Excessive Receptor Editing and Generation of Autoreactive Antibodies in SLE

SLE 中过度的受体编辑和自身反应性抗体的产生

• 批准号: 8059641
• 负责人: Zhixin Zhang
• 金额: $ 29.11万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059641
• 关键词: Adult; Affect; Affinity; African American; Age; Amino Acids; Antibodies; Antibody Affinity; Antibody Repertoire; Antigen Receptors; Antigens; Autoantibodies; Autoimmune Diseases; B-Lymphocytes; Binding; Biological Assay; Cell Lineage; Cells; Charge; Childhood; Chronic; Clonal Deletion; DNA Double Strand Break; Development; Disease; Disease Progression; Employee Strikes; Enzyme-Linked Immunosorbent Assay; Ethnic Origin; Etiology; Female; Flare; Follow-Up Studies; Frequencies; Gender; Gene Expression Profile; Gene Frequency; Generations; Genes; Genetic Recombination; Histones; Human; Immune response; Immunoglobulin Genes; Immunoglobulins; In Vitro; Indirect Immunofluorescence; Inflammatory; Interferons; Ligation; Measures; Mediating; Methods; Mus; Mutate; Newly Diagnosed; Nuclear; Nuclear Antigens; Organ; PTPN6 gene; Pathogenesis; Patients; Peptide Signal Sequences; Peripheral; Plasma Cells; Play; Population; Principal Investigator; Process; Production; Proteins; Receptor Gene; Recombinant Antibody; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Rheumatoid Arthritis; Role; Serum; Signal Transduction; Single-Stranded DNA; Site; Sjogren' s Syndrome; Staging; Stream; Systemic Lupus Erythematosus; Testing; Time; Tissues; V(D)J Recombination; anergy; base; central tolerance; complementarity-determining region 3; crosslink; cytokine; design; ds-DNA; exhaust; follow-up; insight; peripheral blood; prevent; programs; receptor

DESCRIPTION (provided by applicant): Systemic lupus erythematosus (SLE) is characterized by the over production of high affinity autoantibodies against nuclear antigens. The disease associated autoantibodies play important roles in SLE pathogenesis, disease progression, and tissue/organ destruction. To gain insight into the mechanisms responsible for autoantibody generation in SLE, we performed single cell PCR analysis to study the functional antibody repertoires in three active SLE patients. Our initial analyses of 193 IgH genes derived from SLE plasma cells and naove B cells revealed many features of autoantibodies, such as long CDR3 regions with multiple positively charged Arg residues. Strikingly, both the IgH and IgL repertoires in the three active SLE patients showed signs of excessive rather than inefficient receptor editing, with elevated usages of downstream J:5 and J;3 genes in the IgL genes of some patients and significantly elevated frequencies of VH replacement products in the IgH genes in all three patients. Normally, receptor editing is aimed to delete Ig genes encoding self reactive antibodies. However, excessive receptor editing might have undesired consequences. Excessive editing of the IgL genes could exhaust the IgL repertoire with increased usage of the downstream J;3 gene. Such Ig; genes can not be changed by additional recombination. Excessive VH replacement will generate more IgH genes with long CDR3 enriched with charged amino acids, which might directly encode autoantibodies. Indeed, the identified VH replacement footprints preferentially encode charged amino acids within the CDR3 regions. Our initial test of 5 recombinant antibodies derived from VH replacement products showed that 3 of them strongly reacted with nuclear or peri-nuclear antigens. Based on these observations, we hypothesize that excessive receptor editing of IgH and IgL genes occurs in active SLE patients and contributes to the generation of high affinity autoantibodies against nuclear antigens. To test this hypothesis, (1) we will focus on a uniform population of newly diagnosed adult African-American female active SLE patients and perform single cell PCR and real time LM-PCR analyses to determine if excessive receptor editing occurs in these patients in comparison with age, gender and ethnic matched controls. We will perform follow-up studies to determine if excessive receptor editing correlates with disease activities in the same patients. (2) We will express recombinant antibodies using Ig genes derived from the plasma cells and naove B cells of SLE patients and normal controls to determine if accumulated VH replacement products in SLE plasma cells encode high affinity autoantibodies against nuclear antigens. Understanding the mechanism responsible for the generation of high affinity autoantibodies in SLE is timely important, which will allow us to design specific treatments to prevent or reduce autoantibody generation in SLE patients.Systemic lupus erythematosus (SLE) is a devastating disease that affecting 1 million people world wide. Due to the unknown etiology, there is no specific treatment currently available for SLE. The hallmark for SLE is the production of disease associated autoantibodies. Understanding the mechanism of the production and enrichment of autoantibodies during the course of SLE will provide new insight to design specific treatment for SLE patients. The current proposal is based on our recent finding that excessive receptor editing occurs on the IgH and IgL genes in three active SLE patients. Although receptor editing is originally aimed to deleting Ig genes encoding autoreactive antibodies, excessive receptor editing, especially excessive VH replacement might have detrimental consequences. We will directly test the hypothesis if excessive receptor editing of IgH and IgL genes occurs in active SLE patients and thus contributes to the generation of high affinity autoantibodies.

