Excessive Receptor Editing and Generation of Autoreactive Antibodies in SLE

描述(申请人提供)：系统性红斑狼疮(SLE)的特征是产生针对核抗原的高亲和力自身抗体。疾病相关自身抗体在SLE的发病机制、疾病进展和组织/器官破坏中起重要作用。为了深入了解系统性红斑狼疮自身抗体产生的机制，我们进行了单细胞聚合酶链式反应分析，研究了三名活动期SLE患者的功能性抗体谱。我们对来自SLE浆细胞和NAOVE 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，例如带有多个带正电荷的Arg残基的长CDR3区域。值得注意的是，三名活动期SLE患者的IgH和IGL谱带都显示出过度而不是低效的受体编辑迹象，部分患者IGL基因下游J：5和J；3基因的使用量增加，所有三名患者IgH基因中VH替换产物的频率都显著增加。正常情况下，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受欢迎的后果。过度编辑IGL基因可能会随着下游J；3基因使用量的增加而耗尽IGL谱系。这种Ig基因不能通过额外的重组而改变。过度的VH替换会产生更多的IgH基因，其中含有富含带电氨基酸的长CDR3，这可能直接编码自身抗体。事实上，识别出的VH替换足迹优先编码CDR3区域内的带电氨基酸。我们对来自VH替代产品的5个重组抗体的初步试验表明，其中3个抗体与核或核周抗原发生强烈反应。基于这些观察，我们假设在活动期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。为了验证这一假设，(1)我们将关注新诊断的成年非裔美国女性活动期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组相比，这些患者是否发生了过度的受体编辑。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)利用SLE患者和正常人浆细胞和NAOVE B细胞的Ig基因表达重组抗体，以确定SLE浆细胞中累积的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中高亲和力自身抗体产生的机制是非常重要的，这将使我们能够设计出特定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，影响着全球100万人。由于病因不明，目前还没有针对SLE的特效治疗方法。系统性红斑狼疮的特征是产生与疾病相关的自身抗体。了解自身抗体在SLE发病过程中产生和浓缩的机制，将为设计SLE患者的特异性治疗方案提供新的思路。目前的建议是基于我们最近的发现，在三名活动期SLE患者的IgH和IGL基因上发生了过度的受体编辑。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。我们将直接测试假设，如果在活动期的SLE患者中发生了对IgH和IGL基因的过度受体编辑，从而有助于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.