B cell Receptor repertoire, cloning and expression Core

核心C：B细胞受体库、克隆和表达 项目摘要 B细胞表达广泛多样的B细胞抗原受体（BCR）库，其可通过以下方式进一步多样化： 重链恒定区的同种型转换和抗原结合中的体细胞超突变（SHM） 重链和轻链的结构域。应答蛋白质抗原如病毒抗原的B细胞，或 移植抗原，通常在严格选择高亲和力记忆的生发中心扩增 B细胞和抗体分泌细胞（ASC）。相反，B细胞对碳水化合物抗原，如 细菌荚膜成分或血型抗原，很少进入生殖中心，但尽管如此， 基于对各种抗原的反应性和交叉反应性高度选择。克隆扩张的过程 并且选择导致通过BCR序列和抗原特异性相关的B细胞谱系的形成。 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）通常决定了 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 人类因此，本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 和脂肪组织中的一种。重要的是，每个项目将确定生物化学和 单个B细胞产生的抗体的分子特征，决定了 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， 由单个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， 为了加速每个项目的研究活动，核心C将从选定的群体中分选单个B细胞 扩增VH和VL基因片段，将扩增子克隆到IgG表达载体中， 将该载体转染真核细胞，产生重组单克隆抗体。核心C也将 对分选的记忆B细胞和来自相同细胞的ASC进行BCR重链库测序 组织/供者用于对抗原特异性B细胞进行分选。最后，核心C将开发细胞计数微珠 阵列，使每个项目能够测定血清样品、培养上清液和 克隆抗体这些活动将共同促进项目之间的协同作用和合作 并将通过集中需要专门知识的程序来加快研究的步伐， 仪器和标准化试剂的开发和生产为每个单独的项目。

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.