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

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。V/H骨架区域序列(a2同种异型)可能起作用 B细胞选择和有效扩增中的功能作用(S) 附录。我们鉴定了B细胞表面糖蛋白CD5是一种 B细胞表面免疫球蛋白的配体。固定化F(ab‘)2片段 从阑尾细胞裂解液中分离CD5分子。我们提纯了 正常(a2+)和V/H突变型血清中生物素标记的F(ab‘)2片段 动物(a2-)，并将它们用作探针。流式细胞术检测V/HA2+ F(ab‘)2与IgM+B细胞的结合比V/Ha2-F(ab’)2更强。 相互作用以及F(ab‘)2与阑尾生发中心的结合可以 可被抗CD5抗体阻断。细胞附着分析也表明 CD5是B细胞表面免疫球蛋白骨架区域的配体 序列。V/H骨架区域结构与 CD5等内源性配体可能影响维持和选择性 特定B细胞的扩增。这些研究的延伸到人类 B细胞与G.Marti博士和G.Silverman博士合作表明 人免疫球蛋白与表达CD5的细胞系结合。染色结果为 当这些细胞第一次与抗CD5预先孵育时被抑制 抗体。我们从EBV-CLL细胞裂解物中分离出CD5分子 人免疫球蛋白与对甲苯磺酰激活的免疫球蛋白偶联。如果我们 预先孵育的细胞裂解物与抗CD5偶联小球、CD5分子 并不是孤立的。免疫兔制备经典的抗DNP抗体。 研究克隆性V/H和V/L区的半抗原抗体反应 抗体应答过程中生发中心的多样化。 通过收集生发中心的单个细胞 微操作。重抗体和轻抗体的重排基因 对单细胞的链进行聚合酶链式反应扩增和测序。初步 结果显示与克隆相关的序列可能已经多样化 通过超突变和类似基因转化的机制。抗体 似乎识别兔mb-1(CD79a)的基因被产生并 特色化的。这些抗体以及商业上可以买到的 抗CD79b抗体正被用来进一步表征 幼兔阑尾发育的B淋巴细胞。

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. V/H framework region sequences (a2 allotype) probably play functional role(s) in selection and effective expansion of B cells in the appendix. We identified the B-cell surface glycoprotein CD5 as a ligand for B-cell surface immunoglobulin. Immobilized F(ab')2 fragments isolated CD5 molecules from appendix cell lysates. We purified and biotinylated F(ab')2 fragments from sera of normal (a2+) and V/H mutant animals (a2-) and used them as probes. By flow cytometry, V/Ha2+ F(ab')2 bind IgM+ B cells more strongly than V/Ha2- 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 CD5 is a ligand for B-cell surface immunoglobulin framework region sequences. Interactions of V/H framework region structures with endogenous ligands such as CD5 may affect maintenance and selective expansion of particular B cells. An extension of these studies to human B cells in collaboration with Drs. G. Marti and G. Silverman showed that human immunoglobulin binds to CD5-expressing cell lines. Staining was inhibited when these cells were first preincubated with anti-CD5 antibody. We isolated CD5 molecules from EBV-CLL cell lysates using human immunoglobulin coupled to tosyl-activated dynabeads. If we preincubated cell lysates with anti-CD5 coupled beads, CD5 molecules were not isolated. Rabbits were immunized to make classical anti-DNP hapten antibody responses in order to study clonal V/H and V/L region diversification in germinal centers during antibody responses. Individual cells from germinal centers were collected by micromanipulation. The rearranged genes for antibody heavy and light chains in single cells were PCR-amplified and sequenced. Preliminary results show clonally related sequences that have probably diversified by both hypermutation and gene-conversion-like mechanisms. Antibodies that appear to recognize rabbit mb-1 (CD79a) were generated and characterized. These antibodies along with commercially available anti-CD79b are being used to further characterize populations of developing B lymphocytes in the young rabbit appendix.