IMMUNOGLOBULIN HEAVY CHAIN IN B LYMPHOCYTE DEVELOPMENT

B淋巴细胞在高度调控的条件下从造血干细胞发育而来 以免疫球蛋白有序重排为特征的一系列步骤 (IG)基因。这些重新安排是后续进展所必需的 进入下一个发育阶段。特别是，B细胞前体细胞停滞 在S7+c-Kit+CD2+阶段，如果他们无法使功能 膜MU重链蛋白。尽管这些前B细胞还没有 重新排列免疫球蛋白轻链基因后，它们确实表达了两种蛋白质，称为 Gamma5和Vpre=B1，它们用作与之结合的替代轻链 Mu链，形成Ig-型结构。缺乏Gamma5的小鼠 表达在B细胞发育中也表现出阻碍，尽管它不是 同样严重。因此，有人提出这种前B细胞形式的 膜免疫球蛋白发出信号诱导S7+的发育进程 C-Kit+CD2+阶段到随后的S7+c-Kit+阶段。建议数 实验将验证这一假设。我们最近开发了一种系统 用于体外培养前B细胞和高效导入蛋白质 用逆转录病毒载体注入它们。在目标1中，我们将确定是否 将MU重链导入培养的Rag1缺陷的Pre=B细胞将 正如预期的那样，诱导发育成熟。下一步，结构 MU链具有发展功能的要求将是 下定决心。具有特征性关联的粘连蛋白突变形式 Ig-α/Ig-β辅助蛋白及其信号转导特性 将对这些特性进行发育功能测试。如果这些研究 支持Mu链的信号传递功能很重要的假设 对于发展进程，那么在目标2中我们将测试能力 具有Lg-α或Ig-α信号区的各种嵌合蛋白 贝塔被嫁接到他们身上。特别是，我们希望能够介绍一下 转化为具有发育功能的前B细胞嵌合分子 由外源交联剂激活。这样的分子将使我们能够研究 前B细胞中的信号事件被激活。在目标3和目标4中， 将分析信令组件中的变化对 B细胞发育。洛厄尔博士在此SCOR应用程序中的项目3将 产生LYN缺陷小鼠。鉴于Lyn在膜免疫球蛋白中的可能作用 信号，来自这些小鼠的前B细胞的特性将相当 很有趣，将在这个项目中进行分析。这些细胞将是 培养的，如果他们在发育过程中表现出缺陷，我们将 通过引入野生型和改变形式的LYN来补充这一点。在……里面 此外，我们还将介绍多种显性负性突变形式 信号组件(包括Lyn、Syk和RAS)进入培养的Pre-B to 检测它们对Mu Heavey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.