Rabbit Allotypes--structure, Organization And Regulated

兔同种异型——结构、组织和调控

• 批准号: 6506798
• 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/6506798
• 关键词: B lymphocyte; gene expression; gene rearrangement; genetic regulation; gut associated lymphoid tissue; human subject; immunogenetics; immunoglobulin genes; immunoglobulin structure; laboratory rabbit; molecular genetics; monoclonal antibody; nucleic acid sequence; polymerase chain reaction

We study genes of the rabbit immune system by techniques of molecular biology and immunology. The rabbit has a limited number of VH genes that rearrange. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. 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. 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. We recently showed that in contrast to limited combinatorial diversity of its heavy chain, the rabbit can draw upon a diverse set of germline V-kappa genes. In spite of the presence of combinatorial diversity, we found that gene conversion also alters rearranged rabbit V-kappa sequences in splenic germinal centers. We described mechanisms that account for the development of the heterogeneous high affinity anti DNP antibodies that rabbits can produce. We found that in clonal lineages, rearranged V-kappa and VH are further diversified by gene conversion and somatic hypermutation. The positive and negative selection of amino acids in complementarity determining regions observed allows emergence of a variety of different combining site structures. A by-product of the germinal center reaction may be cells with sequences altered by gene conversion that no longer react with the immunizing antigen but are a source of new repertoire. The splenic germinal center would thus play an additional role in adults similar to that of the appendix and other gut associated lymphoid tissues of young rabbits (2). In order to further investigate the development of the early pre-immune repertoire, we studied the kinetics of diversification of heavy and light chains in developing rabbit appendix between 3 and 9-weeks of age. Single B cells collected using hydraulic micromanipulation and laser capture microdissection were lysed, PCR amplified and products directly sequenced. We found that gene conversion of rearranged heavy and light chain sequences was occurring by 4 weeks of age. Somatic mutations occurred in the D regions that lack known conversion donors; they probably also occurred in the V genes. Small clones were found in 4 to 5 -week appendix, but the majority of cells yielded unique, unrelated sequences. By 5.5 weeks, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found in splenic germinal centers where a specific antigen was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed very different amino acid sequences in each complementarity determining region (CDR) including CDR3 whereas dominant clones from spleen underwent few changes in the CDR3. The variety of different combining sites generated by diversification within a single expanding appendix clone suggests that at least some clonal expansion and selection in appendix may not be driven by specific antigens. Rabbit appendix development requires specific normal gut flora. Rather than acting solely as specific antigens, microbial components may contribute to the development of the broad repertoire found in the B cells through indirect effects on appendix B-cell development and diversification (Sehgal et al. Ms in preparation). We compared techniques of manual hydraulic microdissection with techniques of laser capture microdissection (LCM). For these studies, we used both rabbit and human appendix tissues. Because the rabbit mainly rearranges one VH gene, it is a good control for verifying that we have collected a single cell containing a rearranged VH (1). 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 used LCM in a collaborative study on T cells and B cells in mastocytosis patients' tissues (M. Taylor et. al, ms. in preparation) .

本研究运用分子生物学和免疫学技术对兔免疫系统基因进行了研究。兔子有有限数量的VH基因重排。与鸡一样，在大多数兔B淋巴细胞中，最大的3-引物VH 1基因重排。兔阑尾和鸡法氏囊是主要的淋巴器官，其中B细胞抗体库主要通过基因转换样过程在生殖中心发育。在诸如小鼠和人的物种中，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以是一抗库的主要贡献者。在兔中，组合机制对重链多样性的贡献最小，因为只有少数VH基因重排和表达。我们最近发现，与其重链的有限组合多样性相反，兔子可以利用不同的种系V-κ基因。尽管存在组合的多样性，我们发现，基因转换也改变了重排兔V-κ序列在脾germination中心。我们描述的机制，占发展的异质性高亲和力抗DNP抗体，兔可以产生。我们发现，在克隆谱系中，重排的V-kappa和VH通过基因转换和体细胞超突变进一步多样化。观察到的互补决定区中氨基酸的正选择和负选择允许出现各种不同的结合位点结构。生发中心反应的副产物可能是具有通过基因转换改变的序列的细胞，其不再与免疫抗原反应，而是新的库的来源。因此，脾脏生发中心在成年兔中发挥着与幼兔阑尾和其他肠道相关淋巴组织相似的额外作用（2）。为了进一步研究早期免疫前库的发展，我们研究了3和9周龄之间的兔阑尾发育中重链和轻链多样化的动力学。将使用液压显微操作和激光捕获显微切割收集的单个B细胞裂解，PCR扩增并直接测序产物。我们发现，重排的重链和轻链序列的基因转换发生在4周龄。体细胞突变发生在缺乏已知转换供体的D区;它们可能也发生在V基因中。在4至5周的阑尾中发现了小克隆，但大多数细胞产生了独特的、不相关的序列。到5.5周时，回收了一些较大的克隆。来自阑尾的克隆的多样化模式与脾生殖中心中发现的那些明显不同，在脾生殖中心中，特异性抗原驱动扩增和选择过程朝向高亲和力。克隆相关的阑尾B细胞在每个互补决定区（CDR）（包括CDR 3）中产生了非常不同的氨基酸序列，而来自脾的显性克隆在CDR 3中发生了很少的变化。在一个单一的扩张阑尾克隆内的多样化产生的不同的结合位点的多样性表明，至少有一些克隆扩张和选择在阑尾可能不是由特异性抗原驱动。兔阑尾的发育需要特定的正常肠道植物群。微生物组分不仅作为特异性抗原发挥作用，还可能通过对阑尾B细胞发育和多样化的间接影响，促进B细胞中广泛库的发育（Sehgal等人，MS in preparation）。我们比较了手动液压显微切割技术与激光捕获显微切割技术（LCM）。在这些研究中，我们使用了兔和人的阑尾组织。因为兔子主要重排一个VH基因，所以它是验证我们已经收集了含有重排的VH的单个细胞的良好对照（1）。为了完善用于人重链可变区基因重排的PCR扩增和测序的单细胞采集方法，我们采用了基于红外的LCM和另一种基于紫外激光的显微切割系统Leica-LMD采集人阑尾B淋巴细胞。我们还在肥大细胞增多症患者组织中的T细胞和B细胞的合作研究中使用LCM（M. Taylor et. al，Ms. in preparation）。