The Effect And Mechanism Of Enhancing T-cell Activation By MiR34a And Inhibiting B-cell Growth By HLA-G/ILT2 In Acquired Aplastic Anemia

Acquired aplastic anemia(AA)is a kind of bone marrow failure syndrome,characterized by peripheral blood pancytopenia and bone marrow aplasia.Patients with AA often present with symptoms of anemia,purpura or hemorrhage and infection.Especially in severe AA,patients are often in danger for the high incidence rate of organ hemorrhage,severe anemia and infection.AA is happened worldwide.The incidence in our country is higher than in western countries,and most patients were young people.The complicated pathophysiology of AA was still not completely understood,mainly including three aspects,immune dysregulation.hematopoietic stem/progenitor cells and bone marrow mesenchymal stem cells with intrinsic deficits.Some genetic factors were also suggested to be associated with AA in a number of studies.In recent years,a large amount of laboratory and clinical data suggest that immune-mediated suppression of hematopoiesis plays an essential role in the pathogenesis of AA.The abnormally activated T cells in bone marrow attacked the hematopoietic stem/progenitor cells.Furthermore,some putative autoantigens in AA patients could induce immune response and damage the hematopoietic stem/progenitor cells.Although the inciting autoantigens remain elusive,autoantibodies are often detected in the serum,indicating B-cell mediated humoral immune also exert important action in AA.MicroRNAs(miRNAs)are a recently discovered class of naturally occurring short non-coding single-strand RNA molecules with the length of 19 to 25 nucleotides that regulate eukaryotic gene expression post-transcriptionally by loading onto the RNA-induced silencing complex(RISC)to exert repressive function on its target messenger RNA.The role of miRNAs in the regulation of many physiological and pathological processes has been intensely studied in recent years.Some miRNAs,such as miR146a and miR182,play a dominant role in the regulation of the innate and adaptive immune responses,respectively.Many miRNAs are reportedly deregulated in autoimmune diseases,such as systemic lupus erythematosus,rheumatoid arthritis and so on.And rare studies about miRNA on AA were reported.In present study,we performed a microarray analysis of the miRNA expression patterns in BM CD3+ T cells of AA patients and healthy controls to screen for miRNAs differentially expressed in AA.And then we verified the miRNAs level between AA and controls by quantitative real time-polymerase chain reaction(RT-PCR)in 41 patients and 20 healthy individuals and identified the up-regulated miR34a and its corresponding target gene DGKζ in T cells of AA patients.We then generated a murine model of bone marrow failure and explored the possible roles of miR34a and DGKζ in T cell activation in AA.Our work facilitated clarifying the immune mechanism of AA and provide a base for developing new therapeutic target for AA.Human leukocyte antigen(HLA)-G is a kind of non-classic I major histocompatibility complex antigen characterized by low polymorphism,restricted tissue distribution and spliced transcripts which encode four membrane-bound isoforms(mHLA-G,G1-G4)and three soluble protein(sHLA-G.G5-G7).In physiological condition,HLA-G is expressed in fetal trophoblast cells,thymus,cornea,nail matrix,pancreas,and erythroid and endothelial precursors,while in some pathologic situations such as cancers.transplantation,inflammatory and autoimmune disease it could be detected.Several studies have demonstrated that HLA-G interacts with multiple immune cell subsets such as T cells.B cells.NK cells and antigen-presenting cells(APCs)and exerts powerful immune suppression by binding with its receptors,immunoglobulin-like transcript(ILLTs)2(LILRB1/CD85j).ILT4(LILRB2/CD85d)and KIR2DL4(CD158d).It has been reported that HLA-G expressed at high or low level in several pathological conditions,including organ transplantation,tumors,microbial or viral infection,and autoimmune diseases,leading to the impairment of the immune response against tumor cells or pathogens.AA is an immune-mediated disease and no paper has reported the role of HLA-G in AA development until now.In this study,we designed a series of experiments to explore the possible relationship between them and evaluated the role of HLA-G and ILTs in the immunopathology of AA.Part I:Dysregulated miR34a/diacylglycerol kinase ζ interaction enhances T-cell activation in acquired aplastic anemiaObjectiveThe aim of this study was to screen the differentially expressed miRNAs in marrow CD3+ T cells from AA patients by miRNA array analysis.Then expand the sample volume,identify the miRNAs and analyze the relationship between miRNAs and the clinical