Immunoglobulin Constant Region Switch and Hypermutation in the Nurse Shark

描述(申请人提供)：适应性免疫系统建立在淋巴细胞上，表达大量的抗原受体，在对病原体的反应中提供特定的识别。受体多样性是由V(D)J重排和免疫球蛋白(Ig)基因重排后的体细胞超突变所产生的。在高等脊椎动物中，不仅涉及3个Ig基因座的分级激活，而且还涉及成功重排的反馈机制，被认为限制了一个等位基因和一个同型基因的表达(等位基因和同型排除)。这个主要确保每个细胞只有一个受体物种的过程还没有被很好地理解。哺乳动物和鲨鱼是具有基于RAG重组酶的免疫系统的最早脊椎动物的代表，阐明它们共同和不同的调控途径将使我们能够深入了解构建适应性免疫系统的基本原理。初步数据表明，鲨鱼B细胞在细胞表面表达一种轻(L)链类型，还转录其他L链类型。然而，目前还不清楚护士鲨中的70多个L链微环是如何调控的。特异性目的1检测单个鲨鱼B细胞中L链的表达，确定是否存在抗原受体的克隆性表达。一些重(H)链cDNA序列含有V(D)J和C区，与已建立的基因组组织所预期的不同。这种遗传交换发生在躯体内和遥远的免疫球蛋白基因之间；它类似于哺乳动物中存在的H链开关重组。具体目的2是确定15个免疫球蛋白基因之间的连锁关系，并研究鲨鱼免疫球蛋白链转换过程的机制基础。由于这种转换与体细胞超突变同时发生，因此推测两者都是由激活诱导的胞苷脱氨酶启动的。鲨鱼免疫球蛋白的超突变独一无二地包括2-5个碱基对长度的串联变化，有时还包括非模板插入。为了了解这些变化是如何产生的，我们将在特定的目标3中分离处于细胞周期G2/M期的激活的B细胞，寻找那些同时携带突变和亲本V(D)J序列的B细胞。这项拟议的研究是早期脊椎动物免疫球蛋白基因表达长期研究的一部分。了解涉及DNA断裂和修复的B淋巴细胞多样化机制的本质，将有助于深入了解疾病状态的发病机制，包括自身免疫和由异常易位易患的淋巴系统恶性肿瘤。与公共健康相关：我们的鲨鱼模型系统携带大约85个免疫球蛋白基因，这些基因是酶的靶标，在产生抗体多样性的过程中引入缺口或损伤。我们观察到了遥远的基因座之间发生的遗传交换。研究这些现象将有助于确定非等位基因之间的遗传交换参数，并深入了解导致疾病状态的体细胞易位事件的起源。

DESCRIPTION (provided by applicant): The adaptive immune system is founded on lymphocytes expressing a vast array of antigen receptors that provide specific recognition in responding to pathogen. Receptor diversity is generated by V(D)J rearrangement and, in immunoglobulin (Ig) genes, post-rearrangement somatic hypermutation. In higher vertebrates mechanisms involving not only hierarchical activation of the 3 Ig loci but also feedback by a successful rearrangement are thought to restrict expression to one allele and one isotype (allelic and isotype exclusion). This process, which ensures primarily one receptor species per cell, is not well understood. Elucidating shared and divergent regulatory pathways in mammals and in sharks, representatives of the earliest vertebrates with a RAG recombinase-based immune system, will allow us to gain insight into the underlying principles upon which adaptive immune systems are constructed. Preliminary data suggest that shark B cells expressing one light (L) chain type at the cell surface also transcribe other L chain types. However, it is not clear how the more than 70 L chain miniloci in nurse shark are regulated. Specific Aim 1 is to examine L chain expression in single shark B cells and determine whether there is clonal expression of the antigen receptor. Some heavy (H) chain cDNA sequences contain V(D)J in combination with C regions different from what is anticipated from the established genomic organization. This genetic exchange occurs somatically and between distantly located IgH genes; it resembles the H chain switch recombination that exists in mammals. Specific Aim 2 is to determine the linkage relationship among the 15 IgH genes and investigate the mechanistic basis for the IgH chain switching process in sharks. Since the switching occurs at the same time as somatic hypermutation, it is hypothesized that both are initiated activation- induced cytidine deaminase. Hypermutation in shark Ig uniquely includes tandem changes of 2-5 bp stretches and sometimes non-templated insertions. To understand how these changes are generated, we will in Specific Aim 3 isolate activated B cells in G2/M phase of the cell cycle, looking for those carrying both mutated and parental V(D)J sequence. The proposed research is part of long-term studies on Ig gene expression in early vertebrates. Understanding the nature of B lymphocyte diversification mechanisms involving DNA breakage and repair will provide insight into the pathogenesis of disease states that include autoimmunity and lymphoid malignancies predisposed by aberrant translocations. PUBLIC HEALTH RELEVANCE: Our shark model system carries about 85 immunoglobulin genes that are targeted by enzymes introducing nicks or lesions in the course of generating antibody diversity. We have observed genetic exchange taking place between the distantly located loci. Studying these phenomena will help determine parameters for genetic exchange between non-allelic genes and give insight into the origins of somatic translocation events leading to disease states.