Development and maintenance of human glycan and phospholipid antibody repertoires

人聚糖和磷脂抗体库的开发和维护

• 批准号: 10592416
• 负责人: John Franklin Kearney
• 金额: $ 71.89万
• 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/10592416
• 关键词: Affinity; Allergic Disease; Antibodies; Antibody Repertoire; Antibody Specificity; Antigenic Specificity; Antigens; Apoptosis; Atypical lymphocyte; Autoantigens; Autoimmune; Autologous; Automobile Driving; Avidity; B cell repertoire; B cell therapy; B-Cell Antigen Receptor; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; Bacteria; Binding; Biochemical; Biological; Blood; Cadaver; Carbohydrates; Cell Compartmentation; Clone Cells; Cloning; Development; Drug or chemical Tissue Distribution; Epitopes; Evolution; Excision; Exhibits; Gene Expression; Gene Library; Gene Rearrangement; Genetic; Goals; Heterophile Antigens; Homeostasis; Host Defense; Housekeeping; Human; Immune; Immune response; Immune system; Immunity; Immunization; Immunoglobulin Class Switching; Immunoglobulin Gene Rearrangement; Immunoglobulin Genes; Immunoglobulin Isotypes; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Immunotherapy; Incidence; Individual; Infection; Libraries; Maintenance; Mediating; Memory; Memory B-Lymphocyte; Molecular; Mucous Membrane; Mus; Natural regeneration; Organ; Organism; Outcome; Pathogenicity; Patients; Phenotype; Phospholipids; Polysaccharides; Property; Recombinants; Recovery; Role; Sampling; Serum; Shapes; Site; Somatic Mutation; Sorting; Specificity; Surface; T-Lymphocyte; Testing; Tissue Donors; Tissue Transplantation; Tissues; Vaccination; Xenograft procedure; allograft rejection; anti-CD20; chronic infection; clinically relevant; cohort; design; experience; host colonization; host microbiota; human tissue; immune function; indexing; microbiota; microorganism antigen; mouse model; natural antibodies; neoantigens; novel; novel therapeutics; pathogen; prevent; programs; recurrent infection; rituximab; selective IgM deficiency; senescence; transcriptome

Project 1: Development and maintenance of human glycan and phospholipid antibody repertoires Project Summary Natural antibodies (nAbs) exist in the blood of multiple mammalian species in the absence of deliberate immunization. Reactivity of nAbs with epitopes conserved between pathogens and autologous host antigens allow these antibodies to perform dual functions in immunity: providing important host defense against infection and facilitating housekeeping functions important for tissue homeostasis. The mechanisms controlling human nAb development and maintenance, however, are poorly understood. The goal of this project is to define mechanisms controlling maturation of the human natural B lymphocyte repertoire and its tissue distribution. We will achieve this goal by completing a targeted analysis of B cells reactive with conserved carbohydrate and phospholipid T lymphocyte-independent antigens associated with clinically relevant bacteria and xenoantigens. Through this approach, we will test our central hypothesis that the selection of innate-like B cell clonotypes and antigen-specific tuning of the nAb-producing B cell repertoire depend on interactions with autologous antigens and microbial antigens encountered at mucosal surfaces, which together modulate the entry of B cell clonotypes into the memory and antibody-secreting B cell compartments. In the first Specific Aim, we will sort-purify single, indexed carbohydrate- and phospholipid-binding B cells from a cohort of cadaveric human tissue donors. Immunoglobulin gene expression in these B cells will be analyzed together with expressed cellular phenotype to determine the distribution of clonal networks of nAb-producing B cells across B cell compartments in multiple human tissues. We will additionally examine the stability of innate-like B cell clonotypes and specificity of nAb repertoire by longitudinally sampling human blood after anti-CD20 (rituximab) B cell depletion, to determine the extent of antigen-reactive clonal B cell extirpation and clonal repertoire recovery during B cell compartment regeneration. Specific Aim 2 will utilize a novel, high-throughput antibody-cloning and expression platform to express immunoglobulin gene rearrangements from gene amplicon libraries as recombinant Abs and examine their binding properties, including antigen affinity and fine specificity. We will additionally examine the effects of somatic mutation on the binding properties of nAb by assessing the global reactivity of cloned antibodies and germline-reverted clonotypes using mammalian glycan antigen microarrays. This targeted analysis of antigen- reactive human B cells will permit analysis of fine antigen-specificity, affinity, and avidity of B cell clonotypes across human tissues, and the determination of whether these features of the BCR influence the tissue, B cell subset, and immunoglobulin isotype distribution of certain clones. Because nAb antigens are expressed by multiple commensal and pathogenic organisms and are protective in normal immune homeostasis, such findings will facilitate the development of immunotherapies designed to intersect the natural repertoire.

项目1：开发和维持人聚糖和磷脂抗体库 项目摘要 天然抗体（nAb）存在于多种哺乳动物物种的血液中， 次免疫nAb与病原体和自体宿主抗原之间保守表位的反应性 使这些抗体在免疫中发挥双重功能：提供重要的宿主防御， 并促进对组织稳态重要的持家功能。控制人类的机制 然而，对nAb的形成和维持知之甚少。这个项目的目标是定义 控制人天然B淋巴细胞库成熟及其组织分布的机制。我们 将通过完成对与保守碳水化合物反应的B细胞的靶向分析来实现这一目标， 与临床相关细菌和异种抗原相关的磷脂T淋巴细胞非依赖性抗原。 通过这种方法，我们将测试我们的中心假设，即先天性B细胞克隆型的选择和 产生nAb的B细胞库的抗原特异性调节依赖于与自体抗原的相互作用 以及在粘膜表面遇到的微生物抗原，它们共同调节B细胞克隆型的进入 进入记忆和抗体分泌的B细胞区。在第一个特定目标中，我们将分类纯化单个， 来自一组尸体人组织供体的索引碳水化合物和磷脂结合B细胞。 这些B细胞中的免疫球蛋白基因表达将与表达的细胞表型一起分析， 确定产生nAb的B细胞的克隆网络在多个B细胞区室中的分布 人体组织我们还将检查天然样B细胞克隆型的稳定性和nAb的特异性 通过在抗-CD 20（利妥昔单抗）B细胞耗竭后纵向取样人血液，以确定 抗原反应性克隆B细胞摘除的程度和B细胞隔室期间克隆库的恢复 再生Specific Aim 2将利用一种新型的高通量抗体克隆和表达平台， 将来自基因扩增子文库的免疫球蛋白基因重排表达为重组Ab， 它们的结合特性，包括抗原亲和性和良好的特异性。我们还将研究 通过评估克隆抗体的整体反应性，研究体细胞突变对nAb结合特性的影响， 使用哺乳动物聚糖抗原微阵列的种系回复克隆型。对抗原的靶向分析- 反应性人B细胞将允许分析B细胞克隆型的精细抗原特异性、亲和力和亲合力 以及确定BCR的这些特征是否影响组织、B细胞、 亚群和某些克隆的免疫球蛋白同种型分布。因为nAb抗原由 多种病原体和病原体，并在正常的免疫稳态中具有保护作用，这些发现 将促进旨在交叉自然库的免疫疗法的发展。