Rabbit Allotypes--Structure, Organization and Regulated

兔同种异型——结构、组织和调控

• 批准号: 6984922
• 负责人: rose G. mage
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6984922
• 关键词: B lymphocyte; antibody formation; developmental immunology; gene conversion; gene rearrangement; gut associated lymphoid tissue; human tissue; immunogenetics; immunoglobulin genes; immunoglobulin structure; immunoregulation; isoantibody; laboratory rabbit; laser capture microdissection; micromanipulator; molecular genetics; monoclonal antibody; nucleic acid sequence; peptide library; phage display; polymerase chain reaction; technology /technique development; vascular endothelial growth factors

Summary: Rabbit Preimmune Repertoire Development We study genes of the rabbit immune system using techniques of molecular biology and immunology (1). In species such as mouse and human, generation of combinatorial diversity through use of different VH and VL genes in immunoglobulin VHDJH and VLJL rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal as only a few VH genes are rearranged and expressed. This resembles chicken antibody formation. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and also to secondary diversification during immune responses in germinal centers (GCs). We previously showed that diversification patterns in the clones from appendix were strikingly different from those found in splenic GCs where an immunizing antigen was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3 whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual appendix clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign antigens. In collaboration with Dr. Ramit Mehr, we used a novel method for analyzing lineage tree shapes, using terms from graph theory to study diversification in rabbits and chickens. When lineage tree shapes were analyzed to quantify the differences between primary and secondary diversification in rabbits, the analyses indicated that primary diversification appears to occur at a constant rate in the appendix and the type of antigen-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut associated lymphoid tissues may play in early development of mammalian immune repertoires. The data also indicate a higher rate of hypermutation in rabbit during immune responses in splenic GCs, such that the balance between hypermutation and selection tends more towards mutation and less towards selection in rabbit compared to murine GCs (3). Microdissection of Single and Small numbers of Cells for DNA and RNA Analyses In order to collect single cells for PCR amplification and sequencing of rearranged VH genes, we have been using both the infra red based LCM and another UV laser-based microdissection system, Leica-LMD to collect appendix B lymphocytes. We also compared the tedious but successful method of manual hydraulic microdissection with techniques of laser capture microdissection (LCM). For these studies, we used both rabbit and human appendix tissues. Once capability to collect single cells by laser capture microdissection (LCM ) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR, products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for collection of single B cells (2). Both IR and UV lasers have been used for sample collection from tissue sections for genetic analyses. The high peak power densities in nitrogen laser microdissection (337nm) may excite endogenous photosensitizers or some