Accumulation Of Self-reactive Naive B Cells In Primary Immune Thrombocytopenia And A CARD9 Single Nucleotide Polymorphism Rs4077515 Is Associated With Reduced-Susceptibility To Primary Immune Thrombocytopenia

Part I Accumulation of self-reactive(?)aive B cells in primary immune thrombocytopeniaAutoimmune diseases(AIDs)are a collection of diseases that result from failure to sustain tolerance to self-molecules.Primary immune thrombocytopenia(ITP)is a common autoimmune bleeding disease characterized by over-production of anti?platelet antibodies,low platelet counts and subsequent increased bleeding risk,accounting for about one-third of clinical hemorrhagic events.Clinical manifestations of ITP include skin and mucous membrane bleeding,severe cases could occur life-threatening visceral bleeding and intracranial hemorrhage.The pathogenesis of ITP is not clear so far.At present,the mainstream opinion is that defective immune tolerance to self-antigens(mainly platelet glycoproteins GP ?b/?a,GP ?b/? and GP ?a/?a,etc.)play a key role in the pathogenesis of ITP.Anti-platelet antibodies are detectable in approximately 60-70%of ITP patients.Autoantibodies mediate excessive clearance of platelets by the reticuloendothelial system,leading to accelerated platelet destruction in the periphery.Abnormalities in megakaryocyte maturation and apoptosis in the bone marrow result in reduced platelet production.In addition,dysregulated cellular immune is also play an important role in the pathogenesis of ITP,such as cytotoxic T lymphocyte-mediated direct destruction of platelets,helper T cell imbalance(increased Th1/Th2 ratio),regulatory T cell dysfunction also promote the development of ITP.Anti-platelet antibodies are thought to play a key role in the pathogenesis of ITP.However,the underlying mechanism that accounts for autoantibody production is not clear yet.B lymphocyte is a major player in maintaining human immunity by secreting immunoglobulins(antibodies)against antigens.During B cell development,immunoglobulins undergo several mechanisms,including V(D)J recombination,somatic hypermutation,receptor editing,and isotype class switch,to extend its diversity.During these processes,the Ig repertoire is extensively diversified with unpredicted specificities which include auto-reactive one.And antibodies against self-antigens(autoantibodies)may be produced as the by-products.Early developing B cells in bone marrow are highly self-reactive but are efficiently eliminated by tolerance checkpoints and,as a result,they contribute to only a small portion in the mature naive B cell compartment.Failures to establish self-tolerance during early B cell development have been found in some autoimmune diseases,such as systemic lupus erythematosus and rheumatoid arthritis.The impaired early self-tolerance leads to accumulation of peripheral naive B cells,which are subsequently selected and evolved into the disease-related antibody-secreting B cells.In ITP patients,the anti?platelet antibodies are usually isotype-switched IgG and somatically mutated,indicating the late B cell tolerance in the germinal center is defective.However,the status of the early B cell tolerance in ITP is unknown.Known mechanisms of early B cell tolerance include receptor editing,deletion.and anergy.Of them,receptor editing is the predominant mechanism of central tolerance,especially toward membrane antigens,and in general occurs on 20 to 50%of immature B cells.The receptor editing eliminates the unwanted Ig genes by a secondary reco(?)bination of upstream variable(V)segments and downstream joining(J)segments,and thus increases the V-J distance of the Ig genes.Failures of receptor editing on the self-reactive Ig genes were reported to contribute to autoreactive antibodies in multiple autoimmune diseases.Objective:In this study,we characterized the functional Ig repertoire in ITP patients by sequencing and functional experiments,in order to ascertain the status of early B cell tolerance in ITP and its role in the pathogenesis of ITP.And further explore the possible mechanisms that affect early B cell tolerance in ITP patients,for seeking of a new potential therapeutic target.Methods:1.In total,eight patients who were initially diagnosed with ITP according to the established practice consensus were recruited.As control,eight age-matched healthy donors(HDs)without any known autoimmune diseases were recruited.Blood samples were collected before patients were treated.2.Four untreated ITP patients and four age-matched healthy donors(HDs)without any known autoimmune diseases were recruited.10mL of peripheral blood was taken from each subject.B cells were purified by Pan B Cell Isolation Kits.Total RNA was extracted using PAXgene Blood RNA Kit and then reverse transcribed into cDNA.Two rounds of PCR with a multiplex PCR Kit were performed to amplify the V(D)J fragments of Ig heavy chain(IgH)and Ig kappa chain(Ig?)genes for library construction.Next generation sequencing was performed using an Agilent 