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

基本信息

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
项目类别：
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

项目摘要

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常见疾病，其发病率随着人口老龄化而缓慢上升。寻常型天疱疮 (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 (Dsg3) 的抗体，桥粒芯糖蛋白 3 是一种跨膜细胞粘附蛋白，负责将表皮中的角质形成细胞结合在一起。这种疾病会导致皮肤和粘膜出现大面积水疱和糜烂，导致剧烈疼痛、重复感染，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（例如类固醇和 B 细胞耗竭剂（例如利妥昔单抗））进行治疗。由于这些治疗方法具有败血症和死亡等严重副作用，因此临床迫切需要一种更有针对性的方法来治疗该疾病。为了开发针对天疱疮的有效靶向治疗方法，必须了解致病性自身抗体是如何产生的。先前已表明，PV 中的致病性抗 Dsg3 自身抗体仅限于 IgG 同种型，特别是 IgG1 和 IgG4 亚型。 IgG4 在疾病的活动期尤其重要，而 IgG1 在缓解期则显得占主导地位。我们实验室此前已表明，特定患者体内的抗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，目前尚不清楚致病性抗体如何PV 分布在 IgG1 和 IgG4 部分之间，它们如何彼此克隆相关，以及在这些抗体发育过程中的哪个点它们变得具有自身反应性。在本提案中，我们将使用亚型特异性抗体库克隆，以便从一组患有活动性疾病的 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。在该提案的目标 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 克隆是否克隆相关，即 IgG4 克隆是否是共享种系序列的 IgG1 克隆的体细胞突变“子代”。通过使用 PCR 来寻找任何 IgG4 克隆的 IgG1“亲本”，而这些克隆没有通过克隆鉴定出 IgG1 亲属，该方法将得到增强。在我们的第二个目标中，我们将使用成熟的体内泡形成测定、表位作图实验和表面等离振子共振来表征我们的抗 Dsg3 IgG1 和 IgG4 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特定克隆在发育过程中的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IgG4是否是PV的有效治疗靶点，可以有效捕获疾病相关的B细胞群。此外，我们将更好地了解自身抗体如何在真性红斑狼疮中产生，从而更深入地了解自身免疫性疾病的发病机制。