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: 2672363
• 负责人: Ralph C Budd
• 金额: $ 25.34万
• 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/2672363
• 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（根据调查员的摘要改编）：此应用程序是 基于两个新的范式来解释两个有关功能的谜 fas。首先，在LPR小鼠中没有FAS表达的情况下，一种表型 CD4-8-B220+ TCRαβ+细胞的异常种群积累。 范式1提出，这种表型是由高强度TCR引起的 在正常小鼠中导致凋亡的信号。 第二，fas（喜欢 CD28）激活Jun激酶（JNK）并可以诱导细胞凋亡以及 与CD3共刺激静息T细胞，以增强IL-2的产生和 增殖。 范式2表明MAPK和JNK的平衡 确定功能结果； JNK的优势将有利于凋亡， 而MAPK/JNK的平衡挽救了细胞并有利于增殖。 特定的目标1通过使用新的TCR转基因LPR菌株开发范式1 被称为ova-tcr-1 lpr/lpr，识别椭圆蛋白（OVA）肽， siinfekl。 siinfekl变体的管理将授予不同的信号 强度。 中等TCR信号将仅产生CD8+的积累 细胞，而提供高强度信号的肽将促进CD4-8-- 克隆型TCR+细胞。 后者将被删除（通过TUNEL分析检测） 在OVA-TCR-1 +/ +小鼠中，保留在OVA-TCR-1 LPR/LPR小鼠中。 第二个 部分将解决另一个长期存在的争论 是在胸腺或外围产生的。 β2M - / - LPR/LPR的胸腺切除术 小鼠和β2M+/+ LPR/LPR小鼠将进行胸腺移植 相反的基因型。 这将选择性地重新表达i的i 胸腺或外围的CD4-8-T细胞的出现。 特定目标2通过检查JNK的重要性来开发范式2 使用主要的正和负变体激活FAS信号传导 下游C-Jun以及上游RAC和CDC42，Rho家族的成员 激活JNK的GTP结合蛋白。 此外，从凋亡中营救 FA将尝试使用主要的正面MAPK尝试。 最后，这个 模型适用于两个正常的凋亡的免疫情况 扩散以检查MAPK和JNK的水平。 特定目标3扩展 与IL-2的CD3共刺激FAS 2。 预测是 FA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-荧光素酶和 AP-1-环酶转基因小鼠。 最后，一个新颖的替代LPR NFAT结合抑制剂蛋白将被纯化，其抑制剂 使用NFAT-荧光酶/LPR小鼠确认的功能。