Characterization of an Alternative Adaptive Immune System in Hagfish

描述（由申请人提供）：广泛认为是当今存活的最基础的脊椎动物，盲鳗代表了进化的关键点，因此为人类进化之谜提供了关键的一环。 我们的淋巴细胞适应性免疫系统是如何进化的最近在两种现存的无颌脊椎动物七鳃鳗和八目鳗中发现了另一种适应性免疫系统，它们最后一次分享共同的祖先约4.8亿年前。虽然有颌脊椎动物使用基于免疫球蛋白的抗原受体，但无颌脊椎动物使用三种类型的基于富含亮氨酸重复序列（LRR）的可变淋巴细胞受体（VLR）来识别抗原。VLRA和VLRC组装在七鳃鳗胸腺相当于鳃区的两个淋巴细胞谱系，类似于哺乳动物的ab和gd T细胞谱系。VLRB通过B样淋巴细胞组装在造血组织中，B样淋巴细胞经历浆细胞分化并分泌具有结构特征和抗原特异性的多价VLRB抗体，其为生物医学用途提供显著优势。Hagfish细胞表达不同的VLRs尚未确定或功能定义。为了阐明盲鳗的免疫系统，已经制备了对它们的三个VLRs的不变部分具有特异性的单克隆抗体，用于分析盲鳗淋巴细胞的发育、分布和功能，特别强调定义盲鳗VLRB抗体的结构和抗原结合特异性。第一个目的是确定盲鳗VLRA +，VLRB+和VLRC+淋巴细胞的组织分布和基因表达模式。这里的一个主要目的是确定VLRA和VLRB组装发生的主要位点与成熟淋巴细胞增殖和分化的次要位点。第二个目的是表征盲鳗淋巴细胞对抗原、同种异体细胞和有丝分裂原的反应。为了确定为什么盲鳗对免疫表现出弱的体液免疫应答，但具有丰富的循环VLRB蛋白，这些实验将采用在七鳃鳗中引发强VLRB抗体应答的精制免疫方案。初步研究表明，盲鳗VLRA+和VLRC+淋巴细胞优先响应其他七鳃鳗的细胞，并提出实验，以找到无颌脊椎动物的“同种识别元件”。第三个具体目标是构建盲鳗VLRs的酵母表面展示文库，用于受体库的大规模调查，并分离抗原特异性克隆用于结构和功能表征。VLRA和VLRB文库的抗原结合的比较筛选将允许我们检验VLRA和VLRC库被克隆选择用于与加工抗原的反应性的假设。阐明盲鳗免疫系统将有助于我们了解如何和为什么淋巴细胞的发展沿着T-和B-细胞途径发生在脊椎动物的进化，并利用VLRB抗体用于生物医学目的。

DESCRIPTION (provided by applicant): Widely considered the most basal vertebrates alive today, hagfishes represent a pivotal point in evolution and thus offer a key piece to the puzzle of how our lymphocyte-based adaptive immune system evolved. An alternative adaptive immune system has been identified recently in the two extant jawless vertebrates, lampreys and hagfish, which last shared a common ancestor ~480 million years ago. While immunoglobulin- based antigen receptors are used by jawed vertebrates, jawless vertebrates instead use leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) of three types to recognize antigens. VLRA and VLRC are assembled in the lamprey thymus-equivalent gill region by two lymphocyte lineages that resemble mammalian ab and gd T cell lineages. VLRB is assembled in hematopoietic tissues by B-like lymphocytes, which undergo plasma cell differentiation and secrete multivalent VLRB antibodies with structural features and antigen specificities that offer significant advantages for biomedical uses. Hagfish cells expressing the different VLRs have not yet been identified or functionally defined. To elucidate the hagfish immune system, monoclonal antibodies specific for invariant portions of their three VLRs have been made for use in analyzing development, distribution and function of hagfish lymphocytes, with particular emphasis on defining the structure and antigen-binding specificities of hagfish VLRB antibodies. The first aim is to define the tissue distribution and gene expression patterns of hagfish VLRA +, VLRB+ and VLRC+ lymphocytes. A principle objective here is to identify the primary sites in which VLRA and VLRB assembly occurs versus secondary sites in which mature lymphocytes undergo proliferation and differentiation. The second aim is to characterize hagfish lymphocyte responses to antigens, allogeneic cells, and mitogens. To determine why hagfish exhibit weak humoral immune responses to immunization, yet have an abundance of circulating VLRB proteins, these experiments will employ refined immunization protocols that elicit robust VLRB antibody responses in lampreys. Pilot studies imply that the hagfish VLRA+ and VLRC+ lymphocytes preferentially respond to cells from other lampreys, and experiments are proposed to find the 'allo-recognition elements' of jawless vertebrates. The third specific aim is to construct yeast surface display libraries for hagfish VLRs for use in large scale surveys of the receptor repertoires and to isolate antigen-specific clones for structural and functional characterization. Comparative screening of the VLRA and VLRB libraries for antigen binding will allow us to test the hypothesis that the VLRA and VLRC repertoires are clonally selected for reactivity with processed antigen. Elucidation of the hagfish immune system will help us to understand how and why lymphocyte development along T- and B- cell pathways occurred during vertebrate evolution and to exploit VLRB antibodies for biomedical purposes.