histological dyes and cause DNA or RNA damage, perhaps through two photon mechanisms. We looked for diminished yields as evidence of damage when using LMD to isolate B cells. The Leica-LMD pulsed UV-laser system was used to collect single B cells from human appendix tissue sections, and single or multiple B cells from rabbit tissues, which had been immunohistochemically identified. Circles of different radii were used to assess possible loss of efficiency due to UV damage. The frequency of single allele PCR-amplification was compared between LMD and Hydraulic Micromanipulation (HM). Immunoglobulin VDJ PCR products and sequences obtained were indistinguishable for the two methods. We conclude that UV-based lasers can be used to cut cells individually from tissue sections provided the cutting edge is far enough from the cell membrane (>2.0 microns). In addition, with LMD we successfully isolated mRNA from rabbit splenic germinal centers (GC) containing antigen-specific B cells and determined gene sequences after RT-PCR and cloning. Total DNA and mRNA yields from 100-1000 cells collected in clusters were similar for LMD and HM. LMD appears particularly suited for independent collection of specific clusters of cells from anywhere on a slide and contiguous cell clusters from serial sections (2). Rabbit Immune Repertoires for Generation and Humanization of Therapeutic Monoclonal Antibodies The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. A collaboration with Dr. C. Rader and colleagues was established in order to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. Rabbits with the rare b9 and bas allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones is a rabbit/human Fab that binds with a dissociation constant of 1 nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer (5). Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Murine tumor angiogenesis models and receptor-specific antibodies are required to facilitate evaluation of the roles of VEGF receptors in mouse models of human cancer. We developed rabbit antibodies that cross-react with mouse (Flk-1) and human (KDR) VEGFR2. High-affinity, species cross-reactive, VEGFR2-specific Fabs were selected from an antibody phage display library generated from an immunized b9 allotype rabbit. The selected chimeric rabbit/human Fabs were found to bind to KDR and Flk-1 with nanomolar affinity. Three selected Fabs detected KDR expression on human endothelial cells as well as Flk-1 on murine endothelial cells. The availability of anti-VEGFR2 Fab with species cross-reactivity will help to decipher functional role of KDR/Flk-1 in tumor biology as well as facilitate the preclinical evaluation of the suitability of KDR/Flk-1 for drug targeting. This report further underscores our earlier finding that b9 allotype rabbits are excellent sources for generating high affinity cross-reactive antibodies with therapeutic potential (4). Methods: Rabbit immunization, RT PCR, cDNA library construction, PCR and genomic library construction, phage display library construction, immunohistochemistry, microdissection of single cells by hydraulic micromanipulation, laser capture microdissection (LCM) and Leica LMD, single-cell PCR and DNA sequencing, computer analyses of rearranged and germline Ig gene sequences, production of chimeric rabbit/human phage display libraries. Goals and Objectives: A long range objective of this research has been to