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）的特征是过度产生针对核抗原的高亲和力自身抗体。疾病相关性自身抗体在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，以研究三个活动性SLE患者的功能性抗体库。我们初步分析了193个来源于SLE浆细胞和naove B细胞的IgH基因，揭示了自身抗体的许多特征，如长的CDR 3区带有多个带正电荷的Arg残基。引人注目的是，三名活动性SLE患者中的IgH和IgL库都显示出过度而不是低效受体编辑的迹象，一些患者的IgL基因中下游J：5和J：3基因的使用增加，并且所有三名患者中IgH基因中VH替代产物的频率显著增加。通常，受体编辑旨在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具有不期望的后果。IgL基因的过度编辑可能耗尽IgL库，同时增加下游J;3基因的使用。这种IG基因不能通过额外的重组而改变。过量的VH置换将产生更多的IgH基因，其具有富含带电氨基酸的长CDR 3，其可能直接编码自身抗体。事实上，鉴定的VH置换足迹优先编码CDR 3区内的带电氨基酸。我们对5种来自VH置换产物的重组抗体的初步测试显示，其中3种与核或核周抗原强烈反应。基于这些观察结果，我们假设，IgH和IgL基因的过度受体编辑发生在活动性SLE患者中，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）我们将集中于新诊断的成年非裔美国女性活动性SLE患者的统一人群，并进行单细胞PCR和真实的时间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相同患者的疾病活动相关。(2)我们将使用来自SLE患者和正常对照的浆细胞和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。系统性红斑狼疮（Systemic lupus erythematosus，SLE）是一种严重危害人类健康的疾病，目前全球约有100万人患有SLE，因此，了解SLE患者自身抗体产生的机制，对于设计特异性的治疗方案，预防或减少SLE患者自身抗体的产生具有重要意义。由于病因不明，目前尚无针对SLE的特异性治疗方法。SLE的标志是产生疾病相关的自身抗体。了解自身抗体在SLE发病过程中的产生和富集机制，将为SLE患者设计特异性治疗方案提供新的思路。目前的建议是基于我们最近的发现，过度的受体编辑发生在三个活跃的SLE患者的IgH和IgL基因。尽管受体编辑最初旨在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置换可能具有有害后果。我们将直接测试的假设，如果过度的IgH和IgL基因的受体编辑发生在活动性SLE患者，从而有助于产生高亲和力的自身抗体。

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.