Structure and Function of Human Pemphigus Autoantibodies

描述（由申请人提供）：自身免疫性疾病是美国第三大最常见的疾病类别，影响8%的人口。抗体库克隆是一种用于鉴定外周B细胞产生的抗体的最新技术。对HIV或流感感染后人类抗体库的研究已经确定了中和感染的抗体及其发展方式。这些知识是从分析所用的VH基因、体细胞突变模式、表位作图和致病性测定中获得的，并可能最终导致更有效的疫苗接种策略。我们预计，在autoAb介导的疾病中，这些同样的进步可以实现，并且非常需要提高治疗的安全性。寻常天疱疮（PV）是一种潜在的致命性自身免疫性疾病，其中针对皮肤细胞粘附蛋白桥粒芯糖蛋白（Dsg）3的Ab引起皮肤和粘膜的严重起泡。它是一个理想的疾病抗体库克隆，因为抗原是明确定义的，克隆抗体的疾病相关性可以直接通过功能测定。目前，尚不清楚患者体内存在多少不同的抗Dsg 3抗体，抗Dsg 3抗体在患者之间是否相似，以及它们如何发展。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Ab含有两个主要结构域，即可变区和恒定区，其表达分别由VH-DH-JH和CH基因片段的使用控制。病毒感染后的中和Ab通常表现出VH基因限制（如轮状病毒和HIV的VH 1 -46，以及流感的VH 1 -69），可能是因为有限数量的Ab基因区段对于结合特异性抗原表位是最佳的。IgG 4 CH限制在慢性抗原刺激的条件下观察到，例如经历过敏性脱敏的个体和养蜂人。我们的初步数据表明，VH 1 -46和IgG 4是主要的PV抗Dsg 3抗体库，一个令人兴奋的发现，因为它表明，有共同的特点，患者之间的自身免疫反应。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中自身免疫的共同机制。为了检验这一假设，我们将克隆来自PV患者的抗Dsg Ab库，以确定抗Dsg Ab应答是否是寡克隆的、VH和IgG 4限制性的以及在患者中共享的。我们将定义B细胞发育中的早期和晚期分子事件（包括通过VDJ重组、体细胞超突变和类转换重组形成幼稚库），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，而不包括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.