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.

项目总结/摘要 美国国立卫生研究院估计，多达2350万美国人患有自身免疫性疾病，这在很大程度上是由于 由生发中心（GC）衍生的病理性自身抗体。GC B细胞经历反复的体细胞周期， B细胞受体（BCR）基因的超突变和基于BCR-抗原亲和力的选择。随机性质 体细胞超突变的发生允许自身反应性B细胞的出现，但这些细胞死亡，因为它们失去了 抗原特异性，不能被选择。如果没有严格的选择阈值，自身反应性B细胞可能 存活下来并成为长寿的浆细胞。表面BCR转换的调节对于忠实的抗原- 驱动选择在每一轮体细胞超突变和选择期间，必须将预突变的BCR 移除并替换为新突变的BCR，以确保基因编码的BCR指示 选择.未能删除BCR的先前“版本”可能允许选择尚未 测试抗原亲和力。BCR营业额是GC选择当前模型的一个组成部分，但这 从未被完全探索过。 泛素连接酶Itch可预防人类和小鼠的体液自身免疫，但Itch如何限制抗体 生产不清楚。我最近发现瘙痒在B细胞中起作用，限制GC B细胞，浆细胞， 和抗体产生，与其在预防自身免疫中的作用一致。通过分析体细胞突变 在GC B细胞中，我现在表明Itch支持GC B细胞的严格选择。免疫后，我发现 瘙痒缺陷型GC B细胞携带抗原特异性表面BCR，但含有非生产性BCR基因。 这些数据支持瘙痒阻止获得有害BCR突变的B细胞存活的观点， 促进突变后的突变前BCR去除。瘙痒已被证明可调节BCR内化， 运输至幼稚B细胞中的溶酶体。目前尚不清楚Itch如何控制GC中的BCR周转，以及这如何影响GC中的BCR周转。 影响GC选择。在这个提议中，我将检验瘙痒调节GC中表面BCR周转的假设。 B细胞，以确保严格的选择和防止自身抗体的出现。我会用B细胞特异性瘙痒 基因敲除小鼠和分析抗原特异性GC B细胞体细胞突变以模拟GC动力学， 确定痒是如何调节选择的此外，我将定义瘙痒在GC BCR清除中的作用， 使用细胞生物学方法检测GC B细胞中表面BCR去除/置换。到 为了完成这些研究，我与免疫受体生物学、细胞内 贩运、下一代测序和计算建模。这些研究将推动 了解GC中调节选择的机制，并提供一个独特的系统来研究 BCR周转、GC B细胞选择和抗体应答之间的联系。