INTEGRINS AND T CELL DEVELOPMENT AND FUNCTION

整合素与 T 细胞的发育和功能

• 批准号: 2887510
• 负责人: EUGENE E. MARCANTONIO
• 金额: $ 28.57万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887510
• 关键词: T cell receptor; T lymphocyte; animal breeding; biological signal transduction; cytokine receptors; differentiation antigens; extracellular matrix; flow cytometry; genetically modified animals; immunogenetics; integrins; laboratory mouse; leukocyte activation /transformation; phenotype; receptor expression

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.

该提案的广泛目标是确定整合素的角色 T淋巴细胞发育和功能的受体。 一个 已经选择了体内和体外方法的组合。 在 为了测试整联蛋白受体功能在T细胞中的作用 发育，转基因小鼠的产生，主要负面 整合素的形式。具体来说，我们正在使用一个构造 与活性整合素连接的TAC抗原的细胞外结构域 beta1细胞质结构域，我们和许多其他 实验室干扰整联蛋白在组织培养中的功能 细胞。 已选择近端LCK启动子来指导胸腺 该结构在转基因小鼠中的特定表达，导致 胸腺细胞前体的比例和数量增加， 建议在CD4- CD8-正常分化中有一个块（所谓 双阴细胞（DN））。 我们现在表明tac-beta1构造 充当DN中整联蛋白激活的跨性抑制剂 人口。 我们特别表明， 整合素α4Beta1中激活表位的表达。 我们的 假设是该结构减少了A的激活 整联蛋白受体的数量，特别是在DN人群中 整合素通常是胸腺中最活跃的。 独立， 我们将测试整联蛋白在DN与DP的分化中的作用 使用抗体和肽扰动实验的细胞进行 胎儿胸腺器官培养（FTOC），其中胚胎第14天胸腺 裂片是孤立和培养几天的，在此期间 胸腺细胞从完全DN人群中出现，并分化为 DP。 最后，我们将测试TAC-BETA1构建体 通过T细胞受体在培养的中通过T细胞受体激活的抑制剂 小鼠Th2细胞。 如果成功，则使用修改的CD4 发起人，我们将在外围T中高水平表达TAC-BETA1 转基因小鼠的细胞以确定整联蛋白活化的作用 在T细胞功能中。