Structure and Function of Human Pemphigus Autoantibodies

描述(申请人提供)：自身免疫是美国第三种最常见的疾病，影响8%的人口。抗体(Ab)谱系克隆是一项最先进的技术，用于表征外周B细胞产生的抗体。对HIV或流感感染后人类抗体谱系的研究已经确定了中和感染的抗体以及它们是如何发展的。这些知识是通过分析所使用的VH基因、体细胞突变模式、表位定位和致病性分析而获得的，最终可能导致更有效的疫苗接种策略。我们预计，在自身抗体介导的疾病中，可以取得同样的进展，并且非常需要这些进展来提高治疗的安全性。寻常型天疱疮(PV)是一种潜在的致命性自身免疫性疾病，抗皮肤细胞黏附蛋白Desmoglein(DSG)3的抗体可导致皮肤和粘膜严重起泡。这是一种理想的抗体谱系克隆疾病，因为抗原定义良好，克隆的抗体与疾病的相关性可以直接通过功能分析来检验。目前，尚不清楚患者体内存在多少不同的抗Dsg3抗体，患者之间的抗Dsg3抗体是否相似，以及它们是如何发展的。这些信息对于了解疾病的病理生理学和设计更安全的治疗策略是必不可少的。人源抗体具有可变区和恒定区两个主要结构域，其表达分别受VH-DH-JH和CH基因片段的使用控制。病毒感染后的中和抗体通常表现出VH基因的限制(例如轮状病毒和HIV的VH1-46，流感的VH1-69)，这可能是因为有限数量的抗体基因片段是结合特定抗原表位的最佳选择。在慢性抗原刺激条件下观察到IgG4CH限制，例如正在接受过敏脱敏的人和养蜂人。我们的初步数据表明，VH1-46和IgG4在PV抗Dsg3抗体谱系中占主导地位，这是一个令人兴奋的发现，因为它表明患者之间存在自身免疫反应的共同特征。我们假设抗DSG抗体使用一组有限的可变区和恒定区基因，这反映了PV中常见的自身免疫机制。为了验证这一假设，我们将克隆PV患者的抗DSG Ab谱系，以确定抗DSG Ab反应是否是寡克隆的，VH和IgG4限制性的，并在患者中共享。我们将定义B细胞发育的早期和晚期分子事件(包括通过VDJ重组形成NA�ve谱系、体细胞超突变和类开关重组)，这些事件有助于在PV中形成自身反应性抗体。拟议的研究将确定人类对疾病相关抗原的自身免疫是如何发生的，并可能导致只针对疾病特异性抗体的新策略，而不是保护免受感染的有益抗体。

DESCRIPTION (provided by applicant): Autoimmunity is the third most common category of disease in the United States, affecting 8% of the population. Antibody (Ab) repertoire cloning is a state-of-the-art technology for characterizing Abs produced from peripheral B cells. Studies of human Ab repertoires after HIV or influenza infection have identified Abs that neutralize infection and how they develop. This knowledge was gained from analysis of the VH genes used, patterns of somatic mutation, epitope mapping, and pathogenicity assays, and may ultimately lead to more effective vaccination strategies. We anticipate that these same advancements can be made, and are greatly needed to improve the safety of therapy, in autoAb-mediated diseases. Pemphigus vulgaris (PV) is a potentially fatal autoimmune disease in which Abs against the skin cell adhesion protein desmoglein (Dsg) 3 cause severe blistering of the skin and mucous membranes. It is an ideal disease for Ab repertoire cloning because the antigen is well defined and the disease relevance of cloned Abs can be directly tested by functional assays. Currently, it is unknown how many different anti-Dsg3 Abs exist within a patient, whether anti-Dsg3 Abs are similar among patients, and how they develop. This information is essential to understand the pathophysiology of disease and design safer treatment strategies. Human Abs contain two major structural domains, the variable and constant regions, whose expression is governed by VH-DH-JH and CH gene segment usage, respectively. Neutralizing Abs after viral infection often demonstrate VH gene restriction (such as VH1-46 for rotavirus and HIV, and VH1-69 for influenza), presumably because a limited number of Ab gene segments are optimal for binding specific antigenic epitopes. IgG4 CH restriction is observed in conditions of chronic antigen stimulation, such as individuals undergoing allergic desensitization and beekeepers. Our preliminary data suggest that VH1-46 and IgG4 are predominant in the PV anti-Dsg3 Ab repertoire, an exciting finding because it indicates that there are common features of the autoimmune response among patients. We hypothesize that anti-Dsg Abs use a restricted set of variable and constant region genes, which reflects common mechanisms of autoimmunity in PV. To test this hypothesis we will clone anti-Dsg Ab repertoires from PV patients to determine if the anti-Dsg Ab response is oligoclonal, VH- and IgG4-restricted, and shared among patients. We will define early and late molecular events in B cell development (including formation of the na�ve repertoire by VDJ recombination, somatic hypermutation, and class switch recombination) that contribute to the formation of autoreactive Abs in PV. The proposed studies will identify how autoimmunity to a disease-relevant antigen in humans occurs and may lead to novel strategies for targeting only the disease-specific Abs, sparing the beneficial Abs that protect from infection.