Rabbit Allotypes--structure, Organization And Regulated

我们利用分子生物学和免疫学技术研究兔免疫系统的基因。在小鼠和人类等物种中，通过免疫球蛋白VHDJH和VLJL重排中使用不同的VH和VL基因来产生组合多样性可能是一抗库的主要贡献者。在家兔中，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，这与鸡抗体的形成相似。兔阑尾和鸡法氏囊是原发性淋巴器官，其B细胞抗体库主要通过基因转换样过程在生发中心形成。与鸡一样，3-prime most VH1基因在大多数兔B淋巴细胞中被重排。然而，与鸡不同的是，我们最近发现兔子可以利用多种种系V-kappa基因。除了组合多样性外，基因转换还会进一步改变脾脏生发中心重排的V-kappa序列。幼兔阑尾是原代b细胞库发育的动态位点，研究其克隆扩增过程中的多样化模式具有重要意义。对3 ~ 9周龄家兔单根阑尾B细胞进行水力显微操作或激光捕获显微解剖，经PCR扩增和重排，直接测序重、轻链基因产物。基因转换样的变化被观察到发生在3 - 4周龄的序列中。在缺乏已知转化供体的D区发现了体细胞突变，可能也发生在V基因中。对克隆变异的发育也进行了研究。在3 ~ 5周时发现少量的无性系相关阑尾B细胞；到5.5周时，一些较大的克隆恢复了。来自阑尾的克隆的多样化模式与之前在脾生发中心发现的明显不同，在那里免疫抗原驱动着扩张和选择过程向高亲和力方向发展。此外，克隆相关的阑尾B细胞在包括CDR3在内的每个互补决定区（CDR）中产生了不同的氨基酸序列，而来自脾脏的优势克隆的CDR3几乎没有变化。单个克隆内多样化产生的组合位点的多样性表明，至少一些已知需要正常肠道菌群的克隆扩增和选择可能是由微生物成分的间接作用驱动的，而不仅仅是由它们被识别为特定的外来抗原驱动的。b细胞克隆中结合位点的多样性支持了阑尾在产生免疫前库中的作用(3)。为了完善单细胞收集方法，对重排人VH基因进行PCR扩增和测序，我们使用了基于红外的LCM和另一种基于紫外激光的显微解剖系统Leica-LMD来收集人阑尾B淋巴细胞。我们还比较了繁琐但成功的手工液压显微解剖方法与激光捕获显微解剖技术。在这些研究中，我们使用了兔和人的阑尾组织。一旦能够通过激光捕获显微解剖（LCM）收集单个细胞，我们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，并直接比较这两种方法的成功率。细胞碱性裂解，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的每一次重排都是独一无二的，因此该系统使我们能够验证从组织切片中恢复单个淋巴细胞和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细胞的有效替代方法(1)。红外和紫外激光都已用于从组织切片中收集样本进行遗传分析。氮激光显微解剖（337nm）的峰值功率密度可能通过两种光子机制激发内源性光敏剂或某些组织染料，导致DNA或RNA损伤。当使用LMD分离B细胞时，我们寻找产量减少作为损伤的证据。采用Leica-LMD脉冲紫外激光系统分别采集经免疫组织化学鉴定的人阑尾组织切片中的单个B细胞和家兔组织中的单个或多个B细胞。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。比较了LMD和液压微操作（HM）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产物和序列难以区分。我们得出的结论是，只要切割边缘离细胞膜足够远（>2.0微米），紫外激光可以用于从组织切片上单独切割细胞。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）中分离到了mRNA，并通过RT-PCR和克隆确定了基因序列。LMD和HM成组收集的100-1000个细胞的总DNA和mRNA产量相似。LMD似乎特别适合于从载玻片上任何地方独立收集特定的细胞簇和从连续切片中收集连续的细胞簇(2)。兔的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大有希望。与C. Rader博士及其同事合作建立了一项合作，以比较不同的兔子免疫库，以产生和人源化单克隆抗体，这些单克隆抗体与肿瘤血管生成中涉及的抗原具有强亲和力。特别地，我们评估了来自不同kappa轻链同种异体的未选择和选择的嵌合兔/人Fab文库的多样性。具有特殊罕见同种异体的兔子是治疗性单克隆抗体的极佳来源。在选定的克隆中，有一个兔/人Fab，与人和小鼠Tie-2结合的解离常数为1 nM，这将有助于其在人类癌症小鼠模型中的评估（C. Rader等人提交）。

We study genes of the rabbit immune system using techniques of molecular biology and immunology. 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. However, in contrast to chicken, we recently showed that the rabbit can draw upon a diverse set of germline V-kappa genes. In addition to combinatorial diversity, gene conversion further alters rearranged rabbit V-kappa sequences in splenic germinal centers. The young rabbit appendix is a dynamic site for primary B-cell repertoire development and it was of interest to study diversification patterns during clonal expansion. Single appendix B cells from 3 to 9-week old rabbits obtained by hydraulic micromanipulation or laser capture microdissection were lysed, PCR amplified and rearranged heavy and light chain gene products directly sequenced. Gene conversion-like changes were observed to have occurred in the sequences by 3 - 4 weeks of age. Somatic mutations were found in the D regions that lack known conversion donors and probably also occurred in the V genes. The development of clonal variants was also studied. A few small sets of clonally related appendix B cells were found at 3 to 5 weeks; by 5.5 weeks, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found previously in splenic germinal centers where an immunizing antigen was driving the expansion and selection process toward high affinity. In addition, 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 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. This diversity of combining sites within B-cell clones supports the proposed role of appendix in generating the preimmune repertoire (3). In order to perfect the methods to collect single cells for PCR amplification and sequencing of rearranged human VH genes, we have been using both the infra red based LCM and another UV laser-based microdissection system, Leica-LMD to collect human 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 (1). 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). 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. In particular, the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes was evaluated. Rabbits with particular rare 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 (C. Rader et. al. submitted).