INTEGRINS AND T CELL DEVELOPMENT AND FUNCTION

整合素与 T 细胞的发育和功能

• 批准号: 6170957
• 负责人: EUGENE E. MARCANTONIO
• 金额: $ 29.43万
• 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/6170957
• 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.

该提案的总体目标是确定整合素的角色 T淋巴细胞发育和功能中的受体。一个 选择了体内和体外相结合的方法。在……里面 为了测试整合素受体功能在T细胞中的作用 随着转基因小鼠的发展，已经产生了显性阴性的转基因小鼠 整合素的形式。具体地说，我们使用的构造具有 Tac抗原与活性整合素连接的胞外区 β1胞质结构域，已经由我们和其他许多人证明了 实验室在组织培养中干扰整合素功能 细胞。已选择近端的lck启动子来引导胸腺 该结构在转基因小鼠中的特异性表达，导致 胸腺细胞前体细胞比例和数量的增加， 提示CD4-CD8-的正常分化受阻 双阴性细胞(DN))。我们现在展示了Tac-Beta1结构 在糖尿病肾病中作为整合素激活的跨优势抑制物 人口。我们特别指出，有一个减少 整合素α4beta1激活表位的表达。我们的 假设这一构造减少了对 整合素受体的数量，特别是在糖尿病肾病人群中，其 整合素通常是胸腺中最活跃的。独立地， 我们将测试整合素在糖尿病肾病向糖尿病肾病分化中的作用。 使用抗体和多肽扰动实验的细胞，在 胎儿胸腺器官培养(FTOC)，其中胚胎第14天胸腺 叶被隔离并培养数天，在此期间 胸腺细胞从完全的糖尿病肾病群体中分化为 Dp.最后，我们将测试Tac-beta1结构作为跨显性 通过T细胞受体抑制整合素的激活 小鼠TH2细胞。如果这是成功的，那么使用修改后的CD4 启动子，我们将在外周T细胞中高水平表达Tac-β1 确定转基因小鼠细胞整合素活化的作用 在T细胞功能方面。