jointly participate in the occurrence and development of AML.Therefore,we further studied the relationship between SNPs of immune-related genes(including proinflammatory or anti-inflammatory cytokines and key regulators of T cell subsets)and AML,explored some unanticipated functions and pathological mechanisms involved in SNPs in the pathogenesis of AML,and sought for new important immune targets for AML treatment.ObjectivesThe purpose of this study is to explore the relationship between single nucleotide polymorphisms of immunerelated genes and AML susceptibility,susceptibility,prognosis,clinical characteristics,risk stratification and survival analysis.The relationship between different genotypes of immune-related genes and clinical characteristics of AML was analyzed.Combined.with mRNA expression and functional analysis of related genes,possible signaling pathways were further identified to seek new immunotherapy targets for AML.Methods(1)Specimen collection:In this study,bone marrow from 269 newly-diagnosed AML patients and peripheral blood from 200 healthy control were collected from Qilu Hospital of Shandong University.Mononuclear cells were isolated from bone marrow and peripheral blood;DNA and RNA were extracted in mononuclear cells;clinical relevant data of the patients was collected.(2)The AML patients bone marrow and healthy control peripheral blood samples were collected and analyzed for mass spectrometry SNPs typing information.We performed genotyping by primer-extension mass spectrometry using the Sequenom MassARRAY platform at 21 candidate SNPs to provide genotyping accuracy in 269 AML cases and 200 healthy controls.In this study,Hardy-Weinberg equilibrium and minor allele frequency(MAF)were used to evaluate the applicability of the 21 candidate SNPs.(3)RT-PCR detected the expression of IL-12 mRNA in AML mononuclear bone marrow.Results:(1)Association between immunerelated SNPs and AML susceptibility:IL4(rs2070874,rs2243250)and IL22 rs1179251 under the dominant model were significantly associated with AML susceptibility.Univariate logistic regression analysis revealed that only IL4 rs2243250 under dominant model was significantly associated with AML susceptibility after adjusting for age and gender.However,among the above SNPs,there was no SNPs contributing to the AML susceptibility after Bonferroni multiple correction.(2)Association between the percentage of BM blast cells and SNPs in AML:IL2 rs2069762 under recessive model revealed statistically significant difference between high(?42%)and low(<42%)BM blasts groups.(3)Association between WBC count and SNPs in AML:The genotypic frequencies of IL22 rs2227491 under the codominant,dominant,and allelic models were significantly associated with WBC count.In addition,allelic frequencies of IL-4 rs2243250 were significantly associated with WBC count.After Bonferroni multiple correction,genotypic frequencies of IL22 rs2227491 under both dominant and allelic models were also significantly associated with WBC count.After adjusting for age and gender,AML patients carrying the TT genotype of rs2227491 showed a 4.2-fold increased risk of WBC compared with patients carrying major genotype TC/CC in the dominant model.IL22 rs2227491 allelic distribution also showed a statistically significant difference.(4)Association between hemoglobin,lactate dehydrogenase and SNPs in AML:The genotypic frequencies of STAT6 rs324011 and IL12A rs2243115 under both codominant and dominant models revealed statistically significant difference between high and low HGB groups.In addition,allele frequency and genotypic frequencies under the codominant and recessive models of rs2069718 in IFN-y were also significantly associated with HGB.After adjusting for age and gender,AML patients carrying the CC genotype of STAT6 rs324011 showed low level of HGB at diagnosis compared with patients carrying CT/TT.Only the genotypic frequency of IL6R rs2228145 under dominant model was significantly associated with the high level of plasma LDH after adjusting for age and gender.No statistical difference was found between other SNPs and PLT counts.However,no SNPs were significantly associated with HGB or LDH after Bonferroni multiple correction.(5)Association between immunerelated SNPs and WHO classification of AML:genetic frequency of STAT5B rs6503691 under codominant and recessive models was significantly associated with recurrent genetic abnormalities(RGAs).The association between STAT5B rs6503691 and recurrent genetic abnormalities was also statistically significant after Bonferroni multiple correction.Univariate logistic regression analyses adjusting for age and gender showed that when the CC genotype(under codominant model)or CC/CT genotype(under recessive model)was used as a reference,the TT or CT genotypes(under codominant model),and TT(under recessive model)were significantly associated with recurrent genetic abnormalities.(6)Association between immunerelated SNPs and risk stratification of AML:The genetic frequencies of TGFB1 rs 1800469 under codominant,dominant and recessive models were significantly associated with risk stratification.However,after adjusting for age and gender factors TGFB1 rs 1800469 under codominant,dominant and recessive models lost the statistical difference by multiple logistic regression.(7)Overall Survival for AML i...