Disrupted Bone Marrow B-cell Microenviroments And Study Of Therapeutic Mechanisms Of BTK Inhibitor In ITP

Part Ⅰ:Abnormalities of bone marrow B cells and plasma cells in primary immune thrombocytopeniaBackground:Primary immune thrombocytopenia(ITP)is a common autoimmune hemorrhagic disorder,which is characterized by decreased platelet count and increased risk of bleeding.The pathogenesis of ITP is complex and heterogeneous,mainly including excessive platelet destruction mediated by cellular and humoral immunity,and decreased platelet production caused by impaired megakaryocyte maturation.Although many abnormal targets have been revealed,the diagnosis of ITP still lacks"gold standard" and depends on excluding other causes of thrombocytopenia.Meanwhile,only about 2/3 of the patients responded to the first-line drugs.A considerable number of patients could be ineffective or relapsed after combined treatments,and developed into refractory cases.Therefore,it is necessary to further explore the pathogenesis of ITP and provide new strategies for its accurate diagnosis and therapy.As the source of antibodies and lymphocytes that can produce immunologic memory,B cells can not be underestimated in autoimmune diseases.On the one hand,B cells differentiate into plasma cells to produce pathogenic autoantibodies after losing tolerance to autoantigens;on the other hand,the regulatory B cells(Bregs)play anti-inflammatory roles through the immunosuppressive properties.Autoreactive B cells were in a central station in the occurrence of ITP.Previous studies have demonstrated the disrupted B cell subsets in peripheral blood(PB)of ITP patients,but the results were controversial and not comprehensive enough.Researches about bone marrow(BM)mostly focused on the abnormalities of T cells and mesenchymal stem cells,while B cells were rarely reported.BM is the location where B cells develop and mature.Exploring BM microenvironments will help to reveal the abnormal differentiation,chemotaxis and immune tolerance of B cells,so as to further clarify the pathogenesis of ITP.Objectives:(1)To explore the differences of various B-cell subpopulations and plasma cells in BM and PB between ITP patients and healthy controls(HCs),thus to clarify the anomalous development of B cells in ITP.(2)To explore the defective immune regulation of BM B cells in patients with ITP,and verify the feasibility of Breg transfusion in the treatment of ITP through animal models.(3)To explore the abnormal expression and distribution of B-cell related chemokines,cytokines and their receptors in ITP patients,so as to further improve the pathogenesis of ITP.Methods:(1)Sample collection:BM and PB samples of newly diagnosed ITP patients and allogeneic hematopoietic stem cell transplantation donors were collected,and separated into plasma,bone marrow mononuclear cells(BMMCs),and peripheral blood mononuclear cells(PBMCs),respectively.(2)Enzyme-linked immunosorbent assay(ELISA):the concentrations of CXCL13,B-cell activating factor(BAFF),a proliferating-inducing ligand(APRIL),and anti-patelet glycoprotein antibodies in plasma were determined by ELISA kits.(3)Flow cytometry:the frequency of total B cells,immature B cells,naive B cells,memory B cells,Bregs,plasmablasts,short-lived/long-lived plasma cells,and the expression of chemokine/cytokine receptors CXCR5,BAFF-R,BCMA and TACI on B cells were quantified by using multicolor flow cytometry.(4)Quantitative polymerase chain reaction(qPCR):the mRNA level of transcription factors associated with B cell differentiation were detected in PBMCs and BMMCs;the mRNA level of chemokine/cytokine receptors were detected in magnetically sorted B cells.(5)In vitro functional assays of Bregs:Bregs were induced by recombinant human CD40 ligand and CpG-ODN in vitro,then co-cultured with homologous monocytes or CD4+T cells.The inflammatory factors secreted by monocytes and CD4+T cells,and the differentiation of regulatory T cells(Tregs)before and after co-culture were determined.(6)Construction of active ITP murine models:platelets from wild type(WT)C57BL/6 mice were used to immunize homologous CD61 knockout(CD61-KO)mice.The splenocytes of immunized CD61-KO mice were injected into severe combined immunodeficiency(SCID)mice which had received 200cGy irradiation.(7)In vivo functional assays of Bregs:active ITP murine models without any intervention were set as control group,the experimental groups were as follows:active ITP murine models transfused with BM-Bregs from WT mice;active ITP murine models transfused with BM-Bregs from immunized CD61-KO mice.The blood routine test was executed weekly for 4 consecutive weeks.Results:(1)Developmental impairments of B cells in ITP:total B cells were decreased in BM of ITP patients compared with HCs.the mRNA level of transcription factor Pax5,which determines the specificity of B-cell lineage,was much lower in PBMCs and BMMCs of ITP patients than in HCs.