Human B1 Cell Immunoglobulin

人B1细胞免疫球蛋白

• 批准号: 8521076
• 负责人: THOMAS L ROTHSTEIN
• 金额: $ 7.92万
• 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 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8521076
• 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; public health relevance; 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.

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细胞的特征，作为自身免疫性的关键因素。但是，尚未完全确定B细胞促进自身免疫性，尤其是致病自身抗体的精确起源的手段。多年来，一直有人提出，B1细胞可能参与狼疮和相关疾病，但这是不可能评估的，因为缺乏人类B1细胞的标记。现在，这个差距已经填补了我们最近的发现，即人B1细胞共表达CD20，CD27和CD43，并且缺乏CD70的表达。使用手中B1细胞的表型，可以检查B1细胞生成的Ig的性质。人B1细胞产生的Ig具有许多特征，这些特征表明与自身免疫有关系。人B1细胞产生抗DNA自身抗体；人B1细胞表达体形突变的抗体；并且，可以将人B1细胞衍生的抗体用于同种型切口。在狼疮中，所有B细胞中B1细胞的比例不仅增加了2倍，而且表达IgG的B1细胞的比例明显增加16倍（占所有B1细胞的35％）。因此，产生自动反应性IG，体形突变的Ig以及更大（狼疮）或较小（正常）范围的人类B1细胞，同种型开关Ig，表达必要的属性，是自动反应/突变/切换/切换自身抗体的来源，被识别为致病性。然后，我们假设在狼疮B1细胞中，尤其是IgG+ B1细胞中，实际上代表了致病性自身抗体的来源。为了解决这一假设，我们将研究由B1，NAIVE和记忆B细胞产生的Ig，以比较具有正常对照的狼疮患者的B细胞，并将IgG+ B1细胞与非切换的B1细胞进行比较。具体而言，1）我们将通过单细胞分类，PCR扩增和测序评估B1细胞Ig的结构，重点关注VH/VD/VD/VJ/VL使用，体细胞突变，N-区域添加以及CDR3的长度和电荷； 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的曲目，重点介绍了多反应性，自动反应性抗体的频率；和3）我们将通过肾小球自身抗体给药后的肾小球结合/免疫荧光和对肾脏的组织学检查评估B1细胞Ig的致病性，重点介绍抗DNA自动反应性/决策性抗体产生肾脏损伤的能力。我们假设在狼疮中，B1细胞会产生致病性，肾脏生成抗体，这些抗体是体形突变和同型转换的，因此至少代表了至少某些“经典”类型的自动反应性Ig的起源 人们认为这是后中心常规B细胞的权限。这项工作的结果将为狼疮中一个新的，离散的治疗靶标奠定基础，这可能消除了所有成熟B细胞的批发破坏的需求。