描述（由申请人提供）：系统性红斑狼疮（SLE）的特征是针对核抗原的高亲和力自身抗体的过量产生。疾病相关自身抗体在 SLE 发病机制、疾病进展和组织/器官破坏中发挥重要作用。为了深入了解 SLE 中自身抗体生成的机制，我们进行了单细胞 PCR 分析，以研究三名活动性 SLE 患者的功能性抗体库。我们对源自 SLE 浆细胞和幼稚 B 细胞的 193 个 IgH 基因的初步分析揭示了自身抗体的许多特征，例如具有多个带正电荷的 Arg 残基的长 CDR3 区域。引人注目的是，三名活动性 SLE 患者的 IgH 和 IgL 库都显示出过度而不是低效受体编辑的迹象，一些患者 IgL 基因中下游 J:5 和 J;3 基因的使用增加，并且所有三名患者 IgH 基因中 VH 替代产物的频率显着增加。通常，受体编辑的目的是删除编码自身反应性抗体的 Ig 基因。然而，过度的受体编辑可能会产生不良后果。 IgL 基因的过度编辑可能会随着下游 J;3 基因的使用增加而耗尽 IgL 库。这样的免疫球蛋白；基因不能通过额外的重组而改变。过度的VH替换会产生更多带有长CDR3的IgH基因，其中富含带电氨基酸，这些基因可能直接编码自身抗体。事实上，所识别的 VH 替换足迹优先编码 CDR3 区域内的带电氨基酸。我们对源自 VH 替代产品的 5 种重组抗体的初步测试表明，其中 3 种与核或核周抗原发生强烈反应。基于这些观察，我们假设活动性 SLE 患者中发生了 IgH 和 IgL 基因的过度受体编辑，并有助于产生针对核抗原的高亲和力自身抗体。为了检验这一假设，(1)我们将重点关注新诊断的成年非裔美国女性活动性 SLE 患者的统一群体，并进行单细胞 PCR 和实时 LM-PCR 分析，以确定与年龄、性别和种族匹配的对照相比，这些患者中是否发生过度的受体编辑。我们将进行后续研究，以确定过度的受体编辑是否与同一患者的疾病活动相关。 (2)我们将使用源自SLE患者和正常对照的浆细胞和幼稚B细胞的Ig基因表达重组抗体，以确定SLE浆细胞中积累的VH替代产物是否编码针对核抗原的高亲和力自身抗体。了解 SLE 中产生高亲和力自身抗体的机制非常重要，这将使我们能够设计具体的治疗方法来预防或减少 SLE 患者自身抗体的产生。系统性红斑狼疮 (SLE) 是一种毁灭性的疾病，影响着全世界 100 万人。由于病因不明，目前尚无针对 SLE 的特异性治疗方法。 SLE 的标志是产生与疾病相关的自身抗体。了解 SLE 病程中自身抗体产生和富集的机制将为设计 SLE 患者的特异性治疗提供新的见解。目前的提议是基于我们最近的发现，即三名活动性 SLE 患者的 IgH 和 IgL 基因上发生了过度的受体编辑。尽管受体编辑最初的目的是删除编码自身反应性抗体的 Ig 基因，但过度的受体编辑，尤其是过度的 VH 替换可能会产生有害的后果。我们将直接检验该假设是否在活动性 SLE 患者中发生 IgH 和 IgL 基因的过度受体编辑，从而有助于产生高亲和力自身抗体。