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。这种兔子只有有限数量的VH基因可以重排。与鸡一样，VH1基因在大多数兔B淋巴细胞中都有重排。兔阑尾和鸡法氏囊是鸡的初级淋巴器官，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。在小鼠和人类等物种中，通过使用免疫球蛋白VHDJH和VLJL重排中不同的VH和VL基因来产生组合多样性可能是初级抗体库的主要贡献。在兔中，组合机制对重链多样性的贡献很小，因为只有几个VH基因重排和表达。我们最近发现，与其重链有限的组合多样性相反，兔子可以利用一组不同的生殖系V-kappa基因。尽管存在组合多样性，我们发现基因转换也改变了脾生发中心重排的兔V-kappa序列。我们描述了解释兔产生异质高亲和力抗DNP抗体的机制。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而进一步多样化。观察到的互补决定区氨基酸的正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品可能是基因转换改变了序列的细胞，这些细胞不再与免疫抗原反应，但是新谱系的来源。因此，脾生发中心将在成年兔身上发挥类似于幼兔阑尾和其他肠道相关淋巴组织的额外作用(2)。为了进一步研究早期免疫前谱系的发展，我们研究了3-9周龄兔阑尾发育过程中重链和轻链多样化的动力学。用液压显微操作和激光捕获显微切割收集单个B细胞，裂解后进行聚合酶链式反应扩增，产物直接测序。我们发现，重排的重链和轻链序列的基因转换在4周龄时就发生了。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到5周的阑尾中发现了小克隆，但大多数细胞产生了独特的、无关的序列。到5.5周时，一些较大的克隆被回收。来自阑尾的克隆的多样化模式与在脾生发中心发现的显著不同，在那里，一种特定的抗原驱动着扩增和选择过程走向高亲和力。克隆相关的附录B细胞在包括CDR3在内的每个互补决定区(CDR)上产生了非常不同的氨基酸序列，而来自脾的优势克隆在CDR3上几乎没有变化。在一个扩展的阑尾克隆中，多样化产生的不同的结合位点的多样性表明，至少有一些克隆的扩展和选择可能不是由特定的抗原驱动的。兔阑尾的发育需要特定的正常肠道菌群。微生物成分可能通过间接影响附录B细胞的发育和多样化，而不是仅仅作为特定的抗原，促进在B细胞中发现的广泛谱系的发展(Sehga等人)。MS正在准备中)。我们比较了手动液压显微切割技术和激光捕获显微切割技术。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，所以这是一个很好的对照，可以验证我们收集的单个细胞是否包含重排的VH(1)。为了完善人类重排VH基因的单细胞采集方法，我们一直在使用基于红外的LCM和另一种基于UV激光的显微切割系统Leica-LMD来采集人的阑尾B淋巴细胞。我们还使用LCM对肥大细胞增多症患者组织中的T细胞和B细胞进行了合作研究(M.Taylor et.艾尔女士。正在准备中)。

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) .