(2)Disrupted B-cell subsets in ITP:there were lower percentage of CD27-naive B cells and higher proportion of CD27+memory B cells in BM from ITP patients compared to HCs.There was no statistical difference in plasmablasts or short-lived plasma cells between the two groups.Patients who were tested positive for anti-platelet autoantibody had higher frequency of CD19-CD38highCD138+long-lived plasma cells than HCs and antibody-negative patients.(3)Defective number and function of BM Bregs in ITP:The frequencies of CD19+CD24highCD38highBregs in BM and PB of ITP patients were both significantly lower than that of HCs.The ability to produce IL-10 and TGF-β by BM B cells was weaker in ITP patients.Besides,the inhibition of monocytes’ TNF-α secretion and CD4+T cells’ IFN-γ secretion by BM-Bregs from ITP patients was greatly reduced compared with that in HCs,the promotion of Tregs was also compromised in ITP patients.(4)The impacts of Breg transfusion in active ITP murine models:the platelet count of active ITP mice injected with BM-Bregs from immunized CD61-KO mice was significantly higher than that of control mice on days 21 and 28 after splenocytes transfusion.However,the thrombocytopenia in mice injected with BM-Bregs from WT mice was even more serious.(5)Abnormal B-cell chemotactic microenviroments in ITP:the concentrations of CXCL13,BAFF and APRIL in BM plasma from ITP patients were significantly higher than those from HCs,levels of CXCL13 and BAFF in PB plasma were also higher than that of HCs.ITP patients had higher concentrations of APRIL in BM than in their PB plasma.The protein and mRNA levels of CXCR5 and BAFF-R in BM and PB B cells of ITP patients were both increased compared to HCs,while only BM B cells of ITP patients expressed more BCMA than HCs.There was no significant difference in TACI between the two groups.Conclusions:(1)B cells from ITP patients had some developmental impairments at early stage,and tended to be over activated at later stage to differentiate into antibody-secreting cells.(2)The defective quantity and quality of BM-Bregs resulted in imperfect immune tolerance of ITP patients.Transfusion of BM-Bregs from immunized CD61-KO mice could be helpful in alleviating thrombocytopenia in active ITP murine models.(3)The enhanced interaction between CXCL13 and CXCR5 and hyperfunction of BAFF-APRIL systems contributed to the abnormal chemotaxis and distribution of B cells in ITP.Part Ⅱ:Bruton’s tyrosine kinase inhibitor in the treatment of primary immune thrombocytopenia through blocking autoantibody-mediated platelet destruction Background:Primary immune thrombocytopenia(ITP)is an autoimmune disease caused by the dysfunction of a variety of hematopoietic cells,the clinical manifestations of which are heterogeneous.There may be only isolated thrombocytopenia,or accompanied by purpura and ecchymosis in some patients.Gastrointestinal bleeding,urogenital hemorrhage,and even life-threatening intracranial bleeding could be found in severe cases.Glucocorticoids have been the first-line treatments of ITP for many years,but there are lots of adverse events after long-term use.Several second-line drugs have achieved remarkable efficacy,such as rituximab and thrombopoietin receptor agonists(TPO-RAs).However,due to the great complexity of the pathogenesis of ITP,a part of patients still have no response or rapidly relapse,and eventually become refractory ITP.Therefore,it is necessary to explore new therapeutic strategies for those relapsed/refractory ITP patients to improve their quality of lives.Bruton’s tyrosine kinase(BTK)is a non receptor tyrosine kinase of Tec family which is located in the cytoplasm.It is widely expressed in hematopoietic cells except T cells,plasma cells and natural killer(NK)cells.BTK participates in varieties of signal transduction of innate and adaptive immunity,such as B cell receptor(BCR)pathway,toll-like receptor(TLR)pathway,G-protein coupled receptor(GPCR)pathway and many cytokine receptor pathways,thus is closely related to cell survival,proliferation,differentiation,and apoptosis.At present,BTK inhibitor(BTKi)has been widely used in the treatment of hematological malignancies including lymphoma and lymphocytic leukemia.With the emergence of more and more second-generation BTKis with higher selectivity,they have gradually become the therapeutic choices of autoimmune diseases such as rheumatoid arthritis,lupus nephritis and multiple sclerosis.However,there is little basic research on the relationship between BTK and ITP.Platelet destruction mediated by anti-platelet glycoprotein autoantibodies is regarded as the main pathogenesis of ITP.B cells are the sources of autoantibodies and macrophages are the executors of platelet phagocytosis.They are the most important effectors in this pathway.Meanwhile,both B cells and macrophages express BTK.Therefore,we speculated that inhibition of BTK may prevent the destruction of platelets by regulating the phenotype and function of B cells and macrophages.Objectives:(1)To explore whether BTKi can correct the immune dysregulation of B cells in patients with ITP.