INTEGRINS AND T CELL DEVELOPMENT AND FUNCTION

该提案的主要目标是确定整合素的作用， 受体在T淋巴细胞的发育和功能中的作用。 一 已经选择了体内和体外方法的组合。 在 为了检测整合素受体功能在T细胞中的作用， 发展，转基因小鼠已经产生了显性阴性 整合素的形式。具体来说，我们使用一个构造， 与活性整合素连接的Tac抗原的胞外结构域 β 1胞质结构域，这已经被我们和许多其他人所证明， 干扰组织培养中整合素功能的实验室 细胞 近端lck启动子已被选择来指导胸腺 该构建体在转基因小鼠中的特异性表达，导致 胸腺细胞前体的比例和数量增加， 提示阻断了CD 4-CD 8-（所谓的 双阴性细胞（DN））。 我们现在发现Tac-beta1结构 在DN中作为整合素活化的反式显性抑制剂 人口 我们特别表明，有一个减少， 整合素α 4 β 1中活化表位的表达。 我们 一种假设是，这种结构降低了一种 整合素受体的数量，特别是在DN人群中， 整联蛋白通常在胸腺中最活跃。 独立地， 我们将检测整合素在DN向DP分化中的作用， 细胞使用抗体和肽扰动实验，在 胎儿胸腺器官培养（FTOC），其中胚胎第14天胸腺 分离叶并培养数天，在此期间， 胸腺细胞从完全DN群体中出现并分化为 DP. 最后，我们将测试Tac-beta1结构作为一个transdominant 通过T细胞受体的整合素活化的抑制剂 小鼠TH 2细胞 如果这是成功的，那么使用修改的CD 4 启动子，我们将在外周T细胞中高水平表达Tac-β 1。 转基因小鼠的细胞，以确定整合素活化的作用 T细胞功能。

The broad goals of this proposal are to determine the roles for integrin receptors in the development and function of T-lymphocytes. A combination of in vivo and in vitro approaches have been chosen. In order to test the role of integrin receptor function in T cell development, transgenic mice have been produced with a dominant negative form of integrin. Specifically, we are using a construct with the extracellular domain of the Tac antigen connected to the active integrin beta1 cytoplasmic domain, which has been shown by us and many other laboratories to interfere with integrin function in tissue culture cells. The proximal lck promoter has been chosen to direct thymic specific expression of this construct in transgenic mice, leading to an increase in the proportion and number of thymocyte precursors, suggesting a block in the normal differentiation of CD4- CD8- (so called double negative cells (DN)). We now show that the Tac-beta1 construct is acting as a transdominant inhibitor of integrin activation in the DN population. We specifically show that there is a reduction in the expression of an activation epitope in the integrin alpha4beta1. Our hypothesis is that this construct has decreased the activation of a number of integrin receptors, particularly in the DN population, whose integrins are normally the most active in the thymus. Independently, we will test the role of integrins in the differentiation of DN to DP cells using antibody and peptide perturbation experiments, performed in fetal thymic organ culture (FTOC), in which embryonic day 14 thymic lobes are isolated and cultured for several days, during which the thymocytes emerge from a completely DN population and differentiate into DP. Lastly, we will test the Tac-beta1 construct as a transdominant inhibitor of integrin activation via the T cell receptor in cultured mouse TH2 cells. If this is successful, then using a modified CD4 promoter, we will express Tac-beta1 at high levels in the peripheral T cells of transgenic mice to determine the role of integrin activation in T cell function.