FAS REGULATES T CELL DEVELOPMENT/FUNCTION IN 1PR MICE

FAS 调节 1PR 小鼠 T 细胞发育/功能

• 批准号: 2886933
• 负责人: Ralph C Budd
• 金额: $ 26.36万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2886933
• 关键词: CD3 molecule; CD95 molecule; DNA binding protein; MHC class I antigen; T cell receptor; T lymphocyte; apoptosis; biological signal transduction; cell differentiation; genetically modified animals; human tissue; interleukin 2; laboratory mouse; lymphocyte proliferation; thymectomy; thymus; thymus transplantation

DESCRIPTION (Adapted from Investigator's abstract): This application is based on two new paradigms to explain two enigmas regarding the function of Fas. First, in the absence of Fas expression in lpr mice, a phenotypically unusual population of CD4-8- B220+ TCR alpha beta+ cells accumulates. Paradigm 1 proposes that this phenotype results from high intensity TCR signals which, in normal mice, results in apoptosis. Second, Fas (like CD28) activates Jun kinase (JNK) and can induce apoptosis, as well as costimulate with CD3 on resting T cells to augment IL-2 production and proliferation. Paradigm 2 suggests that the balance of MAPK and JNK determines the functional outcome; dominance of JNK will favor apoptosis, whereas a balance of MAPK/JNK rescues cells and favors proliferation. Specific Aim 1 develops Paradigm 1 by use of a new TCR transgenic lpr strain known as OVA-tcr-1 lpr/lpr that recognizes ovalbumin (OVA) peptide, SIINFEKL. Administration of SIINFEKL variants will confer different signal intensities. Moderate TCR signals will yield accumulation of only CD8+ cells, while peptides providing a high intensity signal will promote CD4-8- clonotype TCR+ cells. The latter will be deleted (detected by TUNEL assay) in OVA-tcr-1 +/+ mice and retained in OVA-tcr-1 lpr/lpr mice. The second part will address another long standing debate whether lpr CD4-8- T cells are generated in the thymus or periphery. Thymectomy of beta 2m-/- lpr/lpr mice and beta 2m+/+ lpr/lpr mice, will be followed by thymic transplant of the opposite genotype. This will selectively re-express class I in either the thymus or the periphery, and appearance of CD4-8- T cells monitored. Specific Aim 2 develops Paradigm 2 by examining the importance of JNK activation in Fas signaling using dominant positive and negative variants of downstream c-Jun, and upstream Rac and Cdc42, members of the Rho family of GTP-binding proteins that activate JNK. In addition, rescue from apoptosis by Fas will be attempted using a dominant positive MAPK. Finally, this model is applied to two normal immune situations of apoptosis versus proliferation to examine the levels of MAPK and JNK. Specific Aim 3 extends Paradigm 2 to Fas costimulation with CD3 of IL-2. The prediction is that Fas will enhance activity of transcription factors that use c-Jun, namely AP-1 and NFAT. Functional confirmation will use NFAT-luciferase and AP-1-luciferase transgenic mice. Finally, a novel alternate lpr NFAT-binding suppressor protein will be purified, and its suppressor function confirmed using NFAT-luciferase/lpr mice.

描述（改编自研究者摘要）：本申请是 基于两个新的范式来解释两个谜的功能， 法斯首先，在lpr小鼠中不存在Fas表达的情况下，表型上 CD 4 -8- B220+ TCR α β +细胞的异常群体聚集。 范式1提出这种表型是由高强度TCR 在正常小鼠中，这些信号导致细胞凋亡。 第二，Fas（如 CD 28）激活Jun激酶（JNK）并可诱导细胞凋亡，以及 与静息T细胞上的CD 3共刺激以增加IL-2的产生， 增殖 范式2提示MAPK和JNK的平衡 决定功能结果; JNK的优势将有利于细胞凋亡， 而MAPK/JNK的平衡拯救细胞并有利于增殖。 Specific Aim 1通过使用新的TCR转基因lpr菌株开发Paradigm 1 称为OVA-tcr-1 lpr/lpr，其识别卵清蛋白（OVA）肽， SIINFEKL。 SIINFEKL变体的施用将赋予不同的信号 强度。 中度TCR信号将仅产生CD 8+细胞的积累。 细胞，而提供高强度信号的肽将促进CD 4 -8- 克隆型TCR+细胞。 后者将被删除（通过TUNEL检测） 在OVA-tcr-1 +/+小鼠中，并保留在OVA-tcr-1 lpr/lpr小鼠中。 第二 部分将解决另一个长期存在的争论是否lpr CD 4 -8- T细胞 在胸腺或外周产生。 β 2 m-/- lpr/lpr胸腺切除术 小鼠和β 2 m +/+ lpr/lpr小鼠，随后进行胸腺移植， 相反的基因型 这将选择性地在以下两种语言中重新表达I类： 胸腺或外周，并监测CD 4 -8- T细胞的出现。 具体目标2通过检查JNK的重要性发展了范式2 使用显性阳性和阴性变体的Fas信号转导激活 下游c-Jun，上游Rac和Cdc 42，Rho家族的成员， 激活JNK的GTP结合蛋白。 此外，从细胞凋亡中拯救 将尝试使用显性阳性MAPK。 最后 模型适用于两种正常的免疫情况下的细胞凋亡与 检测MAPK和JNK的水平。 具体目标3延伸 模式2为Fas与IL-2的CD 3共刺激。 预测是， Fas将增强使用c-Jun的转录因子的活性，即 AP-1和NFAT。 功能确认将使用NFAT-荧光素酶， AP-1-荧光素酶转基因小鼠。 最后，一种新颖的替代lpr 将纯化NFAT结合抑制蛋白，并将其抑制因子 使用NFAT-荧光素酶/lpr小鼠确认功能。