define in molecular terms, the organization and regulated expression of rabbit immunoglobulin genes, describe their evolutionary origins, structures and molecular mechanisms leading to the diversification of variable regions. We use genetically defined rabbits in a variety of studies where knowledge of the molecular genetics and biology contributes to the conduct of the study. In addition, from time to time, we study other genes of importance in immune regulation and variable region diversification such as the rabbit recombination activating genes (RAG-1 and RAG-2), the RAD51 family of DNA repair genes and most recently rabbit activation induced deminase (AID).

摘要：兔预免疫指令集的开发 我们利用分子生物学和免疫学技术研究兔子免疫系统的基因 (1)。在小鼠和人类等物种中，通过在免疫球蛋白 VHDJH 和 VLJL 重排中使用不同的 VH 和 VL 基因产生组合多样性可能是一抗库的主要贡献者。在兔子中，组合机制对重链多样性的贡献很小，因为只有少数 VH 基因被重排和表达。这类似于鸡抗体的形成。兔阑尾和鸡法氏囊是主要淋巴器官，其中 B 细胞抗体库主要通过类似基因转换的过程在生发中心发育。与鸡一样，大多数兔 B 淋巴细胞中的 3 素数 VH1 基因发生重排。体细胞超突变和基因转换有助于幼兔阑尾或胚胎和幼鸡法氏囊的初级多样化，也有助于生发中心（GC）免疫反应期间的次级多样化。我们之前表明，来自阑尾的克隆的多样化模式与脾GC中发现的多样化模式显着不同，在脾GC中，免疫抗原驱动着向高亲和力的扩展和选择过程。克隆相关的阑尾 B 细胞在每个互补决定区 (CDR) 中形成不同的氨基酸序列，包括 CDR3，而来自脾脏的显性克隆在 CDR3 中几乎没有变化。各个阑尾克隆内的多样化产生的组合位点的多样性表明，至少有一些已知需要正常肠道菌群的克隆扩增和选择可能是通过微生物成分的间接作用驱动的，而不仅仅是通过将其识别为特定的外来抗原来驱动的。我们与 Ramit Mehr 博士合作，采用了一种分析谱系树形状的新方法，利用图论术语来研究兔子和鸡的多样化。当对家兔的谱系树形状进行分析以量化初级和次级多样化之间的差异时，分析表明初级多样化似乎在阑尾中以恒定的速率发生，并且在脾GC中不存在抗原特异性选择的类型。这支持了这样的观点，即幼兔阑尾中不断扩大的克隆相关 B 细胞正在生成主要组库，并强调了肠道相关淋巴组织在哺乳动物免疫组库的早期发育中可能发挥的重要作用。数据还表明，在脾 GC 的免疫反应过程中，兔子的超突变率较高，因此与鼠 GC 相比，兔子的超突变和选择之间的平衡更倾向于突变，而不是选择 (3)。 用于 DNA 和 RNA 分析的单个和少量细胞的显微切割 为了收集单细胞用于 PCR 扩增和重排 VH 基因测序，我们一直使用基于红外的 LCM 和另一种基于紫外激光的显微切割系统 Leica-LMD 来收集阑尾 B 淋巴细胞。我们还将繁琐但成功的手动液压显微切割方法与激光捕获显微切割 (LCM) 技术进行了比较。在这些研究中，我们使用了兔子和人的阑尾组织。一旦开发出通过激光捕获显微切割 (LCM) 收集单细胞的能力，我们就修改了以前的组织染色和固定方法，以便我们可以通过 HM 和 LCM 从给定的染色组织切片中收集细胞，并直接比较使用这两种方法的成功率。碱裂解细胞，两轮巢式PCR后，回收产物并直接测序。由于在发育中的 B 细胞中形成免疫球蛋白重链编码序列的基因组 DNA 的每次重排都是独特的，因此该系统使我们能够验证从组织切片中回收单个淋巴细胞并扩增单个等位基因的成功率。所开发的方法现已使 LCM 成为 HM 收集单个 B 细胞的有效替代方案 (2)。红外和紫外激光均已用于从组织切片中采集样本以进行遗传分析。氮激光显微切割 (337nm) 中的高峰值功率密度可能会激发内源性光敏剂或某些组织学染料，并可能通过两种光子机制引起 DNA 或 RNA 损伤。当使用 LMD 分离 B 细胞时，我们寻找产量下降作为损伤的证据。采用Leica-LMD脉冲紫外激光系统采集人阑尾组织​​切片中的单个B细胞，以及兔组织中的单个或多个B细胞，并进行免疫组化鉴定。使用不同半径的圆来评估由于紫外线损伤可能造成的效率损失。比较 LMD 和液压显微操作 (HM) 之间的单等位基因 PCR 扩增频率。两种方法获得的免疫球蛋白 VDJ PCR 产物和序列无法区分。我们得出的结论是，只要切割边缘距离细胞膜足够远（> 2.0 微米），基于紫外线的激光可用于从组织切片中单独切割细胞。此外，我们利用LMD成功地从含有抗原特异性B细胞的兔脾生发中心（GC）中分离出mRNA，并在RT-PCR和克隆后确定了基因序列。对于 LMD 和 HM，收集在簇中的 100-1000 个细胞的总 DNA 和 mRNA 产量相似。 LMD 似乎特别适合从载玻片上的任何位置独立收集特定细胞簇以及从连续切片中收集连续细胞簇 (2)。 用于产生治疗性单克隆抗体和人源化的兔免疫库 兔免疫库长期以来一直是诊断性多克隆抗体的丰富来源。现在，它作为治疗性单克隆抗体的来源也具有广阔的前景。与 C. Rader 博士及其同事建立了合作，以便比较不同的兔免疫库，以生成和人源化单克隆抗体，这些抗体与肿瘤血管生成中涉及的抗原具有强亲和力。具有罕见 b9 和 bas 同种异型的兔子是治疗性单克隆抗体的极好来源。所选克隆中的特色是兔/人 Fab，它以 1 nM 的解离常数与人和小鼠 Tie-2 结合，这将有助于在人类癌症小鼠模型中对其进行评估 (5)。血管内皮生长因子（VEGF）及其受体通过刺激肿瘤相关的血管生成来促进实体瘤的生长和转移。需要鼠肿瘤血管生成模型和受体特异性抗体来促进 VEGF 受体在人类癌症小鼠模型中的作用的评估。我们开发了与小鼠 (Flk-1) 和人 (KDR) VEGFR2 发生交叉反应的兔抗体。高亲和力、物种交叉反应、VEGFR2 特异性 Fab 选自免疫 b9 同种异型兔产生的抗体噬菌体展示文库。发现所选择的嵌合兔/人 Fab 以纳摩尔亲和力与 KDR 和 Flk-1 结合。三个选定的 Fab 检测到人内皮细胞上的 KDR 表达以及鼠内皮细胞上的 Flk-1。具有物种交叉反应性的抗 VEGFR2 Fab 的可用性将有助于破译 KDR/Flk-1 在肿瘤生物学中的功能作用，并促进 KDR/Flk-1 药物靶向适用性的临床前评估。该报告进一步强调了我们之前的发现，即 b9 同种异型兔是产生具有治疗潜力的高亲和力交叉反应抗体的极好来源 (4)。 方法：兔免疫、RT PCR、cDNA文库构建、PCR和基因组文库构建、噬菌体展示文库构建、免疫组织化学、通过液压显微操作对单细胞进行显微切割、激光捕获显微切割（LCM）和Leica LMD、单细胞PCR和DNA测序、重排​​和种系Ig基因序列的计算机分析、嵌合体的产生 兔/人噬菌体展示库。 目标和目的：本研究的长期目标是用分子术语定义兔免疫球蛋白基因的组织和调节表达，描述其进化起源、结构和导致可变区多样化的分子机制。我们在各种研究中使用基因定义的兔子，其中分子遗传学和生物学知识有助于研究的进行。此外，我们不时研究在免疫调节和可变区多样化中重要的其他基因，例如兔重组激活基因（RAG-1和RAG-2）、DNA修复基因的RAD51家族以及最近的兔激活诱导脱氨酶（AID）。