Relationships Between Autoimmune IgG1 and IgG4 Repertoires in Pemphigus Vulgaris

寻常型天疱疮自身免疫 IgG1 和 IgG4 库之间的关系

• 批准号: 8649820
• 负责人: Eric Milan Mukherjee
• 金额: $ 4.54万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649820
• 关键词: Adhesions; Adverse effects; Affect; Affinity; Age; American; Antibodies; Antibody Repertoire; Antibody-Producing Cells; Antigens; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Process; B-Lymphocytes; Bacteria; Binding; Biological Assay; Bulla; Categories; Cell Adhesion; Cell Adhesion Molecules; Cells; Cessation of life; Child; Chronic; Clinical; Clonality; Cloning; DNA Sequence; Defect; Development; Disease; Disease remission; Epidermis; Epitope Mapping; Epitopes; Extracellular Domain; Family; Functional disorder; Germ Lines; Health; Human; IgG1; IgG4; Immune; Immune response; Immunoglobulin Class Switching; Immunoglobulin Constant Region; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; Immunosuppression; Immunosuppressive Agents; Incidence; Infection; Laboratories; Left; Longitudinal Studies; Mediating; Memory B-Lymphocyte; Modeling; Molecular; Mucous Membrane; Mutate; Myasthenia Gravis; Onset of illness; Pain; Parents; Pathogenesis; Pathogenicity; Patients; Pemphigus; Pemphigus Vulgaris; Phage Display; Population; Procedures; Production; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Risk; Sclerosis; Second Primary Cancers; Sepsis; Serum; Site-Directed Mutagenesis; Skin; Somatic Mutation; Specificity; Staging; Steroids; Surface Plasmon Resonance; Thrombotic Thrombocytopenic Purpura; United States; Virus; antigen binding; base; cross reactivity; cytokine; desmoglein; desmoglein III; effective therapy; genetic analysis; in vivo; keratinocyte; novel therapeutics; research study; response; rituximab; skin disorder; therapeutic target; variable region gene

DESCRIPTION (provided by applicant): Autoimmune diseases are the third most common category of diseases in the United States, and their incidence is slowly rising as the population ages. Pemphigus vulgaris (PV) is a debilitating autoimmune disease in which B cells produce antibodies against desmoglein-3 (Dsg3), a transmembrane cell adhesion protein responsible for binding keratinocytes together in the epidermis. The disease can cause widespread blisters and erosions in the skin and mucous membranes, leading to severe pain, super-infection, and possibly death. Like most autoimmune diseases, PV is treated using non-specific immunosuppressants like steroids and B-cell depleting agents (e.g. rituximab). Because these treatments have serious side effects like sepsis and death, there is a dire clinical need for a more tailored approach to treating the disease. To develop effective targeted treatments for pemphigus, it is essential to understand how the pathogenic autoantibodies develop. It has been previously shown that the pathogenic anti-Dsg3 autoantibodies in PV are restricted to the IgG isotype, and specifically to the IgG1 and IgG4 subtypes. IgG4, in particular, is important during active stages of the disease, while IgG1 appears dominant during remission. Our laboratory has previously shown that anti-Dsg3 antibodies in a given patient use a limited number of variable region sequences, with shared variable region gene usage among different patients, implying common mechanisms of disease development.. However, it is unknown how pathogenic antibodies in PV are distributed between the IgG1 and IgG4 fraction, how they are clonally related to each other, and at what point during the development of these antibodies that they become autoreactive. In this proposal, we will use subtype-specific antibody repertoire cloning in order to isolate the anti-Dsg3 IgG1 and IgG4 antibodies from a panel of PV patients with active disease and understand their creation and maturation. In Aim 1 of this proposal, we will use a subtype-specific phage display procedure to clone IgG1 and IgG4 from patients. We will then use genetic analysis to determine whether the IgG1 and IgG4 clones are clonally related, i.e. whether the IgG4 clones are somatically mutated "children" of IgG1 clones that share a germ line sequence. This approach will be augmented by using PCR to find IgG1 "parents" of any IgG4 clones for whom IgG1 relatives are not identified through cloning. In our second aim, we will characterize the fine specificities our anti-Dsg3 IgG1 and IgG4 clones using well-established in vivo blister formation assays, epitope mapping experiments, and surface plasmon resonance to quantitate antigen binding affinity. We will also use a site-directed mutagenesis approach to determine at what stage particular clones became reactive during development by reverting somatic mutations to the germline state. Through these experiments, we can evaluate whether IgG4 is a valid therapeutic target for PV that can effectively capture the disease- relevant B cell populations. Furthermore, we will gain a much better understanding of how autoantibodies arise in PV, and therefore a deeper understanding of the pathogenesis of autoimmune disease.

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，其发病率随着人口老龄化而缓慢上升。寻常天疱疮（PV）是一种使人衰弱的自身免疫性疾病，其中B细胞产生针对桥粒芯糖蛋白-3（Dsg 3）的抗体，桥粒芯糖蛋白-3（Dsg 3）是负责将表皮中的角质形成细胞结合在一起的跨膜细胞粘附蛋白。这种疾病会引起皮肤和粘膜的大面积水泡和糜烂，导致剧烈疼痛，双重感染，甚至可能死亡。与大多数自身免疫性疾病一样，PV使用非特异性免疫抑制剂如类固醇和B细胞耗竭剂（例如利妥昔单抗）进行治疗。由于这些治疗具有严重的副作用，如败血症和死亡，因此迫切需要一种更适合治疗这种疾病的方法。为了开发有效的天疱疮靶向治疗方法，了解致病性自身抗体是如何发展的至关重要。先前已经表明，PV中的致病性抗Dsg 3自身抗体限于IgG同种型，并且特别限于IgG 1和IgG 4亚型。特别是IgG 4在疾病的活动期是重要的，而IgG 1在缓解期占主导地位。我们的实验室先前已经表明，给定患者中的抗Dsg 3抗体使用有限数量的可变区序列，不同患者之间具有共享的可变区基因使用，这意味着疾病发展的共同机制。然而，目前尚不清楚PV中的致病性抗体如何分布在IgG 1和IgG 4组分之间，它们如何彼此克隆相关，以及在这些抗体发展过程中的什么时候它们成为自身反应性的。在本提案中，我们将使用亚型特异性抗体库克隆，以便从一组活动性疾病PV患者中分离抗Dsg 3 IgG 1和IgG 4抗体，并了解它们的产生和成熟。在本提案的目标1中，我们将使用亚型特异性噬菌体展示程序从患者中克隆IgG 1和IgG 4。然后，我们将使用遗传分析来确定IgG 1和IgG 4克隆是否是克隆相关的，即IgG 4克隆是否是共享生殖系序列的IgG 1克隆的体细胞突变“子代”。这种方法将通过使用PCR来寻找任何IgG 4克隆的IgG 1“亲本”来增强，其中IgG 1亲属未通过克隆来鉴定。在我们的第二个目标中，我们将使用完善的体内水疱形成测定、表位作图实验和表面等离子体共振来表征我们的抗Dsg 3 IgG 1和IgG 4克隆的精细特异性以定量抗原结合亲和力。我们还将使用定点诱变方法来确定在发育过程中特定克隆在哪个阶段通过将体细胞突变恢复到种系状态而变得具有反应性。通过这些实验，我们可以评估IgG 4是否是PV的有效治疗靶标，其可以有效捕获疾病相关的B细胞群体。此外，我们将更好地了解自身抗体是如何在PV中产生的，从而更深入地了解自身免疫性疾病的发病机制。