characteristics of patients.Furthermore,we determined the target gene of this miRNA and built a murine model of bone marrow failure to clarify the role of miRNA and its target gene in T-cell activation in AA and elucidate the underlying molecular mechanism.Materials and methods1.Sample collection:bone marrow sample were extracted from 49 AA patients and 28 healthy controls,including 33 severe AA patients and 16 moderate AA patients.2.Microarray experiments:CD3+ cells in bone marrow mononuclear cells(BMMCs)were positively selected from three SAA patients and three healthy controls using an immunomagnetic activated cell sorting system.Affymetrix GeneChip microRNA 2.0 Array was used for detection of miRNA expression in the six samples.3.Verify miRNAs:miR34a expression in bone marrow mononuclear cells(BMMCs)was determined by RT-PCR and analyze the correlation between miR34a and patients’ clinical characteristics.4.Determine the miR34a expression in marrow naive T cells:naive T cells and non-naive T cells were sorted by immunomagnetic activated cell sorting system,and then RT-PCR was used to determine the miR34a expression in the sorted T cells.5.Identify the target gene of miR34a in AA:The expression of DGKζ which might be the target gene of miR34a was analyzed by RT-PCR and western blot.6.Functional assay in vitro was performed to explore the role of miR34a in marrow T-cell activation in AA.6.1 The BMMCs obtained from AA patients were transfected with LV3-miR34a inhibitor and LV3-NC.Cells with GFP were observed under fluorescence microscope and collected 5 days after transfection.6.2 Analyze the T cell activation by flow cytometry(FCM):the collected cells from 6.1 were stained with anti-CD4,CD8.CD69 and CD25 mAbs,and analyze CD4 and CD8 T-cell activation in the LV3-miR34a inhibitor and LV3-NC group.7.The effect of miR34a deficiency on mouse lymphohematopoiesis was analyzed by comparing the cellular composition in blood,BM,spleen,and lymph node between miR34a-/-and wild-type mice.8.Functional assay in vitro was performed to explore the role of miR34a in T-cell activation in mice:Lymph node(LN)cells and splenocytes from miR34a-deficient and wild-type mice were stimulated with anti-CD3 and anti-CD28 mAbs.8.1 T-cell activation assay:CD25 and CD69 expression were analyzed by flow cytometry.8.2 T-cell proliferation assay:5-Bromo-2-deoxyuridine(Brdu)and carboxyfluorescein diacetate succinimidyl ester(CFSE)were used to evaluate T-cell proliferation by flow cytometry.8.3 DGKC protein expression and phosphorylated ERK1/2 in T cells after being stimulated were determined by western blot.9.Induction of a murine model of bone marrow failure:Recipient CB6F1 mice were pre-irradiated with 5 Gy total body irradiation(TBI).Four to six hours later,mice were injected with MHC-mismatched LN(5 × 106 wild-type or miR34a-/-)cells through the lateral tail vein.Compare the bone marrow hematopoietic function and T-cell proliferation in the two transfusion group.Results1.MiRNA microarray results:A total of 16 miRNAs were upregulated and 15 miRNAs were downregulated.2.A significant upregulation of miR34a was verified in AA patients.The miR34a expression showed a negative correlation with peripheral blood neutrophil or reticulocyte counts in AA patients,and was higher in SAA than in MAA groups.No difference was observed in naive T cells and non-naive T cells.3.Referring to previous studies and using a target prediction and validation program.miRWalk 2.0,7 potential target genes of miR34a were chose to examine further in AA patients and healthy individuals.Of these,the mRNA level of DGKC in AA patients was much lower than that in controls,and was negatively correlated with the level of miR34a.4.The protein level of CD69 on T-cells transfected with miR34a inhibitor lentivirus was lower than that on cells transfected with control lentivirus,indicating miR34a down-regulation decreases activation of T cells from AA patients.5.Lymphohematopoietic cellularity in miR34a-deficient mice:Deletion of miR34a resulted in a mild decrease in CD4+ cells in peripheral blood and BM,and had no major effect on other lymphohematopoietic cell types.6.MiR34a deficiency attenuates T cell activation and DGKζ down-regulation in response to TCR stimulation:CD4+ and CD8+ lymph node cells from miR34a-/-mice expressed lower levels of CD69 and CD25 than those from wild-type mice after TCR stimulation.In miR34a-/-LN cells,DGKζ mRNA levels decreased to a lesser extent than in wild-type cells.Besides,miR34a-deficient T cells are hypoproliferative.MiR34a-deficient CD4+ and CD8+ LN T cells showed reduced Brdu incorporation compared with that of wild-type cells after stimulation with anti-CD3 and anti-CD28 mAbs.CD8+ miR34a-deficient T cells divided more slowly than wild-type T cells.7.MiR34a