B cell Receptor repertoire, cloning and expression Core

B 细胞受体库、克隆和表达核心

• 批准号: 10592411
• 负责人: Troy D Randall
• 金额: $ 75.83万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592411
• 关键词: Acceleration; Adipose tissue; Affinity; Antibodies; Antibody Affinity; Antibody Specificity; Antigens; Atlases; B cell repertoire; B-Cell Antigen Receptor; B-Lymphocytes; Bacterial Capsules; Binding; Biochemical; Biological Assay; Blood Group Antigens; Carbohydrates; Cell Lineage; Cells; Characteristics; Clonal Expansion; Cloning; Coupled; Development; Donor Selection; Eukaryotic Cell; Family suidae; Fluorochrome; Genes; Genetic Vectors; Goals; HLA Antigens; Heterophile Antigens; Histocompatibility Antigens; Human; Human body; Immunity; Immunoglobulin Class Switching; Immunoglobulin Constant Region; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulin-Secreting Cells; Incubated; Individual; Light; Location; Lymphoid Tissue; Maps; Memory B-Lymphocyte; Molecular; Monoclonal Antibodies; Mucous Membrane; Normal tissue morphology; Organ; Organ Donor; Polysaccharides; Population; Procedures; Process; Production; Proteins; Reagent; Recombinant Antibody; Recombinants; Research; Research Activity; Research Personnel; Sampling; Serum; Services; Sorting; Specificity; Standardization; Structure of germinal center of lymph node; Tissues; Viral Antigens; Viral Proteins; antigen binding; cross reactivity; expression vector; human tissue; instrumentation; programs; reagent standardization; synergism

Core C: B cell Receptor repertoire, cloning and expression Core Project Summary B cells express a widely diverse repertoire of B cell antigen receptors (BCRs) that can be further diversified by isotype-switching of heavy chain constant regions and by somatic hypermutation (SHM) in the antigen-binding domains of both heavy and light chains. B cells responding to protein antigens, such as viral antigens or transplant antigens, are often expanded in the germinal center, which stringently selects for high affinity memory B cells and antibody-secreting cells (ASCs). In contrast, B cells responding to carbohydrate antigens, such bacterial capsule components or blood group antigens, rarely enter the germinal center, but are nonetheless highly-selected based on reactivity and cross-reactivity to various antigens. The processes of clonal expansion and selection leads to the formation of B cell lineages that are related by BCR sequence and antigen-specificity. Importantly, the characteristics of the BCR (V gene use, SHM, isotype, affinity, cross-reactivity) often determine the functions of individual B cells, the tissues in which they reside, and the effector activities of the antibodies they produce. However, we have a limited understanding of antigen-specific B cells in the non-lymphoid tissues of humans. Therefore, the projects of this U19 will characterize B cells specific for different types of antigens (glycans in Project 1, viral antigens in Project 2 and xenoantigens in Project 3), obtained from lymphoid, mucosal and adipose tissues of normal human donors. Importantly, each project will determine the biochemical and molecular characteristics of antibodies made by individual B cells, determine the clonal relationships between antigen-specific B cells in different locations and use cytometric bead arrays to characterize the specificities, cross-reactivities and affinities of antibodies made by individual B cells. In order to achieve these goals and accelerate the research activities of each project, Core C will sort individual B cells from selected populations into 384-well plates, amplify VH and VL gene segments, clone the amplicons into IgG expression vectors, transfect the vectors into eukaryotic cells and produce recombinant monoclonal antibodies. Core C will also perform BCR heavy chain repertoire sequencing on sorted memory B cells and ASCs from the same tissues/donors used by the projects to sort antigen-specific B cells. Finally, Core C will develop cytometric bead arrays to enable each project to assay the reactivity profiles of serum samples, culture supernatants and monoclonal antibodies. Together, these activities will promote synergy and cooperation between projects and will accelerate the pace of research by centralizing procedures that require specialized expertise and instrumentation and by standardizing reagent development and production for each of the individual projects.

核心 C：B 细胞受体库、克隆和表达核心 项目概要 B 细胞表达广泛多样的 B 细胞抗原受体 (BCR)，这些受体可以通过以下方式进一步多样化： 重链恒定区的同种型转换以及抗原结合中的体细胞超突变（SHM） 重链和轻链的结构域。 B 细胞对蛋白质抗原做出反应，例如病毒抗原或 移植抗原，通常在生发中心扩增，严格选择高亲和力记忆 B 细胞和抗体分泌细胞 (ASC)。相反，B 细胞对碳水化合物抗原做出反应，例如 细菌荚膜成分或血型抗原，很少进入生发中心，但仍然存在 根据对各种抗原的反应性和交叉反应性进行高度选择。克隆扩增的过程 选择导致 B 细胞谱系的形成，这些谱系与 BCR 序列和抗原特异性相关。 重要的是，BCR 的特征（V 基因使用、SHM、同种型、亲和力、交叉反应性）通常决定 单个 B 细胞的功能、它们所在的组织以及抗体的效应活性 他们生产。然而，我们对非淋巴组织中抗原特异性 B 细胞的了解有限 人类的。因此，这个U19的项目将表征对不同类型抗原具有特异性的B细胞 （项目1中的聚糖，项目2中的病毒抗原和项目3中的异种抗原），从淋巴、粘膜获得 和正常人类供体的脂肪组织。重要的是，每个项目都将确定生化和 单个 B 细胞产生的抗体的分子特征，确定之间的克隆关系 不同位置的抗原特异性 B 细胞，并使用细胞计数珠阵列来表征特异性， 单个 B 细胞产生的抗体的交叉反应性和亲和力。为了实现这些目标并 加速每个项目的研究活动，Core C将从选定群体中分选单个B细胞 进入384孔板，扩增VH和VL基因片段，将扩增子克隆到IgG表达载体中， 将载体转染真核细胞并产生重组单克隆抗体。核心C也将 对来自同一细胞的排序记忆 B 细胞和 ASC 进行 BCR 重链库测序 项目使用的组织/捐赠者来分选抗原特异性 B 细胞。最后，Core C 将开发细胞计数珠 阵列，使每个项目能够测定血清样本、培养物上清液和 单克隆抗体。这些活动将共同促进项目之间的协同与合作 并将通过集中需要专门知识和技术的程序来加快研究步伐 仪器仪表以及标准化每个单独项目的试剂开发和生产。