DGK in T Cell Regulation and Tolerance

项目主任/首席调查员(最后、第一、中间)：Gajesski，Thomas F.1R01 AI080745-01A1 修订后的摘要部分 有证据表明，外周免疫耐受的一种机制是通过T细胞无能的过程发生的。无能I细胞在TCR/CD28介导的RAS激活过程中存在缺陷。最近的数据表明，二酰基甘油激酶(DGK)家族脂蛋白激酶的上调表达，特别是DGK-u，参与了抑制血管生成的过程。 (I) J&lt；-0 L]‘ 广告 .TiU，0 0(Q 0-0 鳕鱼 C-0 得了- RasGRP介导的RAS在无能状态的激活。与这些变化相关的是转录调节因子EGR2的表达增加，我们已经在假定的DGK-Ci启动子中确定了EGR结合位点。这项建议的主要目标是详细了解DGKs和EGR2在控制T细胞激活和外周血细胞中的作用 ._0 5A‘ 首席运营官 U�r 拖 宽容。在第一个特定目标中，将阐明DGK-U的转录调控，重点是EGR2。使用CAR TG系统，将使用新的工具对初级T细胞进行遗传操作。这包括转录报告腺病毒载体， ..。 OUP 用于EGR2表达的腺病毒，以及用于在外周I细胞条件删除EGR2的Cre腺病毒。支持性数据将来自芯片分析和基因表达谱。在……里面 O-0 第二个具体目标是DGK-u酪氨酸磷酸化蛋白的功能作用 ：e、 �c‘ N-0 0-0 AUX。 BE O03�-‘ 胸腺后I细胞室中的靶向基因。T细胞的激活特性，体内的自身免疫和无能敏感性，以及可能改善的体内肿瘤排斥反应将被讨论。总而言之，这项工作将详细描述控制外周耐受的关键途径，为开发可操作的药理学药物铺平道路。 (吉普 M=‘(Dm 3m03 A�Flu` ..，-‘1 已经确定了。将使用腺病毒载体和CAR TG T细胞在体外对DGK-z突变进行功能分析，并将在体内研究表达关键DGK-u突变的转基因小鼠体内免疫功能的变化。在第三个具体目标中，将研究外周T细胞中EGR2直接消除对外周耐受的影响。这些实验将利用Cre-腺病毒的方法有条件地删除 _=c� L17 疾病情况下的免疫耐受性。 111 CDG‘O V、12 划桨 首席运营官 COB

Program Director/Principal Investigator (Last, First, Middle): Gajewski, Thomas F. 1R01 AI080745-01A1 Revised Abstract Section Evidence suggests that one mechanism of peripheral immunologic tolerance occurs through the process of T cell anergy. Anergic I cells have been characterized to have a defect in TCR/CD28mediated Ras activation. Recent data have indicated that upregulated expression of lipid kinases of the diacylglycerol kinase (DGK) family, in particular DGK-u, participate in the suppression of (Ti j-<-0 L]' ADZ .Ti U,0 0(Q 0-0 COD c-0 'D- RasGRP-mediated Ras activation in the anergic state. Correlating with these changes is increased expression of the transcriptional regulator EGR2, and we have identified EGR-farnily binding sites in the putative DGK-ci promoter. The major goal of this proposal is to gain a detailed understanding of DGKs and EGR2 in controlling T cell activation and peripheral ._0 5a' coo u�r tow tolerance. In the first specific aim, the transcriptional regulation of the DGK-u will be elucidated, with a focus centered on EGR2. Novel tools for the genetic manipulation of primary T cells will be employed using the CAR Tg system. This includes transcriptional reporter adenoviral vectors, ... OUP adenoviruses for EGR2 expression, and a Cre adenovirus for conditional deletion of EGR2 in peripheral I cells. Supportive data will come from ChIP assays and gene expression profiling. In O-0 the second specific aim, the functional role of DGK-u tyrosine phosphorylabon will :E, �c' N-0 0-0 aux. be o03�-' targeted genes in the post-thymic I cell compartment. T cell activation properties, in vivo autoimmunity and anergy susceptibility, and possible improved tumor rejection in vivo will be addressed. In total, this work will characterize in detail a critical pathway in the control of peripheral tolerance, paving the way for development of pharmacologic agents to manipulate (gyp m='(Dm 3m03 a�fl` .., -'1 ascertained. Mutants of DGK-z will be analyzed functionally using adenoviral vectors and CAR Tg T cells in vitro, and transgenic mice expressing key DGK-u mutants in peripheral T cells will be studied for altered immune function in vivo. In the third specific aim, the consequence of elimination of EGR2 directly in peripheral T cells on peripheral tolerance will be investigated. These experiments will take advantage of the Cre-adenovirus approach to delete conditionally _=c� l17 immunologic tolerance in disease situations. 111 CDG'O V,12 oar coo COB