FAS REGULATES T CELL DEVELOPMENT/FUNCTION IN 1PR MICE

描述(改编自调查者摘要)：此应用程序是 基于两个新的范式来解释关于细胞外营养因子功能的两个谜 Fas.首先，在LPR小鼠中没有Fas表达的情况下，一种表型 异常的CD4-8-B220+TCRα-β+细胞聚集。 范式1提出，这种表型是高强度TCR的结果 在正常小鼠中，导致细胞凋亡的信号。第二，Fas(喜欢) CD28)激活Jun激酶(JNK)，并可诱导细胞凋亡，以及 与CD3共刺激静息T细胞增加IL-2的产生和 扩散。范式2表明，MAPK和JNK的平衡 决定功能结果；JNK的主导地位将有利于细胞凋亡， 然而，MAPK/JNK的平衡拯救了细胞，有利于细胞增殖。 特异性目标1利用一种新的TCR转基因LPR株发展为范式1 被称为OVA-TCR-1 LPR/LPR，识别卵清蛋白(OVA)肽， 西因费克。对SIINFEKL变体的管理将发出不同的信号 强度。适度的TCR信号将只产生CD8+的累积 细胞，而提供高强度信号的多肽将促进CD4-8- 克隆型TCR+细胞。后者将被删除(用TUNEL法检测) 在OVA-TCR-1+/+小鼠体内，并保留在OVA-TCR-1 LPR/LPR小鼠体内。第二 部分将解决另一个长期存在的争论，即LPR CD4-8-T细胞 是在胸腺或外周产生的。β2M-/-LPR/LPR胸腺切除术 小鼠和Beta 2M+/+LPR/LPR小鼠，随后将进行胸腺移植 相反的基因。这将有选择地在以下两种情况中重新表示I类 胸腺或外周，并监测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 Fas信号通路中显性正、负变异体的激活 下游c-jun，上游Rac和cdc42，Rho家族成员 激活JNK的GTP结合蛋白。此外，从细胞凋亡中解救出来 将使用显性阳性MAPK尝试通过Fas。最后，这一点 模型被应用于两种正常的免疫情况，即细胞凋亡和 检测MAPK和JNK的表达水平。《特定目标3》扩展 范式2至Fas与IL-2的CD3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-荧光素酶和 AP-1-荧光素酶转基因小鼠。最后，提出了一种新的替代车牌识别算法 将提纯NFAT结合抑制蛋白，并将其抑制蛋白 用NFAT-荧光素酶/LPR小鼠证实其功能。

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.