(2)To explore whether BTKi can inhibit the destruction of platelets by macrophages.(3)To explore the therapeutic effects and safety of BTKi on ITP murine models,so as to lay the foundation for its clinical application.Methods:(1)Separation and in vitro culture of B cells:peripheral blood mononuclear cells(PBMCs)were separated from patients with active ITP by Ficoll-density gradient centrifugation,CD19+ B cells were sorted by magnetic beads and inoculated in 24-well culture plate.Anti-human IgM was used to stimulate BCR.The concentrations of BTKi were set as InM,10nM,100nM and 1μM,the control group was treated with DMSO of equal volume.BTKi needs to be pre-incubated with B cells for 1hr before any stimulation in culture.(2)Determination of B-cell surface markers:after 24hr of culture,the expression of a series of activation and costimulation molecules including CD69,CD86,CD80,CD40,MHC class I molecules(HLA-A,B,C),and MHC class Ⅱmolecules(HLA-DR/DP/DM/DQ)were detected by flow cytometry.(3)Apoptosis and proliferation:after 48hr of culture,the supernatant was collected for cytokine determination,and the degree of apoptosis was quantified through annexin V-PI double staining.As for proliferation,the expression of nucleus protein Ki67 was detected by flow cytometry after 72hr of culture.Another way to evaluate proliferation was to stain B cells with CFSE before culture,and the fluorescence intensity of CFSE was examined by flow cytometry after 72hr of culture.(4)B cell differentiation:IgM and CD40L were supplemented in culture medium to assist B cells differentiating into plasma cells.After 72hr of culture,the expression of CD27,CD38 and CD138 were quantified by flow cytometry.(5)Macrophage induction and Fcγ receptor(FcγR)activation:CD 14+monocytes from PBMCs of ITP patients were sorted by magnetic beads,macrophages were induced by recombinant human macrophage-colony stimulating factor(rhM-CSF)for 7 days.Macrophages were pre-incubated with BTKi for 1hr before stimulated with anti-human IgG-Fc,the culture supernatant was collected after 4 hr of stimulation.(6)In vitro phagocytosis of platelets by macrophages:platelets from healthy donors were isolated and stained with 5-chloromethylfluorescein diacetate(CMFDA),then were opsonized with anti-human CD41a antibodies.After 1hr co-culture of platelets and macrophages,the fluorescence intensity of CMFDA in macrophages was acquired on a flow cytometer.(7)BTKi in the treatment of active ITP murine models:the active ITP murine models were constructed by wild type mice-immunized CD61-KO mice and severe combined immunodeficiency(SCID)mice.The BTKi powder was suspended in 0.5%CMC-Na and was intragastrically administered to ITP mice in 10mg/kg once a day.The control group was treated with equivalent 0.5%CMC-Na once a day.Blood routine test was executed once a week for 4 consecutive weeks.The serum anti-CD61 antibodies were detected on days 14 after splenocytes transfusion,B cells and plasma cells in peripheral blood,spleen,and bone marrow were quantified by flow cytometry on days 28 after splenocytes transfusion.(8)Enzyme-linked immunosorbent assay(ELISA):the concentrations of cytokines in culture supernatant of B cells and macrophages,and in serum of active ITP murine models were detected by ELISA kits.Results:(1)BTKi inhibited the expression of activation markers downstream of BCR pathway and reduced cytokine secretion of B cells from ITP patients in vitro:BTKi at 1nM-1μM inhibited the expression of CD69 in a dose-dependent manner,while only significantly inhibited CD86 at 100 nM and 1μM.Compared with the control group which was treated with DMSO,BTKi reduced the expression levels of HLA-DM,HLA-DP,HLA-DQ,and HLA-DR in B cells,but had no effect on CD40,CD80 or HLA-A,B,C.Futhermore,BTKi could down-regulate the secretion of TNF-α and IL-6 by B cells stimulated with IgM.(2)BTKi promoted apoptosis and inhibited proliferation of autoreactive B cells from patients with ITP in vitro:although no significant effect of BTKi on apoptosis was found in PBMCs without any stimulation,BTKi from 1nM to 1μM all increased the percentages of early apoptosis(Annexin V+PI-)and late apoptosis(Annexin V+PI+)cells in IgM-activated B cells.In terms of proliferation,the positive rate of Ki67 in B cells treated with BTKi was decreased obviously compared with DMSO-treated cells,and the fluorescence intensity of CFSE was higher in BTKi-treated B cells.Therefore,it was confirmed that the proliferation of BCR-activated B cells was suppressed by inhibition of BTK.