Analysis of class switch recombination and somatic hypermutation in patients affected with autosomal dominant hyper-IgM syndrome type 2

Autosomal recessive form of hyper-IgM syndrome type 2 (AR-HIGM2) is secondary to mutations affecting both alleles of AICDA gene encoding activation-induced cytidine deaminase, characterized by defects of immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in most of the patients. We herein report the immunological phenotype of seven patients carrying a single heterozygous R190X mutation in AICDA. Variable defect in in vivo CSR inherited as an autosomal dominant (AD) trait strongly suggests that this heterozygous AICDA mutation causes HIGM (AD-HIGM2). In AD-HIGM2 B cells, CSR was consistently found impaired in vitro. However, in contrast to AR-HIGM2, the CSR-induced double-stranded DNA breaks in the switch region of IgM heavy chain gene were detected. The SHM frequency in V regions of IgM heavy chain gene in B cells was normal in all (but one patient). The characteristics of the AD-HIGM2 phenotype indicate that the AID C-terminal region may be involved in DNA repair machinery required for CSR.     

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis

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Activation-induced deaminase contributes to the antibody-independent role of B cells in the development of autoimmunity

B cells contribute to autoimmunity both as secretors of pathogenic antibodies and through the activation of autoreactive T cells. B cells and antibodies acquire higher affinity to self-antigen through a process known as immunoglobulin hypermutation or SHM. The contribution of SHM to pathogenic antibody development in lupus has been established in various autoimmune mouse models and by examining antibodies from patients. However, its role in the antibody-independent contribution of B cells to autoimmunity has not been examined. Herein, we generate lupus-prone MRL/lpr mice with a limited IgM-only B cell repertoire, no secreted antibodies and no SHM. This enabled us to isolate the role of somatic hypermutation in B cell-mediated autoimmunity. We found that SHM-deficiency correlated with a reduction in autoreactive B cells, a decrease in T cell activation and a decrease in kidney lymphocytic infiltration. These data establish AID as an important contributor to the antibody-independent role of B cells in autoimmunity.     

Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies

Humoral immunodeficiency caused by mutations in the Wiskott-Aldrich syndrome protein (WASp) is associated with failure to respond to common pathogens and high frequency of autoimmunity. Here we addressed the question how deficiency in WASp and the homologous protein N-WASp skews the immune response towards autoreactivity. Mice devoid of WASp or both WASp and N-WASp in B cells formed germinal center to increased load of apoptotic cells as a source of autoantigens. However, the germinal centers showed abolished polarity and B cells retained longer and proliferated less in the germinal centers. While WASp-deficient mice had high titers of autoreactive IgG, B cells devoid of both WASp and N-WASp produced mainly IgM autoantibodies with broad reactivity to autoantigens. Moreover, B cells lacking both WASp and N-WASp induced somatic hypermutation at reduced frequency. Despite this, IgG1-expressing B cells devoid of WASp and N-WASp acquired a specific high affinity mutation, implying an increased BCR signaling threshold for selection in germinal centers. Our data provides evidence for that N-WASp expression alone drives WASp-deficient B cells towards autoimmunity.     

Rheumatoid arthritis and anti-thyroid antibodies

The aim of this study was to evaluate the quality of B cell responses in patients with Inflammatory Bowel Disease (IBD) and healthy individuals of different ages, vaccinated with the pandemic (p)2009 influenza vaccine. The in vivo response was measured by the hemagglutination inhibition (HAI) assay, which represents the most established correlate with vaccine protectiveness. The in vitro response was measured by activation-induced cytidine deaminase (AID) in cultures of vaccine-stimulated PBMC. Both responses are somewhat impaired in IBD patients undergoing anti-TNF-α treatment but these are much more decreased in IBD patients undergoing treatment with anti-TNF-α and immunosuppressive (IS) drugs. These latter patients had in vivo and in vitro B cell responses similar to those of elderly individuals. Moreover, as we have previously demonstrated in healthy subjects, the in vitro response to the polyclonal stimulus CpG may be used as a biomarker for subsequent vaccine response and AID activation is correlated with the serum response in IBD patients, as it is in healthy individuals. These results altogether indicate that IBD patients on anti-TNF-α and IS have significantly impaired in vivo and in vitro B cell responses, as compared to those on monotherapy. This is the first report to demonstrate that B cell defects, as measured by the autonomous AID reporter, in IBD patients contribute to reduced humoral responses to the influenza vaccine, as we have previously shown for elderly individuals.     

Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE.     

AID induces intraclonal diversity and genomic damage in CD86+ chronic lymphocytic leukemia cells

The activation‐induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off‐target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B‐cell malignancies. Although it has been shown that AID is expressed in B‐cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B‐cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sμ), showing ongoing AID activity in vivo during disease progression. This AID‐mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ‐H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.     

抗核抗体和抗核抗体谱联合检测诊断自身免疫性疾病的临床价值

目的：探讨自身免疫性疾病(AID)患者血清抗核抗体(ANA)和抗核抗体谱(ANAs)的表达及临床意义。方法选取2013年1月至2015年4月海南省第三人民医院收治的194例AID患者和103例非AID患者(对照组),根据美国风湿病学会的诊断标准将194例AID患者分为系统性红斑狼疮(SLE)组、混合性结缔组织病(MCTD)组、干燥综合征(SS)组、硬皮病(PSS)组、类风湿性关节炎(RA)组和过敏性紫癜(AP)组。采用间接免疫荧光法(IIF)和免疫印迹法(IBT)检测所有患者的ANA和ANAs,并进行统计分析。结果女性患者检出ANA、Ul-SnRNP、抗dsDNA、抗SSA、抗SSB、抗核小体和抗组蛋白的阳性率均明显高于男性,差异均有统计学意义(P<0.05);在不同AID患者中ANA检测均呈阳性,其中SLE组阳性率最高,达93.5%,其次分别为MCTD组(85.0%)、SS组(79.2%)、PSS组(69.2%)、RA组(35.8%)、AP组(20.0%),均明显高于对照组(4.9%),差异均有统计学意义(P<0.05)。RA组主要表达抗组蛋白(47.7%),明显高于对照组(1.0%)。SLE组主要表达抗SmDl (37.9%)、抗dsDNA (66.1%)、抗SSA (41.9%）、抗核小体(77.4%)和抗组蛋白(37.1%),均明显高于对照组,差异均有统计学意义(P<0.05)。SS组主要表达抗SSA (58.3%)和抗SSB (50.0%),均明显高于对照组。MCTD组主要表达Ul-SnRNP (35.0%)、抗SSA (60.0%)和抗SSB (30.0%),均明显高于对照组。PSS组和AP组主要表达抗Scl-70(46.1%和10.0%),均明显高于对照组,差异均有统计学意义(P<0.05);130例ANA阳性患者检出97例ANAs阳性,符合率74.6%;64例ANA阴性患者检出58例ANAs阴性,符合率为90.6%;AID患者ANA核型分布以核颗粒型和核均质型为主,其他核型较少见;核颗粒型多为抗SSA (48.5%)、抗SSB (23.7%)和抗组蛋白(26.8%),核均质型多为抗dsDNA (28.9%)、抗核小体(29.9%)和抗组蛋白(25.7%)。结论 ANA可辅助诊断自身免疫性疾病,但缺乏特异性,联合抗核抗体谱检测对AID的诊断有重要意义。     

抗核抗体谱的检测在临床应用中的研究进展

自身免疫性疾病（autoimmune diseases,AID）确切的发病机制目前尚不清楚,且临床症状和体征不典型,对其诊断和鉴别诊断较复杂,但由于该类疾病都存在一些特异性的自身抗体,所以对自身免疫性疾病的诊断不仅依赖于临床表现,而且很大程度上取决于免疫学实验诊断,特别是实验室的抗核抗体（antinuclear antibody,ANA）检测[1].通过对ANA类型及其滴度的检测,能为自身免疫性疾病的诊断、分型、治疗和预后判断提供重要的依据,本文就ANA和ANA谱的检测方法及其临床应用做一综述.