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(申请人提供)：自身免疫性疾病是美国第三种最常见的疾病，随着人口老龄化，其发病率正在缓慢上升。寻常型天疱疮(PV)是一种衰弱的自身免疫性疾病，B细胞产生针对桥粒蛋白-3(Dsg3)的抗体，Dsg3是一种跨膜细胞黏附蛋白，负责将表皮中的角质形成细胞结合在一起。这种疾病会在皮肤和粘膜中引起广泛的水泡和侵蚀，导致剧烈疼痛、双重感染，甚至可能死亡。像大多数自身免疫性疾病一样，PV使用非特异性免疫抑制药，如类固醇和B细胞耗尽剂(如美罗华)进行治疗。由于这些治疗方法有严重的副作用，如败血症和死亡，临床上迫切需要一种更有针对性的方法来治疗这种疾病。为了开发有效的针对天疱疮的靶向治疗方法，了解致病自身抗体是如何发展的至关重要。已有研究表明，PV的致病性抗Dsg3自身抗体仅限于Ig G亚型，尤其是Ig G1和Ig G4亚型。尤其是IgG4，在疾病的活动期很重要，而在缓解期，IgG1似乎占主导地位。我们的实验室以前已经证明，在给定的患者中，抗Dsg3抗体使用有限数量的可变区序列，不同患者之间共享可变区基因，这意味着疾病发展的共同机制。然而，目前尚不清楚PV中的致病抗体如何分布在IgG1和IgG4组分之间，它们如何相互克隆相关，以及在这些抗体的发展过程中，它们在什么时候会产生自身反应。在这个方案中，我们将使用亚型特异性抗体库克隆，以从一组活动性疾病的PV患者中分离出抗Dsg3 IgG1和IgG4抗体，并了解它们的产生和成熟。在这项提案的目标1中，我们将使用亚型特异性噬菌体展示程序来克隆患者的IgG1和IgG4。然后，我们将使用遗传分析来确定IgG1和IgG4克隆是否与克隆相关，即IgG4克隆是否是具有相同生殖系序列的IgG1克隆的体细胞突变“子代”。这一方法将通过使用聚合酶链式反应来寻找任何通过克隆无法确定其免疫球蛋白1亲缘关系的免疫球蛋白G4克隆的“父母”而得到加强。在我们的第二个目标中，我们将使用成熟的体内水泡形成试验、表位定位实验和表面等离子共振来表征我们的抗Dsg3 IgG1和IgG4克隆的良好特异性，以定量抗原结合亲和力。我们还将使用定点突变方法，通过将体细胞突变恢复到生殖系状态来确定特定克隆在发育过程中在哪个阶段变得具有反应性。通过这些实验，我们可以评估IgG4是否是治疗PV的有效靶点，能够有效地捕获与疾病相关的B细胞群。此外，我们将更好地了解自身抗体是如何在PV中产生的，从而对自身免疫性疾病的发病机制有更深的理解。