Characterization of the Lamprey Adaptive Immune System

描述（由申请人提供）：最近在现存的无颌脊椎动物中发现了第二种适应性免疫系统。七鳃鳗和盲鳗的淋巴细胞经过模块化亮氨酸-富重复（LRR）遗传单位的体细胞重组组装，产生一个估计与哺乳动物抗体库一样大的潜在可变淋巴细胞受体（VLR）库；也就是说,> 1014。在免疫应答中产生的抗原特异性VLRs可能具有许多实际用途，类似于在各种诊断测试、蛋白质纯化、抑制细胞内蛋白质、病原体鉴定和病原体抑制中为抗体所建立的用途。为了实现这一潜力，本提案的实验旨在表征海洋七鳃鳗vlr介导的适应性免疫系统的功能和结构特性。主要目的是(i)确定七鳃鳗对颗粒抗原（炭疽芽孢杆菌、鼠伤寒沙门氏菌和人红细胞）和可溶性抗原（BcIA炭疽蛋白及其c端结构域）的细胞和体液免疫反应的免疫要求、动力学和记忆潜力；（ii）在实验中表征可溶性VLR的分子性质，以验证VLR作为预先形成的多价蛋白复合物从单个淋巴细胞释放的假设，（iii）鉴定单个抗原特异性VLR携带淋巴细胞并分离它们，以表征其VLR产物的抗原结合亲和力和结构，以及（iv）检查VLR适应性免疫系统对模型病原体的保护性免疫的潜力。美国沙门氏菌感染。这些实验包括携带VLR淋巴细胞的鉴定和分离，通过荧光活化细胞分选进行培养，电镜分析，cDNA文库的生成，通过亲和柱和快速蛋白液相色谱（FPLC）等色谱方法分离VLR的生化分析，VLR cDNA测序和重组VLR蛋白的表达，研究VLR半胱氨酸在VLR转导细胞系中的突变，以研究它们在形成VLR多聚体的二硫键中的潜在作用，并通过表面等离子体共振分析VLR抗原结合亲和力。总之，最近人们了解到，无颌鱼拥有一种适应性免疫系统，其特点是它们的淋巴细胞产生一种非常广泛的抗原特异性受体，这种受体在结构上与我们的抗体不同。由于这些可变淋巴细胞受体（VLR）与我们的抗体一样多样，并且可能具有相对稳定的物理结构，因此它们在检测和抑制危险病原体（如炭疽芽孢杆菌）方面非常有用。本文提出的实验将定义VLRs的生物学和结构特征，以探索其与健康相关的潜在用途。

DESCRIPTION (provided by applicant): A second type of adaptive immune system has been discovered very recently in the extant jawless vertebrates. Lymphocytes in the lamprey and hagfish undergo somatic recombinatorial assembly of modular leucine-rich-repeat (LRR) genetic units to generate a potential variable lymphocyte receptor (VLR) repertoire estimated to be as large as the antibody repertoire in mammals; i.e., >1014. The antigen-specific VLRs that are generated in response to immunization could have many practical uses similar to those established for antibodies in a variety of diagnostic tests, protein purification, inhibition of intracellular proteins, pathogen identification, and pathogen inhibition. Toward the realization of this potential, the experiments in this proposal are designed to characterize functional and structural properties of the VLR-mediated adaptive immune system in the sea lamprey. The primary aims are to (i) define the immunization requirements, kinetics, and memory potential in the lamprey cellular and humoral immune responses to particulate antigens (Bacillus anthracis exosporium, Salmonella typhimurium, and human erythrocytes) and to soluble antigens (BcIA anthrax protein and its C-terminal domain), (ii) characterize the molecular nature of soluble VLRs in experiments designed to test the hypothesis that VLRs are released from individual lymphocytes as preformed multivalent protein complexes, (iii) identify individual antigen-specific VLR-bearing lymphocytes and isolate them in order to characterize the antigen-binding affinity and structure of their VLR products, and (iv) examine the potential of the VLR adaptive immune system for protective immunity against a model pathogen, S. typhimurium. These experiments involve VLR-bearing lymphocyte identification and isolation by fluorescence activated cell sorting for culture, electron microscopic analysis, generation of cDNA libraries, biochemical analysis of VLRs separated by chromatographic methods including affinity columns and fast protein liquid chromatography (FPLC), VLR cDNA sequencing and expression of recombinant VLR protein, mutation of VLR cystines in VLR transduced cell lines to examine their potential role in disulfide linkage to form the VLR multimers, and analysis of VLR antigen-binding affinity by surface plasmon resonance. In summary, it has recently been learned that jawless fish possess an adaptive immune system that features the production by their lymphocytes of a very large spectrum of antigen-specific receptors that differ structurally from our antibodies. Since these variable lymphocyte receptors (VLR) are as diverse as our antibodies and may have a relatively stable physical structure, they could prove very useful in detecting and inhibiting hazardous pathogens, like Bacillus anthracis. The experiments proposed here will define biological and structural features of the VLRs in order to explore their potential for health-related uses.