Human B1 Cell Immunoglobulin

人B1细胞免疫球蛋白

• 批准号: 8284733
• 负责人: THOMAS L ROTHSTEIN
• 金额: $ 20.93万
• 依托单位: FEINSTEIN INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-02 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8284733
• 关键词: Address; Affect; Anti-DNA Antibodies; Antibodies; Antibody-Producing Cells; Appearance; Ascaridil; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; Binding; Biological Assay; CD27 Antigens; Cell Separation; Cells; Characteristics; Charge; DNA; Diagnostic; Disease; Enzyme-Linked Immunosorbent Assay; FarGo; Foundations; Frequencies; Functional disorder; Goals; Hand; Human; Immune response; Immunity; Immunofluorescence Immunologic; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulins; Individual; Kidney; Length; Lupus; MS4A1 gene; Mature B-Lymphocyte; Measures; Mediating; Memory B-Lymphocyte; Mus; Mutate; Nature; Pathogenicity; Patients; Phenotype; Play; Population; Population Heterogeneity; Property; Reporting; Rheumatoid Arthritis; Role; Sampling; Somatic Mutation; Sorting - Cell Movement; Source; Structure; Structure of germinal center of lymph node; Subgroup; Testing; Time; Work; autoreactive B cell; expression cloning; in vivo; neglect; pathogen; success; therapeutic target; therapy design; tositumomab

DESCRIPTION (provided by applicant): This project concerns human B1 cells, and the immunoglobulin (Ig) produced by this B cell subpopulation. In recent years the success of B cell depletion therapies has raised the profile of B cells as key contributors to autoimmunity. However, the means by which B cells promote autoimmunity, and especially the precise origin of pathogenic autoantibodies, have not been fully worked out. For many years it has been suggested that B1 cells might be involved in lupus and related illnesses, but this has been impossible to evaluate because identifying markers for human B1 cells have been lacking. This gap has now been filled with our recent finding that human B1 cells co-express CD20, CD27 and CD43, and lack expression of CD70. With the phenotype of B1 cells in hand it is possible to examine the nature of B1 cell-generated Ig. Human B1 cells produce Ig with many features that suggest a relationship to autoimmunity. Human B1 cells produce anti-DNA autoantibody; human B1 cells express antibody that is somatically mutated; and, human B1 cell- derived antibody can be isotype-switched. And in lupus, not only is the proportion of B1 cells among all B cells increased 2-fold, but the fraction of B1 cells that express IgG is markedly increased 16-fold (to 35% of all B1 cells). Thus, human B1 cells that produce autoreactive Ig, somatically mutated Ig, and to a greater (lupus) or lesser (normal) extent, isotype-switched Ig, express the necessary attributes to be a source of autoreactive/mutated/switched autoantibody that is recognized as pathogenic. We hypothesize then that in lupus B1 cells, particularly IgG+ B1 cells, do, in fact, represent a source of pathogenic autoantibody. To address this hypothesis, we will study Ig produced by B1, naive, and memory B cells, comparing B cells from lupus patients with normal controls, and comparing IgG+ B1 cells with non-switched B1 cells. Specifically, 1) We will evaluate the structure of B1 cell Ig by single cell sorting, PCR amplification, and sequencing, focusing on VH/VD/VJ/VL usage, somatic mutation, N-region addition, and CDR3 length and charge; 2) We will evaluate the repertoire of B1 cell Ig by expression cloning, antibody purification, HEp-2 immunofluorescence, ELISA assay, and autoantigen array, focusing on the frequency of polyreactive, autoreactive antibodies; and, 3) We will evaluate the pathogenicity of B1 cell Ig by glomerular binding/immunofluorescence and histological examination of kidneys after murine in vivo autoantibody administration, focusing on the capacity of anti-DNA autoreactive/polyreactive antibodies to produce renal damage. We hypothesize that in lupus, B1 cells generate pathogenic, nephritogenic antibodies that are somatically mutated and isotype-switched, and thus represent the origin of at least some of the "classical" type of autoreactive Ig that has been thought to be purview of post-germinal center conventional B cells. The results of this work will lay the foundation for a new and discrete therapeutic target in lupus that may eliminate the need for wholesale destruction of all mature B cells. PUBLIC HEALTH RELEVANCE: The recent successful use of B cell depletion therapy shows that B cells play an important role in autoimmune diseases. Newly discovered human B1 cells are a unique subpopulation of B cells capable of making autoantibodies and thus could be directly involved in initiating autoimmunity; further, we have found that human B1 cells are expanded in patients with lupus autoimmunity. The goal of the present proposal is to gather evidence indicating that B1 cells are a source of autoantibodies in lupus and thereby contribute to lupus disease, which will provide the foundation for new targeted therapies designed to eliminate the "bad" pathogenic B1 cells without affecting the majority population of B cells needed for immunity against infectious pathogens.

