The evolution of B cell selection and affinity maturation in ectotherms

项目摘要/摘要 亲和力成熟增加了B细胞受体与其靶抗原的结合强度，是一种 影响疫苗效力的基本机制、自身免疫性疾病和 免疫疗法。从历史上看，这一过程被认为只发生在吸热型脊椎动物(即鸟类和 哺乳动物)，发生在称为生发中心的特化结构中，生发中心形成于哺乳动物的B细胞毛囊中 脾和淋巴在免疫反应过程中。在这里，B细胞将其免疫球蛋白基因突变为 产生更多不同的受体。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，它们是达尔文选择B细胞的完整抗原 克隆人。最近，尽管缺乏生发中心和真正的滤泡树突状细胞，但 明确地表明，冷血脊椎动物能够有一定程度的亲和力使其抗体成熟。 回应。这一发现导致了一种假设，即原始的B细胞选择结构或生发中心 前体，先于哺乳动物中存在的复杂而严格的选择结构。 该项目将研究模式生物护士鲨的B细胞选择和亲和力成熟 属于现存最古老的分类学类群(软骨鱼类)，拥有B细胞受体，T细胞受体， 和主要组织相容性复合体。此前已经证明，护士鲨既能做到这两点 亲和力成熟和免疫记忆。此外，护士鲨有一种免疫球蛋白类 只含有不与轻链结合的重链，IgNAR，适合B细胞受体 曲目分析。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个研究途径来解决：第一，位点 将在护士鲨的脾中鉴定B细胞的选择，然后评估它们的细胞结构，B细胞 和细胞因子转录组，通过免疫荧光染色、激光捕获显微切割和 高通量测序。其次，护士鲨的原代和亲和力成熟的B细胞库将是 通过噬菌体展示对抗原特异性IgNAR克隆进行测序和分离；这些将是 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 选择用于长期免疫保护，并确定维持长寿B细胞的信号细胞因子 鲨鱼队。这些目标的结果将提供对B细胞选择的进化的更好的理解，体细胞 高度突变，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.