(3)BTKi inhibited B cell differentiation of ITP patients in vitro:BTKi at 100 nM and 1μM significantly reduced the proportion of CD27-CD38 double positive cells in CD19+ B cells compared with control group.The number of CD 13 8-positive plasma cells was decreased after incubated with BTKi at all concentrations.(4)BTKi inhibited FcγR-mediated phagocytosis and cytokine secretion by macrophages of ITP patients:the phagocytosis of platelets by monocyte-derived macrophages was decreased significantly after incubation with BTKi.The secretion of proinflammatory cytokines induced by FcyR activation was also inhibited by BTKi,including TNF-α,IL-6,and IL-1β.(5)Immunomodulatory effects of BTKi on active ITP murine models:BTKi could alleviate thrombocytopenia in early stage.On days 14,21 and 28 after splenocytes transfusion,the platelet count of mice administered with BTKi was significantly higher than that in control mice.Serum anti-CD61 autoantibodies in BTKi group was much lower than that in the control group.The frequencies of total B cells in peripheral blood and GL-7+germinal center B cells in splenocytes in BTKi-treated mice were all reduced compared to control mice.The plasma cells in spleen and bone marrow of BTKi group were both much lower than that in control group.Besides,the levels of IL-6 and IL-1β in serum of BTKi mice were significantly lower than those in control mice.Conclusions:(1)BTKi could promote B cell apoptosis,reduce B cell proliferation,proinflammatory cytokine secretion and plasma cell differentiation of ITP patients in vitro through inhibiting BCR activation.(2)BTKi suppressed phagocytosis of platelets and proinflammatory factors secretion by macrophages of ITP patients in through inhibiting FcyR activation.(3)BTKi could rapidly increase the platelet count of active ITP murine models and improve the survival rates through reducing the differentiation of plasma cells,production of autoantibodies,and secretion of proinflammatory cytokines.Part Ⅲ:Clonal hematopoiesis in the differential diagnosis,clinical characterization,and prognosis of primary immune thrombocytopenia Background:Clonal hematopoiesis(CH)is described as expansion of a clonal blood cell population with somatic mutations.CH is not morbid hematopoiesis or malignant expansion,but does have some specific biological characteristics including competitive expansion advantage and unbalanced hematopoietic function.CH was firstly raised in a study of X-chromosome inactivation among elderly healthy women,and has been extensively identified in various clinical settings as the wide use of high-throughput sequencing technologies,such as hematological diseases,solid tumors,and cardiovascular diseases.The aging of hematopoietic stem cells and environmental pressures together lead to CH,which has become the pathological basis of various hematological malignancies.With the deepening understanding of CH,a new definition appeared:clonal hematopoiesis of indeterminate potential(CHIP)refers to CH defined by recurrent mutations in driver genes associated with hematological malignancies,detected at a variant allele frequency(VAF)≥ 0.02 in adults with no history of hematological malignancies.CHIP is considered to be more clinically significant.CHIP carriers has a 0.5%-1%risk per year of progression to hematologic neoplasia,and a increased all-cause mortality.Primary immune thrombocytopenia(ITP)is a hemorrhagic disease mediated by a variety of immune disorders with the incidence rate of about 5-10/100000.The precise diagnosis and treatments have always been a difficult problem.Hereditary thrombocytopenia,atypical aplastic anemia(AA)and hypoplastic myelodysplastic syndrome(hMDS)could be misdiagnosed sometimes.So far,the association between CH and ITP remains unclear.Exploring the prevalence of CH in ITP may play a guiding role in its differential diagnosis;drawing the CH profile of ITP could be useful in predicting disease severity and treatment responsiveness.Objectives:(1)To explore the prevalence of CH and the profile of mutations in ITP,demonstrating the role of targeted gene sequencing in the differential diagnosis between ITP and other atypical hematological diseases accompanied with thrombocytopenia.(2)To identify the common mutations and types in ITP,draw CH profile of patients with ITP.