描述（由申请人提供）：该项目涉及人类 B1 细胞以及该 B 细胞亚群产生的免疫球蛋白 (Ig)。近年来，B 细胞耗竭疗法的成功提高了 B 细胞作为自身免疫关键贡献者的地位。然而，B细胞促进自身免疫的方式，特别是致病性自身抗体的确切来源，尚未完全阐明。多年来，人们一直认为 B1 细胞可能与狼疮和相关疾病有关，但这一直无法评估，因为一直缺乏识别人类 B1 细胞的标记。我们最近的发现填补了这一空白，即人类 B1 细胞共表达 CD20、CD27 和 CD43，但缺乏 CD70 的表达。有了 B1 细胞的表型，就可以检查 B1 细胞产生的 Ig 的性质。人类 B1 细胞产生的 Ig 具有许多特征，表明与自身免疫有关。人类B1细胞产生抗DNA自身抗体；人类 B1 细胞表达体细胞突变的抗体；并且，人 B1 细胞衍生的抗体可以进行同种型转换。在狼疮中，不仅 B1 细胞在所有 B 细胞中的比例增加了 2 倍，而且表达 IgG 的 B1 细胞比例也显着增加了 16 倍（占所有 B1 细胞的 35%）。因此，产生自身反应性 Ig、体细胞突变 Ig 以及较大（狼疮）或较小（正常）程度的同种型转换 Ig 的人类 B1 细胞表达作为自身反应性/突变/转换自身抗体来源的必要属性，即被认为是致病的。我们假设狼疮 B1 细胞，特别是 IgG+ B1 细胞，实际上代表了致病性自身抗体的来源。为了验证这一假设，我们将研究 B1、初始 B 细胞和记忆 B 细胞产生的 Ig，将狼疮患者的 B 细胞与正常对照进行比较，并将 IgG+ B1 细胞与非转换 B1 细胞进行比较。具体来说，1）我们将通过单细胞分选、PCR扩增和测序来评估B1细胞Ig的结构，重点是VH/VD/VJ/VL使用、体细胞突变、N区添加以及CDR3长度和电荷； 2) 我们将通过表达克隆、抗体纯化、HEp-2免疫荧光、ELISA测定和自身抗原阵列评估B1细胞Ig的库，重点关注多反应性、自身反应性抗体的频率； 3）小鼠体内给予自身抗体后，我们将通过肾小球结合/免疫荧光和肾脏组织学检查来评估B1细胞Ig的致病性，重点关注抗DNA自身反应性/多反应性抗体产生肾损伤的能力。我们假设，在狼疮中，B1 细胞产生致病性、肾炎性抗体，这些抗体发生体细胞突变和同种型转换，因此代表至少一些“经典”类型自身反应性 Ig 的起源 这被认为是生发中心后传统 B 细胞的权限。这项工作的结果将为狼疮的新的、离散的治疗靶点奠定基础，该靶点可能消除大规模破坏所有成熟 B 细胞的需要。 公共健康相关性：最近 B 细胞耗竭疗法的成功使用表明 B 细胞在自身免疫性疾病中发挥着重要作用。新发现的人类B1细胞是能够产生自身抗体的独特B细胞亚群，因此可能直接参与启动自身免疫；此外，我们发现狼疮自身免疫患者体内的人类 B1 细胞扩增。本提案的目标是收集证据，表明 B1 细胞是狼疮自身抗体的来源，从而导致狼疮疾病，这将为新的靶向治疗奠定基础，旨在消除“坏”致病性 B1 细胞而不影响大多数 B 细胞群需要对传染性病原体产生免疫力。