Characterization of Lamprey B cells and Antibodies

描述(申请人提供)：拟议的研究建立在我们之前的发现基础上，表明互动的T和B样淋巴细胞是无颌脊椎动物和有颌骨脊椎动物适应性免疫系统的基本特征，尽管无颌脊椎动物(七鳃鳗和七鳃鳗)通过使用不同的富亮氨酸重复序列(LRR)供体序列将不完整的生殖系VLR基因(VLRA、VLRA和VLRC)重组转化为完全组装的VLR基因，从而产生可变的淋巴细胞受体(VLR)用于抗原识别。最近的研究表明，虽然VLRA基因的组装与胞苷脱氨酶1(CDA1)在淋巴上皮胸腺鳃区域的表达一致，但VLRB和CDA2的一致表达主要发生在造血组织中。本文对七鳃鳗类VLRB淋巴细胞的发育、分布和功能进行了全面的分析。总体目标是更好地了解无颌脊椎动物的B细胞生物学，特别是产生VLRB抗体多样性的机制，并将七鳃鳗抗体系统用于生物医学目的。一种新开发的有效的七鳃鳗免疫方案将与随后从免疫动物中高通量筛选VLRB cDNA文库相结合，以获得与具有生物医学意义的模式抗原具有高结合亲和力的VLRB特异性单抗。第一个特异性目的是阐明VLRB抗体亲和力成熟的序列多样性和在七鳃鳗B细胞对模式蛋白抗原的初次免疫和加强免疫应答过程中的可能性。第二个特定目的是确定七鳃鳗CDA2的结构、功能潜力和表达，作为VLRB系细胞分化状态和组织定位的函数。第三个具体目标是生产具有新特异性的重组VLRB抗体，用作医学上重要的感染性病原体和肿瘤细胞类型的诊断试剂。这些相互关联的研究将为无颌骨和有颌骨脊椎动物替代B细胞类型的进化提供洞察力，并将产生针对人类感染病原体和肿瘤细胞抗原的新型单抗，用于诊断和疾病监测。

DESCRIPTION (provided by applicant): The proposed studies build on our previous findings indicating that interactive T- and B-like lymphocytes are basic features of the adaptive immune system in both jawless and jawed vertebrates, although jawless vertebrates (lampreys and hagfish) generate variable lymphocyte receptors (VLR) for antigen recognition by recombinatorial conversion of incomplete germline VLR genes (VLRA, VLRA and VLRC) into fully assembled VLR genes using diverse leucine-rich repeat (LRR) donor sequences. Recent studies indicate that while VLRA gene assembly coincides with expression of cytidine deaminase 1 (CDA1) within the lymphoepithelial thymoid gill region, coincident VLRB and CDA2 expression instead occurs primarily in hematopoietic tissues. A comprehensive analysis is proposed to elucidate the development, distribution and function of the B-like VLRB lymphocytes in lampreys. The overall goals are to better understand B cell biology in a jawless vertebrate, in particular the mechanism of generating VLRB antibody diversity, and to harness the lamprey antibody system for biomedical purposes. An efficient newly-developed scheme for lamprey immunization will be coupled with subsequent high-throughput screening of VLRB cDNA libraries from immunized animals to obtain specific monoclonal VLRB antibodies with high binding affinity for model antigens of biomedical significance. The first specific aim is to elucidate the sequence diversity and possibility of VLRB antibody affinity maturation during lamprey B cell responses to primary and booster immunizations with model protein antigens. The second specific aim is to determine the lamprey CDA2 structure, functional potential, and expression as a function of the differentiation status and tissue localization of VLRB lineage cells. The third specific aim is to produce recombinant VLRB antibodies with novel specificities for use as diagnostic reagents for medically important infectious agents and tumor cell types. These interrelated studies will provide insight into the evolution of the alternative B cell types f jawless and jawed vertebrates and will also yield novel monoclonal lamprey antibodies with specificity for infectious agents and tumor cell antigens of humans for use as diagnostic and disease-monitoring reagents.