(3)To explore the promoters of CH in ITP,and to work out whether CH is associated with disease severity and therapeutic effects,thus clarify the role of CH in the prognosis of ITP.Methods:(1)Sample collection:peripheral blood samples(EDTA anti-coagulation)of 105 ITP patients,128 AA patients and 86 hMDS patients were collected in 8 hospitals in Shandong province.The basic information and clinical characteristics of patients were recorded,and the patients were followed up.(2)DNA extraction:MagMAXTM DNA multi sample ultra 2.0 kit was used to extract the DNA of total leukocytes after erythrolysis of whole blood,and the DNA samples were quantified by spectrophotometer.(3)Amplicon sequencing:38 genes closely related to the pathogenesis of myeloid hematological malignancies were selected for next-generation sequencing.Sequence was executed on an illumina MiseqDx PE250 platform with 90%coverage.Bases of sequence with Phred score Q<20 and population variants whose frequencies exceeded 1%were ruled out.The variants were annotated by the Ensembl GRch38.The detection threshold for mutations was determined as VAF ≥ 1%,and the cut-off value for defining CH was VAF≥ 2%,while VAF>40%were discarded for the possibility of germline variant.(4)Data analysis:the correlation between sequencing results and misdiagnosis rate,patients’ age,gender,disease duration and severity,chronic clinical complications,and treatment responsiveness was analyzed.Results:(1)The misdiagnosed cases and patients with VAF<2%or>40%were excluded.Finally,102 ITP patients,126 AA patients and 80 hMDS patients were enrolled in the present study.The prevalence of CH in ITP(10.78%)was significantly lower than that in AA(19.05%,OR 5.99,95%confidence interval 5.01-7.16,P<0.001)and hMDS(68.75%,OR 20.34,95%confidence interval 16.71-24.77,P<0.001).(2)Gender was not found to be the influencing factor of CH in all 3 diseases.The prevalence of CH was increased with the growth of age in ITP and AA,but not in hMDS.The prevalence of CH in ITP patients over 65 years old was higher than that under 65 years old(27.27%vs.6.25%,P=0.005).(3)The percent of patients with multiple mutations in ITP(9.09%)were less than AA(16.67%)and hMDS(23.64%).The VAF values of mutated genes in ITP were significantly lower than that in AA(5.64%± 1.80%vs.12.46%± 8.27%,P=0.008)and hMDS(5.64%± 1.80%vs.16.41%± 11.54%,P=0.002).(4)In ITP group,more than half of the patients had mutations of DNMT3A,followed by ASXL1/TET2(18.2%)and SRSF2/TP53(9.1%).By contrast,the most frequently mutated genes were DNMT3A/ASXL1/TET2(16.67%),BCOR(12.50%),and PIGA/U2AF1(8.33%)in AA patients.Similarly,gene mutations in hMDS were concentrated in DNMT3A/ASXL1/SF3B1(18.18%),U2AF1(10.91%),TET2(9.09%),and TP53(7.27%).Single-nucleotide variants were dominant in all types of mutations,among which transitions were the most prevalent.(5)There was no obvious correlation between CH in ITP patients and several chronic conditions including disease duration,hypertension,diabetes,anti-platelet glycoprotein autoantibodies,cerebral infarction,and coronary heart disease.Moreover,no difference was observed in blood cell indices between CH-harboring group and non-CH group in ITP patients.However,CH was positively correlated with bleeding severity as CH-ITP patients were 6.95 times more likely to develop severe bleeding than those without CH(P=0.008).(6)Initial response rate in ITP patients with CH was lower compared to patients without CH(54.5%vs.80.6%,P=0.054).Patients who failed the first-line treatments were more likely to be resistant to subsequent second-line drugs,and the percentage of refractory cases in CH positive group was significantly higher than that in the non-CH group(36.4%vs.4.5%,P=0.002).Conclusions:(1)The prevalence of CH in ITP was much lower than that in AA and hMDS,the mean VAF value of the mutated genes was also significantly lower than that in AA and hMDS.Therefore,targeted gene sequencing could be helpful in distinguishing atypical AA and hMDS from refractory ITP,so as to further improve the accuracy of differential diagnosis.(2)The most common mutations in CH-ITP were DNMT3A,TET2 and ASXL1,the most prevalent type of mutation was transition,which was similar in AA and hMDS.(3)The occurrence of CH in ITP was related to aging,but not to gender,disease duration or anti-platelet autoantibodies.CH-ITP patients tended to have lower platelet count and higher risk of severe bleeding compared with non-CH patients.Therefore,targeted gene sequencing was helpful in judging disease severity.(4)Initial response rate in ITP patients with CH was lower compared to patients without CH,the proportion of refractory cases in patients with CH was significantly higher than that in non-CH patients.Therefore,detection of CH might indicate a poor treatment responsiveness,and targeted gene sequencing could be helpful in predicting the prognosis of ITP.