Itch Ubiquitin Ligase and Germinal Center B cells

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，这在很大程度上是由 由生发中心(GC)衍生的病理性自身抗体。GC B细胞经历反复循环的体细胞 B细胞受体(BCR)基因的高度突变，以及基于BCR-抗原亲和力的选择。随机性 体细胞的超突变可以产生自体反应性B细胞，但这些细胞死亡是因为它们失去了 抗原特异性强，未被选择。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，成为长寿的浆细胞。调节表面BCR周转对忠诚的抗原至关重要- 驱动选择。在每一轮体细胞高突变和选择过程中，突变前的bcr必须 移除并替换为新突变的BCR，以确保基因编码的BCR口述 选择。如果不能删除BCR的以前版本，则可能会允许选择尚未 进行了抗原亲和力测试。BCR周转是当前GC选择模型的一个组成部分，但这 从未被充分探索过。 泛素连接酶之痒可预防人类和小鼠的体液自身免疫，但之痒如何限制抗体 生产情况尚不清楚。我最近确定了Itch在B细胞中的作用，以限制GC B细胞，浆细胞， 和抗体的产生，与其在预防自身免疫中的作用相一致。通过体细胞突变分析 在GC B细胞中，我现在展示了Itch支持对GC B细胞的严格选择。在免疫后，我发现 瘙痒缺陷GC B细胞，具有抗原特异性表面BCR，但含有非生产性BCR基因。 这些数据支持这样一种观点，即瘙痒可以防止获得有害bcr突变的B细胞存活和 促进突变后去除突变前的bcr。瘙痒已被证明调节BCR内化和 在幼稚的B细胞中运输到溶酶体。目前尚不清楚Itch如何控制GC中的BCR周转，以及这是如何实现的 影响GC选择。在这项提议中，我将测试瘙痒调节GC中表面BCR周转的假设 确保严格选择B细胞，防止出现自身抗体。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细胞体突变以模拟GC动力学和 确定Itch如何调整选择。此外，我将定义瘙痒在GC BCR去除和 用细胞生物学方法检测GC B细胞表面bcr的去除/替换。至 完成这些研究后，我与细胞内免疫受体生物学的专家们形成了合作关系 贩运、下一代测序和计算模型。这些研究将推动 了解GC中的选择调节机制，并提供一个独特的系统来研究 BCR周转、GC B细胞选择和抗体反应之间的联系。

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.