2100 Bioanalyzer.The VDJtools was used to post-analyze features of the Ig genes,including the basic statistics,segment usages,repertoire overlaps,diversity analysis,and repertoire clustering.VH replacement products were identified by searching the VH replacement "footprints" in the Ig heavy chain(IgH)V-D junctions using VH Replacement Footprint Analyzer-I(VHRFAI).3.Purified peripheral-blood mononuclear cells(PBMCs)from four newly-diagnosed ITP patients and four HDs were stained with anti-CD 10,CD 19,CD27 and IgM antibodies.Single naive B cells(CD10-CD19+CD27-IgM+)were directly sorted in 96-well PCR plates at one cell per well using a FACS Aria III.All samples were frozen immediately on dry ice for future studies.4.Immunoglobulin genes were amplified by a one-step RT-PCR and a nested PCR.PCR products were subcloned into corresponding IgGI,IgK,or Ig? expression vectors.Resulting constructs were then sequenced and analyzed by the IMGT/V-QUEST program and VHRFAI.Paired in-frame IgH and Ig?/Ig? genes from the same cell were co-transfected into HEK 293T cells using lipofectamine 2000 for antibody production and purification.5.Platelet membrane protein was purified and attached to ELISA plates.The ITP/HD-derived rmAbs were added as the primary antibody.Anti-platelet ELISA was used to detect the reactivity to platelets of the above rmAbs.6.Platelet immunofluorescence test was used to compare the reactivity to platelets of the rmAbs derived from ITP patients and HDs,separately.7.Flow cytometry was performed to further verify the reactivity to platelets of the ITP/HD-derived rmAbs.8.GP ?b/?a,double-stranded DNA(dsDNA),lipopolysaccharide(LPS)and recombinant insulin were used as antigens,respectively.GP ?b/?a-reactive and polyreactive ELISA were performed to test the GP ?b/?a-reactivity and polyreactivity of the rmAbs.Results:1.Peripheral B cell clonality is altered in ITP patients.No significant difference was found between the final analyzed sequences of IgH and IgK or between those derived from HDs and ITP patients.However,ITP patients have fewer IgH clone types and fewer IgH diversities than HDs.2.Ig repertoire is changed in ITP patients.Use of downstream VK genes(VK4-1 to 3-20)and the upstream JK genes(JK1-3)were elevated in ITP patients.And we also found that VK-JK pair distances were reduced in ITP patients compared to in HDs.Elevated use of downstream VK and upstream JK genes and reduced VK-JK distances suggest that insufficient receptor editing on IgK occurred in these ITP patients.3.We have obtained 290 IgH gene sequences,217 IgK sequences,and 134 Ig?sequences derived from the peripheral naive B cells.ITP naive B cell-derived IgK genes revealed a profound tendency with use of downstream VK genes(VK4-1 to 3-20)and upstream JK genes(JK1-3)compared to the HD-derived IgK genes.The VK-JK pair distances were also significantly reduced in ITP patients compared to those in HDs.4.In HDs,only 1.94%(2/1 03)naive B cell-derived rmAbs wei positively platelet-reactive(platelet+).By comparison,14.53%(1 7/11 7)of the ITP-derived rmAbs were platelet+,significantly more than in HDs.Anti-platelet naive B cells are accumulated in ITP patients,indicating defective early B cell tolerance.5.ITP patients had significantly higher frequencies of GP ?b/?a positive(GP?b/?a+)or polyreactive naive B cells than HDs.Moreover,platelet+rmAbs and GP ?b/?a+/polyreactive rmAbs predominantly overlapped with each other.6.We compared the CDR3s derived from platelets and platelet-rmAbs and found that Platelet+rmAbs frequently have>2 positive charge-IgH CDR3s and>12 AA-Ig? CDR3s.The results showed that IgH CDR3s with more positively charged AAs and longer IgK CDR3s preferentially encode platelet+ rmAbs.Conclusions:1.Accumulation of anti-platelet naive B cells was found in patients with primary ITP,indicating a defective early B-cell tolerance.2.The defective early B-cell tolerance in ITP may be due to insufficient receptor editing in immunoglobulin genes.Significance:Our study identified the defective early B-cell tolerance in ITP through next generation sequencing of ITP/HD-derived Ig repertoire,sanger sequencing of Ig genes of naive B cells and a series of functional experiments.And we suspected the defective early B-cell tolerance in ITP may be due to insufficient receptor editing in immunoglobulin genes.This finding helps us to understand the status of central B-cell tolerance in ITP,which contributes to further improve the understanding of the pathogenesis of ITP.Part ? A CARD9 Single Nucleotide Polymorphism rs4077515 is associated with reduced susceptibility to primary immune thrombocytopeniaPrimary immune thrombocytopenia(ITP),formerly known as idiopathic or immune thrombocytopenic purpura,is a clinically common acquired autoimmune disorder characterized by a transient or persistent decrease in platelet count.It is important to understand the pathophysiology of primary ITP for seeking of more effective cinical interventions.Pathogenesis of ITP includes accelerated