Excessive Receptor Editing and Generation of Autoreactive Antibodies in SLE

描述（由申请人提供）：系统性红斑狼疮（SLE）的特点是针对核抗原的高亲和力自身抗体过量产生。疾病相关自身抗体在SLE发病机制、疾病进展和组织/器官破坏中起重要作用。为了深入了解SLE自身抗体产生的机制，我们进行了单细胞PCR分析，研究了3例活动性SLE患者的功能性抗体库。我们对来自SLE浆细胞和原始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CDR3长区域具有多个带正电荷的精氨酸残基。引人注目的是，在三名活动性SLE患者中，IgH和IgL都显示出过度而不是低效的受体编辑迹象，下游J:5和J的使用升高；部分患者的IgL基因中有3个基因，所有3例患者的IgH基因中VH替代产物的频率均显著升高。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意想不到的后果。过度编辑IgL基因可能耗尽IgL库，增加下游J的使用；3基因。这样的搞笑;基因不能通过额外的重组而改变。过量的VH替换将产生更多具有长CDR3的IgH基因，这些基因富含带电氨基酸，可能直接编码自身抗体。事实上，鉴定的VH替代足迹优先编码CDR3区域内的带电氨基酸。我们对从VH替代产品中获得的5种重组抗体的初步测试表明，其中3种抗体与核或核周围抗原有强烈反应。基于这些观察结果，我们假设在活动性SLE患者中发生了过度的IgH和IgL基因受体编辑，并有助于产生针对核抗原的高亲和力自身抗体。为了验证这一假设，(1)我们将集中研究新诊断的成年非洲裔美国女性活动性SLE患者的统一人群，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组相比，这些患者中是否存在过度的受体编辑。我们将进行后续研究，以确定在同一患者中过度的受体编辑是否与疾病活动相关。(2)我们将利用来自SLE患者和正常对照的浆细胞和中核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是及时重要的，这将使我们能够设计特异性治疗来预防或减少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的标志是疾病相关自身抗体的产生。了解SLE过程中自身抗体的产生和富集机制将为SLE患者设计特异性治疗方案提供新的见解。目前的建议是基于我们最近的发现，在三个活动性SLE患者中，过度的受体编辑发生在IgH和IgL基因上。虽然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，特别是过度的VH替换可能会产生有害的后果。我们将直接验证活动期SLE患者是否存在过度的IgH和IgL基因受体编辑，从而导致高亲和力自身抗体的产生。

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.