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) .

我们通过分子生物学和免疫学技术研究兔免疫系统的基因。兔子的重新排列的VH基因数量有限。与鸡一样，在大多数兔B淋巴细胞中，大多数VH1基因都重新排列。 Fabricius的Rabbit附录和鸡肉囊是原发性淋巴器官，在该器官中，B细胞抗体库主要通过基因转换过程在生发中心发展。在小鼠和人类等物种中，通过在免疫球蛋白VHDJH中使用不同的VH和VL基因来产生组合多样性，而VLJL重排可能是主要抗体库的主要促成者。在兔子中，组合机制对重链多样性的贡献很小，因为只有几个VH基因被重新排列和表达。我们最近表明，与其重链的有限组合多样性相反，兔子可以利用一组种系V-kappa基因。尽管存在组合多样性，但我们发现基因转化率也改变了脾发芽中心中重新排列的兔V-kappa序列。我们描述了兔子可以产生的异质高亲和力抗DNP抗体的发展的机制。我们发现，在克隆谱系中，重新排列的V-kappa和VH通过基因转化和体细胞超夸rive。在互补性决定区域中观察到的氨基酸的阳性和阴性选择允许出现各种不同的组合位点结构。生发中心反应的副产品可以是细胞，其序列因基因​​转化而改变，而基因转化不再与免疫抗原反应，而是新曲目的来源。因此，脾生发中心将在类似于附录和其他肠道相关的年轻兔子淋巴组织类似的成年人中发挥额外的作用（2）。为了进一步调查早期免疫前曲目的发展，我们研究了在3至9周之间发展兔附录中重链和轻链多样化的动力学。使用液压微观液和激光捕获微分解收集的单个B细胞被裂解，PCR扩增并直接测序产物。我们发现，重排重链和轻链序列的基因转化发生在4周龄时。体细胞突变发生在缺乏已知转换供体的D区域。它们可能也发生在V基因中。在4至5周的附录中发现了小克隆，但是大多数细胞产生了独特的无关序列。到5.5周，回收了一些较大的克隆。附录的克隆中的多样化模式与在脾发芽中心发现的不同不同，在脾发芽中心中，特定的抗原正在推动膨胀和选择过程朝向高亲和力。克隆相关的附录B细胞在每个互补性确定区域（CDR）中形成了非常不同的氨基酸序列，包括CDR3，而Spleen的主要克隆在CDR3中发生了很少的变化。单个扩展的附录克隆中多元化产生的各种组合位点表明，在附录中至少某些克隆扩张和选择可能不会由特定的抗原驱动。兔附录开发需要特定的普通肠道菌群。微生物成分不仅是作为特定的抗原作用，还可以通过间接对附录B细胞发育和多样化的影响（Sehgal etal。MS进行制备中），有助于在B细胞中发现的广泛曲目的发展。我们将手动液压微解剖的技术与激光捕获微分解技术（LCM）进行了比较。对于这些研究，我们同时使用了兔子和人类附录组织。由于兔子主要重新排列一个VH基因，因此可以验证我们收集了一个包含重新排列VH的单个单元（1）是一个良好的控制。为了完善收集单个细胞进行PCR扩增和测序的方法的方法，我们一直在使用Infra Red基LCM和另一个基于UV激光的微型解剖系统Leica-LMD收集人类附录B淋巴细胞。我们还在肥大细胞增多症患者组织中对T细胞和B细胞的合作研究中使用了LCM（M. Taylor等，MS。制备中）。