peripheral platelet destruction by the activated reticuloendothelial system and impaired platelet production in the bone marrow,which is related to defects in immune tolerance.The impaired self-tolerance leads to abnormal humoral and ce ular responses.Desiccation of cellular immunity is considered important in ITP pathophysiology.Dysregulation of T cell subgroup proportion(elevated T helper(Th)1/Th2 ratio)suggests that ITP is a Thl dominated disease.Th1 polarization and inflammatory cytokine abnormalities due to gene polymorphisms plays a role in the clinical features of ITP was well as in the response to treatment.Caspase-recruitment domain family member 9(CARD9)is an inflammation-related molecule,which can trigger inflammatory cytokine cascade.It mediates production of pro-inflammatory cytokines,which drive Th1 and Th17 responses.Polymorphisms(SNPs),as the most common form of genetic variants,affect the human genome and mediate individual susceptibility to some diseases.Recently published genome-wide association studies have demonstrated that the SNP rs4077515 in CARD9 in the human genome is strongly linked to the Development of several human autoimmune inflammatory diseases,including ankylosing spondylitis,rheumatoid arthritis.IgA nephropathy,Crohn's disease,ulcerative colitis and inflammatory bowel disease(IBD).Nevertheless,polymorphism does not affect the susceptibility to candidemia or recurrent vulvovaginal Candidiasis.However,to our knowledge,there exist few studies attempting to clarify the link between CARD9 rs4077 5 1 5 polymorphism and ITP.With regard of participation of dysregulated Th subsets in initiation and progression of ITP,as well as the important role of CARD9 in inflammatory cytokine production and Th polarization,we hypothesize that CARD9 polymorphism may be involved in the Pathogenesis of ITP.Objective:Taken together,established studies have investigated the associations between CARD9 rs4077515 polymorphism and several common autoimmune inflammatory diseases.And the results showed that the polymorphism played a protective,risky or meaningless role in different diseases.ITP is also a typical autoimmune inflammatory disease.Herein our Study was designed to explore whether there is an association between CARD9 rs4077515 polymorphism and primary ITP in the Chinese Han population.Methods:1.Based on the inclusion and exclusion criteria of the ITP International Working Group(I WG),we consecutively recruited 294 patients with ITP from 2007 to 2016 at the Department of Hematology,Qilu Hospital.Meanwhile,a total of 324 age-matched healthy participants were enrolled in our study.Peripheral venous blood from all participants were collected.In addition to disease susceptibility,ITP patients were further stratified in terms of severity,corticosteroid sensitivity,and refractoriness,to explore the link between CARD9 rs40775 15 polymorphism and more complex clinical data.2.Genomic DNA was extracted and purified from whole blood of above samples.The CARD9 gene polymorphism was amplified by polymerase chain reaction(PCR)from extracted DNA.Genotyping was performed by Sanger sequencing.3.The genotype data of ITP patients and healthy participants were obtained by sequencing.Then the chi-squared(?2)test or the Fisher's exact test were used to.nalyze the association between CARD9 rs4077515 polymorphism and ITP susceptibility,severity,corticosteroid sensitivity,and refractoriness.For the results with statistical significance,logistic regression analysis was further applied to detect the strength of correlation.Results:1.For CARD9 rs4077515,the AA rather than GG genotype was significantly associated with a decreased risk of susceptibility to ITP under the codominant model.This polymorphism indicated a protective effect.In accordance with the above finding,ITP patients carrying the GG/AG genotypes of CARD9 rs4077515 showed a 6.183-fold increase in susceptibility to ITP compared to patients carrying the minor genotype AA under the recessive model.Thus,the GG/AG genotypes of CARD9 rs4077515 greatly increased ITP susceptibility.2.ITP patients carrying the AG and AA/AG genotypes showed a decreased risk of developing severe ITP compared with patients carrying the major genotype GG under the codominant and dominant models.As a result,for CARD9 rs407751 5 polymorphism,the A allele may protect patients from severe ITP compared with the G allele.3.No association was detected between CARD9 rs4077515 polymorphism and sensitivity to corticosteroid therapy and refractoriness of ITP patients.Conclusions:1.The existence of the allele A,the mutant A A genotype and the heterozygous AG genotype of CARD9 rs4077515 play a protective role in ITP.2.CARD9 rs4077515 polymorphism had no effect on corticosteroid sensitivity or refractoriness of ITP.Significance:Our studies have demonstrated that CARD9 rs4077515 polymorphism is associated with reduced susceptibility to and severity of ITP and may serve as a predictive factor to monitor the progression of ITP.