deletion impairs T cell function in murine model of BM failure:Infusion of wild-type LN cells resulted a more significant decrease in the percentage and the total number of LSK cells compared to mice that received miR34a-deficient LN cells.Furthermore,infused wild-type LN cells expanded more vigorously in F1 recipient BMs than miR34a-deficient LN cells.Conclusion1.MiRNAs were differentially expressed in marrow T cells from AA patients and healthy individuals.indicating that miRNA may involve in the T-cell mediated immune dysregulation in AA.2.Restroing miR34a expression in marrow T cells from AA patients greatly decreased T-cell activation.3.In the murine model of bone marrow failure miR34a gene deletion also showed decreased T-cell activation and proliferation.4.The increased miR34a in AA targeted DGKζ and obviously enhanced T-cell activation,suggesting a potential therapeutic target for AA.Part II:HLA-G-ILT2 interaction contributes to suppression of marrow B-cell growth in acquired aplastic anemiaObjectiveThe aim of this study was to verify the expression pattern of HLA-G,ILT2,and ILT4 in peripheral blood and bone marrow from AA patients,explore the role of HLA-G-ILT2 interaction in marrow B cells from AA patients and the possible molecular mechanism,finaly provide new way to research on the immune pathophysiology of AA.Materials and methods1.Sample collection:46 AA patients and 28 healthy individuals were enrolled,including 31 sever AA patients and 15 moderate AA patients.Plasma and mononuclear cells were prepared from bone marrow and peripheral blood.2.The level of soluble HLA-G in plasma from bone marrow and peripheral blood were examined by enzyme-linked immune sorbent assay(ELISA).3.The expression of HLA-G mRNA,ILT2 mRNA,and ILT4 mRNA in mononuclear cells from peripheral blood and bone marrow were determined by RT-PCR.4.The protein level of mHLA-G,ILT2,and ILT4 in mononuclear cells from peripheral blood and bone marrow were determined by FCM.5.ILT2 level in different stage of BM B cells was analyzed by FCM.in which mAbs targeted surface IgD(sIgD),sIgM,ILT2 and CD 19 were used to stain BMMCs.6.Functional assay in vitro:CD19+ cells in BMMCs were positively selected from healthy controls using an immunomagnetic activated cell sorting system.CD19+cells were cultured on a confluent layer of OP9 stromal cells in MEMa with 20%FCS.IL-7,SCF,and Flt3-L.Cells were treated with recombinant human HLA-G protein or BM supernatant with high sHLA-G level from AA patients as well as blocking antibody,anti-ILT2 or anti-HLA-G.Brdu incorporation was analyzed by flow cytometry after staining with anti-CD 19 fluorescence-labeled mAbs.Results1.The level of sHLA-G in BM supernatant from AA patients was significantly higher than from healthy controls,but no difference was observed between SAA group and MAA group.In PB plasma the level of sHLA-G was much lower than in BM supernatant,and there were no difference between the AA patients and controls.2.The ILT2 mRNA expression in BMMCs from AA patients was much higher than inhealthy controls.There were no significant change in HLA-G and ILT4 mRNA level from AA patients BMMCs compared with controls.3.The percentage of CD 19+ B lymphocytes was significantly lower in BMMCs from AA patients than from control subjects accompanied with the higher CD8+lymphocytes proportion.Further,the ILT2-expressing cells among CD19+ marrow B lymphocytes in AA patients were much more than in healthy controls.4.The proportion of mature B cells among CD19+ B lymphocytes in BM from A A patients was significantly higher than from healthy subjects,while percentage of pro－B plus pre-B cells was much lower.ILT2-expressing cells in immature B cells and pro-B plus pre-B cells from AA patients was much more than from controls,in mature B cells ILT2 expression was similar between the two groups.5.The marrow B cells from healthy individuals co-cultured with HLA-G protein and AA patients’ supernatant showed obviously decreased Brdu incorporation rate compared with control group.Anti-HLA-G mAb and anti-ILT2 mAb could reverse the effect and increase the Brdu incorporation rate.Conclusion1.The aberrant expression of HLA-G and ILT2 in bone marrow from AA patients indicates HLA-G and ILT2 might play a role in the development of immune dysregulation in AA.2.The binding of sHLA-G and ILT2 on the surface of marrow B cells could induce suppression of marrow B-cell growth,suggesting in AA high-level sHLA-G in marrow plasma interacted with the increased ILT2 on the surface of pro plus pre and immature B cells and inhibit B-cell growth,and finally pro plus pre and immature B cells decrease while the proportion of mature B cells increased.3.Blocked interaction between HLA-G and ILT2 facilitates normalizing the amount of marrow B cells.