IG GENETICS--ONTOGENY AND DIFFERENTIATION OF CELLS OF THE RABBIT IMMUNE SYSTEM

IG遗传学--兔免疫系统细胞的个体发育和分化

• 批准号: 2566694
• 负责人: R 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/2566694
• 关键词: B cell receptor; B lymphocyte; CD antigens; CD5 molecule; apoptosis; cell differentiation; developmental genetics; flow cytometry; gene expression; gene rearrangement; gut associated lymphoid tissue; immunocytochemistry; immunogenetics; immunoglobulin M; immunoglobulin genes; laboratory rabbit; leukopoiesis

We used techniques of immunogenetics and molecular biology to study rabbit immunoglobulins, and other genes including RAG-1 and RAG-2, which are necessary for gene rearrangements to occur during lymphocyte development. We investigated the development of anatomical sites such as appendix follicles and germinal centers in gut-associated lymphoid tissues and the regulated expression and sequence diversification of Ig genes during lymphoid cell development. Whereas B cells with rearranged VH1 predominate in normal rabbits, in homozygous Alicia mutant rabbits (ali/ali) the VH1 gene is deleted and B cells with upstream VH genes rearrange. We found differences between appendix development in normal and ali/ali rabbits based on immunohistochemistry, analyses of cell proliferation and apoptotic death. The development of the appendix in mutants appears to be retarded compared to normals. As populations of B cells bearing VHa2-like epitopes develop in mutant animals, appendix development appears more like normals. A higher proportion of B cells expressing the a2 allotype may receive strong signals to survive rather than undergo apoptosis. VHa2-positive B cells express high levels of Bcl-2 protein compared to a2-negative B cells. This suggests that B cells with FR allotypic motifs may become resistant to programmed cell death via the Bcl-2 pathway. The a2 allotype probably plays functional role(s) in selection and effective expansion of B cells in the appendix. We have now identified CD5 as a ligand for B-cell surface immunoglobulin. Immobilized F(ab')2 fragments isolate CD5 molecules from appendix cell lysates. We purified and biotinylated F(ab')2 fragments from sera of normal (a2+) and VH mutant animals (a2-) and used them as probes. By flow cytometry, VHa2+ F(ab')2 bind IgM+ B cells more strongly than VHa2- F(ab')2. This interaction as well as F(ab')2 binding to appendix germinal centers can be blocked by anti-CD5 antibodies. Cell attachment assays also suggest that the B cell-surface glycoprotein CD5 is a ligand for B cell surface immunoglobulin framework region sequences. Interactions of VH framework region structures with endogenous ligands such as CD5 may affect maintenance and selective expansion of particular B cells.

我们使用了免疫遗传学和分子生物学技术来研究 兔免疫球蛋白，以及包括rag-1和rag-2在内的其他基因，它们 是在淋巴细胞中发生基因重排所必需的 发展。我们调查了像这样的解剖地点的发展 作为肠相关淋巴组织中的阑尾滤泡和生发中心 组织与免疫球蛋白的调控表达和序列多样化 淋巴样细胞发育过程中的基因。而重排的B细胞 VH1在正常兔和纯合子Alicia突变兔中占优势 (ALI/ALI)VH1基因缺失，具有上游VH基因的B细胞 重新安排。我们发现正常情况下阑尾发育不同 并对ALI/ALI兔进行免疫组织化学、细胞分析 增殖和凋亡性死亡。中附录的发展 与正常人相比，突变者似乎反应迟缓。作为人口数量 携带VHA2样表位的B细胞在突变动物体内发育，附录 发展看起来更像是常态。更高比例的B细胞 表达a2同种异型可能会收到强烈的生存信号 而不是经历细胞凋亡。VHA2阳性的B细胞高水平表达 Bcl2蛋白与a2阴性B细胞的比较。这表明B 具有FR异型基序的细胞可能对程序性细胞产生抵抗力 通过Bcl2途径死亡。A2同种异型可能起作用。 S在阑尾B细胞选择和有效扩增中的作用。 我们现在已经确定CD5是B细胞表面免疫球蛋白的配体。 固定化F(ab‘)2片段从阑尾细胞分离CD5分子 裂解物。我们从猪血清中提纯并生物素标记了F(ab‘)2片段 正常(a2+)和VH突变动物(a2-)，并用它们作为探针。通过 流式细胞仪检测，VHA2+F(ab‘)2与IgM+B细胞的结合强度高于VHA2- F(ab‘)2.这种相互作用以及F(ab’)2与附录的结合 生发中心可被抗CD5抗体阻断。细胞附着 分析还表明，B细胞表面糖蛋白CD5是一种配体 对于B细胞表面免疫球蛋白框架区序列。 VH骨架区域结构与内源性配体的相互作用 例如CD5可能会影响特定数据维护和选择性扩展 B细胞。