Itch Ubiquitin Ligase and Germinal Center B cells

痒泛素连接酶和生发中心 B 细胞

• 批准号: 9977374
• 负责人: Emily K. Moser
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977374
• 关键词: Affinity; American; Antibodies; Antibody Formation; Antibody Response; Antigens; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmunity; B-Cell Antigen Receptor; B-Lymphocytes; B-cell receptor repertoire sequencing; Binding; Biological Response Modifiers; Biology; Cell Count; Cell Surface Receptors; Cells; Cellular biology; Collaborations; Computational Biology; Computer Models; Confocal Microscopy; Data; Development; Endocytosis; Ensure; Excision; Failure; Flow Cytometry; Foundations; Future; Generations; Goals; Hemagglutinin; Human; Immune response; Immunization; Immunize; Immunoglobulin Somatic Hypermutation; Impairment; Influenza A virus; Kinetics; Knockout Mice; Label; Ligands; Link; Lysosomes; Modeling; Molecular; Molecular Target; Mus; Mutate; Mutation; Mutation Analysis; Nature; Neutralization Tests; Pathologic; Pathology; Pathway interactions; Plasma Cells; Proteins; Proteomics; Pruritus; RNA vaccine; Receptor Gene; Regulation; Research; Risk; Role; Serum; Shapes; Signal Transduction; Somatic Mutation; Specificity; Structure of germinal center of lymph node; Surface; System; Techniques; Testing; Therapeutic; Training; UBB gene; United States National Institutes of Health; Variant; antigen binding; antigen test; autoreactive B cell; base; cross reactivity; crosslink; deep sequencing; design; functional outcomes; neutralizing antibody; next generation sequencing; novel therapeutics; prevent; programs; receptor internalization; recruit; response; risk minimization; therapeutic target; trafficking; ubiquitin ligase; ubiquitin-protein ligase

PROJECT SUMMARY/ABSTRACT The NIH estimates that up to 23.5 million Americans suffer from autoimmune disease, driven in large part by germinal center (GC)-derived pathologic autoantibodies. GC B cells undergo iterative rounds of somatic hypermutation of B cell receptor (BCR) genes, and selection based on BCR-antigen affinity. The random nature of somatic hypermutation allows emergence of autoreactive B cells, but these cells die because they have lost antigen specificity, and fail to be selected. Without a stringent selection threshold, autoreactive B cells may survive and become long-lived plasma cells. Regulation of surface BCR turnover is critical for faithful antigen- driven selection. During each round of somatic hypermutation and selection, the pre-mutated BCR must be removed and replaced with the newly-mutated BCR to ensure that the genetically encoded BCR dictates selection. Failure to remove previous “versions” of the BCR could allow selection of a BCR that has not been tested for antigen affinity. BCR turnover is an integral component of the current model of GC selection, yet this has never been fully explored. The ubiquitin ligase Itch prevents humoral autoimmunity in humans and mice, but how Itch limits antibody production is unclear. I have recently determined that Itch functions in B cells to limit GC B cells, plasma cells, and antibody production, aligning with its role in preventing autoimmunity. Through analysis of somatic mutations in GC B cells, I now show that Itch supports stringent selection of GC B cells. After immunization, I identified Itch deficient GC B cells bearing antigen-specific surface BCRs, yet containing nonproductive BCR genes. These data support the idea that Itch prevents survival of B cells acquiring detrimental BCR mutations and promotes pre-mutated BCR removal after mutation. Itch has been shown to regulate BCR internalization and trafficking to lysosomes in naïve B cells. It is unknown how Itch controls BCR turnover in GCs, and how this impacts GC selection. In this proposal I will test the hypothesis that Itch regulates surface BCR turnover in GC B cells to ensure stringent selection and prevent the emergence of autoantibody. I will use B cell specific Itch knockout mice and analysis of antigen-specific GC B cell somatic mutations to model GC dynamics and determine how Itch regulates selection. Additionally, I will define the role of Itch in GC BCR removal and replacement using cell biology approaches to probe surface BCR removal/replacement in GC B cells. To complete these studies, I have formed collaborations with experts in immunoreceptor biology, intracellular trafficking, next generation sequencing, and computational modeling. These studies will advance the understanding of mechanisms regulating selection in GCs, and provide a unique system in which to study the link between BCR turnover, GC B cell selection, and antibody responses.

项目摘要/摘要 NIH估计，多达2350万美国人患有自身免疫性疾病，在很大程度上驱动 由生发中心（GC）衍生的病理自身抗体。 GC B细胞经历体细胞的迭代弹 B细胞受体（BCR）基因的超数和基于BCR抗原亲和力的选择。随机性质 躯体超突击允许自动反应性B细胞的出现，但是这些细胞死亡，因为它们已经丢失 抗原特异性，无法选择。没有严格的选择阈值，自动反应性B细胞可能 生存并成为长寿命的浆细胞。表面BCR的调节对于忠实的抗原至关重要 驱动选择。在每一轮的躯体超成名和选择期间，预先突破的BCR必须为 删除并用新熔化的BCR替换，以确保一般编码的BCR指示 选择。未能删除BCR的先前“版本”可能允许选择尚未的BCR 测试了抗原亲和力。 BCR营业额是当前GC选择模型的组成部分，但是 从未被充分探索。 泛素连接酶瘙痒可防止人类和小鼠的体液自身免疫，但瘙痒如何限制抗体 生产尚不清楚。我最近确定瘙痒在B细胞中的功能以限制GC B细胞，浆细胞， 和抗体产生，与其在预防自身免疫性中的作用保持一致。通过分析体突变 在GC B细胞中，我现在表明瘙痒支持严格选择GC B细胞。免疫后，我确定了 具有抗原特异性表面BCR的瘙痒不足的GC B细胞，但含有非生产性BCR基因。 这些数据支持以下观点：瘙痒可以防止B细胞的生存，从而加剧有害的BCR突变和 突变后促进预先突破的BCR去除。瘙痒已被证明可以调节BCR内在化和 在幼稚的B细胞中运输到溶酶体。不知道瘙痒如何控制GC中的BCR失误，以及如何控制BCR 影响GC选择。在此提案中，我将测试以下假设，即瘙痒调节GC中的表面BCR更新 B细胞以确保严格选择并防止自身抗体的出现。我将使用B细胞特定的瘙痒 敲除小鼠和抗原特异性GC B细胞体细胞突变的分析，以建模GC动力学和 确定瘙痒如何调节选择。此外，我将定义瘙痒在GC BCR去除中的作用，并且 使用细胞生物学方法替换，以探测GC B细胞中探测表面BCR的去除/替换。到 完成这些研究，我已经与免疫受体生物学专家建立了合作 贩运，下一代测序和计算建模。这些研究将推动 了解GC中调节选择机制的理解，并提供了一个独特的系统来研究 BCR转换，GC B细胞选择和抗体反应之间的联系。