IMMUNOGLOBULIN HEAVY CHAIN IN B LYMPHOCYTE DEVELOPMENT

B淋巴细胞从造血干细胞发育而来， 一系列步骤，以免疫球蛋白的有序重排为特征 (Ig)基因. 这些重排是后续进展所必需的 到下一个发展阶段。 特别是B细胞前体 在S7+ c-kit+ CD 2+阶段，如果他们不能使功能性 膜μ重链蛋白。 虽然这些前B细胞还没有 重组了它们的IG轻链基因后，它们确实表达了两种蛋白质，称为 γ 5和Vpre=B1，其作为替代轻链与以下物质联合收割机结合 mu链，形成Ig型结构。 缺乏γ 5的小鼠 表达也表现出B细胞发育的阻滞，尽管它不是 一样严重。 因此，有人提出，这种前B细胞形式的 膜IG发送信号以诱导从S7+的发育进展 c-kit+ CD 2+阶段至随后的S7+ c-Kit+阶段。 拟议 实验将验证这一假设。 我们最近开发了一个系统 用于体外培养前B细胞并有效地引入蛋白质 用逆转录病毒载体植入体内 在目标1中，我们将确定 将μ重链引入培养的Rag 1缺陷型pre=B细胞将 诱导发育成熟 其次，结构 对mu链具有发展功能的要求将是 测定 具有特征性关联的突变形式的μ链， Ig-α/Ig-β辅助蛋白和特征性信号传导 将测试性能的发育功能。 如果这些研究 支持mu链信号功能重要的假说 为了发展进步，那么在目标2中，我们将测试以下能力： 具有Ig-α或IG-α的信号传导区的各种嵌合蛋白质， 被植入了贝塔 特别是，我们希望能够介绍 进入具有发育功能的前B细胞嵌合分子， 通过外源交联活化。 这样的分子将使我们能够研究 前B细胞中激活的信号事件。 在目标3和4中， 将分析信号成分的改变对 B细胞发育。 Lowell博士在SCOR申请中的项目3将 产生Lyn-deficient小鼠。 考虑到林恩在膜IG中的可能作用， 信号，这些小鼠的前B细胞的特性将非常 这很有趣，并将在本项目中进行分析。 这些细胞将 培养，如果他们表现出可能的发展缺陷，我们将 通过引入野生型和改变形式的林恩来补充。 在 此外，我们还将引入多种显性负突变形式， 信号传导组分（包括林恩、Syk和Ras）进入培养的前B细胞， 检查它们对μ重链诱导的发育成熟的影响。

B lymphocytes develop from hematopoietic stem cells in a highly regulated series of steps, characterized by ordered rearrangements of immunoglobulin (Ig) genes. These rearrangements are required for subsequent progression to the next developmental stage. In particular, B cell precursors arrest at an S7+ c-kit+ CD2+ stage if they are unable to make a functional membrane mu heavy chain protein. Although these pre-B cells have not yet rearranged their Ig light chain genes, they do express two proteins, called gamma5 and Vpre=B1, which serve as surrogate light chains to combine with mu chain and forms an Ig-type structure. Mice deficient for gamma5 expression also exhibit a block in B cell development, although it is not as severe. Thus, it has been proposed that this pre-B cell form of membrane Ig sends a signal to induce developmental progression from the S7+ c-kit+ CD2+ stage to the subsequent S7+ c-Kit+ stage. The proposed experiments will test this hypothesis. We have recently developed a system for culturing pre-B cells in vitro and efficiently introducing proteins into them with retroviral vectors. In Aim 1, we shall determine whether introducing mu heavy chain into cultured Rag1-deficient pre=B cells will induce developmental maturation, as expected. Next, the structural requirements for mu chain to have developmental function will be determined. Mutant forms of muchain with characterized associations with the Ig-alpha/Ig-beta accessory proteins and with characterized signaling properties will be tested for developmental function. If these studies support eh hypothesis that the signaling function of mu chain is important for developmental progression, then in Aim 2 we shall tests the abilities of various chimeric proteins that have signaling regions of lg-alpha or Ig- beta grafted onto them. In particular, we hope to be able to introduce into the pre-B cells chimeric molecules with developmental function that is activated by exogenous crosslinking. Such molecules will allow us to study the signaling events activated in the pre-B cell. In Aims 3 and 4, alterations in signaling components will be analyzed for their effects on B cell development. Dr. Lowell' Project 3 in this SCOR application will generate Lyn-deficient mice. Given the likely role of Lyn in membrane Ig signaling, the properties of pre-B cells from these mice will be quite interesting and will be analyzed in this project. These cells will be cultured and, if they exhibit may defect in development, we shall complement that by introducing wild type and altered forms of Lyn. In addition, we shall introduce a variety of dominant negative mutant forms of signaling components (including Lyn, Syk, and Ras) into cultured pre-B to examine their effect on mu heavey chain-induced developmental maturation.