Structure and Function of Human Pemphigus Autoantibodies

描述（由申请人提供）：自身免疫是美国第三大常见疾病，影响8%的人口。抗体（Ab）库克隆是鉴定外周血B细胞产生的抗体的最新技术。对HIV或流感感染后的人类抗体库的研究已经确定了中和感染的抗体及其如何发展。这些知识是通过对所使用的VH基因、体细胞突变模式、表位定位和致病性分析获得的，并可能最终导致更有效的疫苗接种策略。我们期待这些同样的进步可以实现，并且非常需要提高自身抗体介导疾病治疗的安全性。寻常型天疱疮（Pemphigus vulgaris， PV）是一种潜在致命的自身免疫性疾病，其中针对皮肤细胞粘附蛋白粘连蛋白（Dsg） 3的抗体引起皮肤和粘膜严重起泡。它是一种理想的抗体库克隆疾病，因为抗原定义明确，克隆抗体的疾病相关性可以通过功能分析直接检测。目前尚不清楚患者体内存在多少种不同的抗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至关重要。人抗体包含可变区和恒定区两大结构域，其表达分别受VH-DH-JH和CH基因片段使用的支配。病毒感染后中和的抗体通常表现出VH基因限制（如轮状病毒和HIV的VH1-46，流感的VH1-69），可能是因为有限数量的Ab基因片段最适合结合特定的抗原表位。在慢性抗原刺激条件下观察到IgG4 CH限制，例如进行过敏性脱敏的个体和养蜂人。我们的初步数据表明，VH1-46和IgG4在PV抗dsg3抗体库中占主导地位，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自身免疫的共同机制。为了验证这一假设，我们将克隆PV患者的抗dsg Ab基因库，以确定抗dsg Ab反应是否寡克隆，是否受VH-和igg4限制，是否在患者之间共享。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、体细胞超突变和类开关重组形成的原始库），这些事件有助于PV中自身反应性抗体的形成。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，并可能导致仅针对疾病特异性抗体的新策略，而保留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.