The evolution of B cell selection and affinity maturation in ectotherms

项目总结/摘要 亲和力成熟增加了B细胞受体对其靶抗原的结合强度，并且是一种免疫调节剂。 影响疫苗效力、自身免疫性疾病和 免疫疗法历史上，这一过程被认为仅发生在吸热脊椎动物（即鸟类和哺乳动物）中。 哺乳动物），发生在称为生发中心的专门结构中，生发中心形成于哺乳动物的B细胞滤泡中。 脾脏和淋巴结的免疫反应。在这里，B细胞突变其免疫球蛋白基因， 产生更多样的受体。然后B细胞在生发中心内迁移，与T细胞相互作用 滤泡辅助细胞和滤泡树突状细胞，为B细胞的达尔文选择提供完整的抗原 克隆最近，尽管缺乏生发中心和真正的滤泡树突状细胞， 明确表明，冷血脊椎动物能够在一定程度上的亲和力成熟其抗体， 应答这一发现导致了一个假设，即原始B细胞选择结构，或生殖中心， 前体，在哺乳动物中存在的复杂的严格选择结构之前。 本计画将探讨模式生物护士鲨的B细胞选择与亲和力成熟 属于现存最古老的分类群（软骨鱼纲），具有B细胞受体，T细胞受体， 和主要组织相容性复合体。之前已经证明护士鲨能够同时 亲和力成熟和免疫记忆。此外，护士鲨的免疫球蛋白 仅由不与轻链结合的重链组成，IgNAR，非常适合于B细胞受体 剧目分析这些特点使护士鲨成为研究B细胞进化的理想模型 选择和亲和力成熟。 B细胞选择的演变将通过三种研究途径来解决：首先， 的B细胞选择将在护士鲨脾脏中鉴定，然后评估它们的细胞结构，B细胞 库和细胞因子转录组，通过免疫荧光染色，激光捕获显微切割， 高通量测序。第二，护士鲨原代和亲和成熟B细胞库将被 通过测序和经由噬菌体展示分离抗原特异性IgNAR克隆来分析;这些将被 测序以评估克隆多样性，然后表达为重组蛋白以评估其结合亲和力。 最后，护士鲨的记忆反应将被表征，以确定克隆的多样性和亲和力 选择长期免疫保护，并确定维持长寿的B细胞的信号细胞因子， 鲨鱼这些目标的结果将提供对B细胞选择、体细胞选择和细胞周期的进化的更好理解。 超突变和亲和力成熟。

Project Summary/Abstract Affinity maturation increases the binding strength of the B cell receptor for its target antigen, and is a fundamental mechanism affecting vaccine efficacy, autoimmune diseases, and the development of immunotherapies. Historically, this process was believed to occur only in endothermic vertebrates (i.e. birds and mammals), taking place in specialized structures called germinal centers, which form in the B cell follicles of the spleen and lymph nodes during an immune response. Here B cells mutate their immunoglobulin genes to produce a greater diversity of receptors. The B cells then migrate within the germinal center to interact with T follicular helper cells and follicular dendritic cells, which present intact antigen for Darwinian selection of B cell clones. More recently, and despite an absence of germinal centers and true follicular dendritic cells, it has been definitively shown that cold-blooded vertebrates are capable of some level of affinity maturing their antibody responses. This finding leads to the hypothesis that a primordial B cell selection structure, or germinal center precursor, preceded the complex stringent selection structures present in mammals. This project will investigate B cell selection and affinity maturation in the nurse shark, a model organism that belongs to the oldest extant taxonomic group (Chondrichthyes) to possess B cell receptors, T cell receptors, and major histocompatibility complex. It has previously been demonstrated that nurse sharks are capable of both affinity maturation and immunological memory. Additionally, the nurse shark has an immunoglobulin class consisting of only heavy chain that does not associate with light chain, IgNAR, well suited for B cell receptor repertoire analysis. These characteristics make the nurse shark an ideal model to study the evolution of B cell selection and affinity maturation. The evolution of B cell selection will be addressed through three avenues of investigation: first, the sites of B cell selection will be identified in the nurse shark spleen, then assessed for their cellular architecture, B cell repertoire, and cytokine transcriptome through immunofluorescent staining, laser capture microdissection, and high throughput sequencing. Second, the nurse shark primary and affinity matured B cell repertoires will be analyzed by sequencing and isolation of antigen-specific IgNAR clones via phage display; these will be sequenced to evaluate clone diversity, then expressed as recombinant proteins to assess their binding affinity. Finally, the nurse shark memory response will be characterized to determine the diversity and affinity of clones selected for long-term immune protection and identify the signaling cytokines that maintain long-lived B cells in sharks. The results of these aims will provide a greater understanding of the evolution of B cell selection, somatic hypermutation, and affinity maturation.