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的疾病类别，随着人口年龄的增长，它们的发病率正在缓慢上升。 Pemphigus dulgaris（PV）是一种使人衰弱的自身免疫性疾病，其中B细胞对Desmoglein-3（DSG3）产生抗体，这是一种跨膜细胞粘附蛋白，负责在表皮中结合角质形成细胞。该疾病会引起皮肤和粘膜的广泛水泡和侵蚀，导致严重的疼痛，超级感染以及可能的死亡。像大多数自身免疫性疾病一样，使用非特异性免疫抑制剂（例如类固醇和B细胞耗尽剂（例如利妥昔单抗））对PV进行治疗。由于这些疗法具有严重的副作用，例如败血症和死亡，因此对治疗疾病的更量身定制的方法存在巨大的临床需求。为了开发有效的针对性治疗，必须了解病原体自身抗体的发展至关重要。以前已经显示，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。特别是在疾病的活动阶段，IgG4很重要，而IgG1在缓解过程中似乎显着。我们的实验室先前已经表明，给定患者中的抗DSG3抗体使用有限数量的可变区域序列，共享可变区域基因在不同患者之间的使用，这意味着疾病发展的共同机制。但是，未知PV中的致病性抗体在IgG1和IgG4级别之间分布在其他情况下，在这些过程中它们在其他方面分布在其他情况下，并且在这些过程中的发展是在这些过程中，并且在这些过程中的发展是在这些过程中，并且在这些过程中均在这些过程中，并且在这些过程中均在这些过程中，并且在这些过程中的发展是在这些过程中，并且在这些抗体中均在这些过程中构成了这些抗体，这些抗体是在这些过程中且在这些抗体中的分布，并且在这些抗体中均在这些过程中，并且在这些抗体中均在这些过程中，并且在这些抗体中均在这些过程中，并且在这些抗体中均在这些过程中构成。自动反应性。在此提案中，我们将使用亚型特异性抗体曲目克隆来分离抗DSG3 IgG1和IgG4抗体，从一组活性疾病的PV患者中，并了解其创造和成熟。在该提案的目标1中，我们将对患者的克隆IgG1和IgG4使用亚型特异性噬菌体显示程序。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克隆是否是具有共享生殖线序列的IgG1克隆的体外突变的“儿童”。通过使用PCR查找任何IgG4克隆的IgG1“父母”，该方法将得到增强。在第二个目标中，我们将使用良好的体内水泡形成测定法，表位映射实验和表面等离子体共振来定量抗原结合亲和力，来表征我们的抗DSG3 IgG1和IgG4克隆的良好特异性。我们还将使用定位定向的诱变方法来确定在哪个阶段特定的克隆在发育过程中通过将躯体突变恢复到种系状态的发育过程中。通过这些实验，我们可以评估IgG4是否是PV的有效治疗靶标，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在PV中的出现，因此对自身免疫性疾病的发病机理有了更深入的了解。