Biochemical/Molecular Changes Upon Naive T Cell Priming

初始 T 细胞启动后的生化/分子变化

• 批准号: 6511465
• 负责人: THOMAS F GAJEWSKI
• 金额: $ 27.92万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511465
• 关键词: Adenoviridae; CD28 molecule; Coxsackievirus; T cell receptor; T lymphocyte; apoptosis; biological signal transduction; cell differentiation; cytokine; cytoskeletal proteins; gene expression; genetically modified animals; laboratory mouse; microarray technology; protein biosynthesis; protein kinase; transcription factor; transfection

DESCRIPTION (Provided by the Applicant): Naive CD8+ T lymphocytes lack effector function and undergo a differentiation process to exit the naive state and to acquire a primed effector phenotype. Preliminary results indicate that this transition is a complex and highly regulated process, involving receptor-mediated signal transduction, cell cycle progression, chromatin remodeling, and changes in gene expression. Evidence suggests that the naive state may be actively maintained, raising the possibility of a genetic program to place a brake on differentiation. In contrast, primed T-cells gain effector function, as well as altered signal transduction pathways and a more dynamic actin cytoskeleton, suggesting that new genes become expressed to mediate these processes. The overall goal of this application is to identify the molecular phenotype of naive and primed effector CD8+ T-cells, and to study the functional role of identified molecules in normal T-cell populations. The principal model will utilize 2C TCR transgenic mice crossed to a Rag2 background, to extinguish endogenous TCR gene rearrangement and ensure a naive phenotype. Mechanistic experiments in normal, post-thymic, T-cells will be enabled by a novel Coxsackie and adenovirus receptor (CAR) transgenic mouse and adenoviral transduction. The first specific aim is to identify genes and proteins that are differentially expressed between naive and primed effector CD8+ T-cells, by: I) examination of candidates by Western blotting, ii) analysis of expression of defined genes using Affymetrix microarrays, and iii) identification of unknown genes by subtractive library cloning. The second specific aim is to optimize the CAR transgenic mouse system for transduction of normal, post-thymic T-cells. Transgenic mice expressing CAR have been constructed using an LckICD2 promoter/enhancer cassette. Optimization of adenoviral transduction will be performed, and interbreeding with 2C/Rag2-/- mice will be carried out for studies of naive and primed CD8+ T-cells. The third specific aim is to analyze the function of differentially expressed signaling molecules and transcription factors linked to MAP kinase pathways in normal T-cells using CAR transgenic mice. Preliminary results have shown that several proteins involved in MAP kinase signaling become markedly up-regulated upon T-cell priming. Wildtype and mutant Ras, FosB, MKK7, JNK1, JNK2, and other candidates will be introduced into naive and primed CAR transgenic T-cells. Effects on early T-cell differentiation, as well as on apoptosis, TCR sensitivity, CD28 independence, and magnitude of cytokine production, will be examined. The fourth specific aim is to investigate the functional requirement for modulators of actin cytoskeleton dynamics in normal T-cells. Preliminary results have revealed that expression of several molecules associated with cytoskeletal dynamics is increased tremendously upon differentiation from the naive to the primed effector state. Wildtype and mutant a-Pak, Raci, and other candidates will be introduced into primed effector T-cells, and potential effects on TCR capping, conjugate formation, cell spreading, cytolysis, and cytokine production will be investigated. In addition, gelsolin4 -/- mice will be examined for functional defects in the T-cell compartment. The results of these experiments will provide invaluable information regarding the molecular and biochemical changes that accompany T-cell differentiation, pointing the way to new potential targets for manipulating immune responses in vivo.

描述（申请人提供）：幼稚的CD8+ T淋巴细胞缺乏效应器 功能并经历差异化过程，以退出天真状态 获取启动效应子表型。初步结果表明这是 过渡是一个复杂且高度监管的过程，涉及 受体介导的信号转导，细胞周期进程，染色质 重塑和基因表达的变化。证据表明天真 国家可以积极维护，从而提高了遗传程序的可能性 将刹车置于差异化。相反，底漆的T细胞增益效应器 功能，以及改变的信号转导途径和更动态的 肌动蛋白细胞骨架，表明新基因表达以介导这些基因 过程。该应用的总体目标是确定分子 天真和启动效应子CD8+ T细胞的表型，并研究 鉴定分子在正常T细胞种群中的功能作用。这 主要模型将利用2C TCR转基因小鼠越过RAG2 背景，以扑灭内源性TCR基因重排并确保天真 表型。正常，胸腔后，T细胞的机械实验将是 由新颖的coxsackie和腺病毒受体（CAR）转基因小鼠和 腺病毒转导。第一个具体目的是识别基因和 在幼稚和引发效应子之间差异表达的蛋白质 CD8+ T细胞，作者：i）通过Western blotting检查候选人，ii） 使用Affymetrix微阵列和III分析确定基因的表达 通过减法库克隆识别未知基因。第二个 具体目的是优化汽车转基因小鼠系统以转导 正常的，胸膜后T细胞。表达汽车的转基因小鼠已经 使用LCKICD2启动子/增强剂盒构建。优化 将进行腺病毒转导，并与2C/rag2 - / - 杂交 小鼠将进行幼稚和底漆CD8+ T细胞的研究。这 第三个具体目的是分析差异表达的功能 信号分子和与MAP激酶途径相关的转录因子 使用CAR转基因小鼠的正常T细胞。初步结果表明 MAP激酶信号涉及的几种蛋白质显着上调 在T细胞启动时。野生型和突变ras，fosb，mkk7，jnk1，jnk2和其他 候选人将被引入幼稚和底漆的转基因T细胞中。 对早期T细胞分化以及凋亡，TCR的影响 灵敏度，CD28独立性和细胞因子产生的大小将是 检查。第四个特定目的是调查功能要求 用于正常T细胞中肌动蛋白细胞骨架动力学的调节剂。初步的 结果表明，与 分化与 天真的启动效应子状态。野生型和突变体A-pak，RACI和其他 候选人将被引入启动效应子T细胞，并潜在 对TCR上限，结合形成，细胞扩散，细胞解析和影响的影响 将研究细胞因子的产生。此外，gelsolin4 - / - 小鼠将 在T细胞室中检查功能缺陷。结果 这些实验将提供有关分子的宝贵信息 以及伴随T细胞差异化的生化变化，指向道路 在体内操纵免疫反应的新潜在靶标。