FAS REGULATES T CELL DEVELOPMENT/FUNCTION IN LPR MICE

描述(改编自调查者摘要)：此应用程序 探讨天然抑制物Fas和Fas对免疫功能的影响 半胱氨酸天冬氨酸氨基转移酶-8(FLICE)被称为FLIP(FLICE抑制蛋白)。它还画出了 过度表达FliP的小鼠和Fas缺陷的LPR小鼠之间的相似之处。这个 初步研究表明，可溶性Fas配体(FasL)可与 原代T细胞抗CD3增殖和产生IL-2的研究 依赖于caspase的时尚。T细胞Fas共刺激也上调ERK 和核因子-kappaB活性。这很可能是通过物理翻转来调节的 Flio与Raf-1(ERK途径的上游调节因子)的关联，以及 TRAF-1和RIP(与核因子-kappaB通路相连)。因此，Flip具有一个 双重功能不仅通过抑制Fas介导的细胞死亡 与caspase-8竞争，它也发出积极的信号来增强TCR信号。 目标1检查是否确实需要caspase-8的翻转切割 增强ERK和NF-kappaB活性。Flip包含两个潜在的caspase 裂解位点。这些都被突变了，突变的翻转将稳定 分别转染T、B细胞系和逆转录病毒载体 初级T细胞。不可分裂翻转应该与内源性翻转竞争 与caspase-8或DISC上的FADD结合(死亡诱导信号复合体) 减少ERK和NF-kappaB的活化。目标2研究了导致 转基因(TG)小鼠体内CD8+细胞的耗竭及其结果 从FLIP诱导的TCR信号增加和细胞选择性过早死亡 CD8+T细胞。相反的情况在缺乏Fas的LPR小鼠中被考虑 表情。TCR-TG小鼠OT-1已被培育成Flip-TG和LPR小鼠，并 卵白蛋白多肽(OVAp)将用于监测ERK和NF-kappaB的激活， 增殖、体内细胞周期和死亡。目标3检查翻转是否 过度表达或Fas缺陷导致“”错选“” 胸腺细胞，T细胞，在阳性选择中存活下来，但随后没有遇到 外周有任何合适的多肽/MHC结合，通常 被细胞凋亡消除，但保留在过度表达翻转或 缺少Fas。这种“错误选择”的CD8+T细胞可能是 在LPR小鼠体内蓄积CD4-8-T细胞。目标4研究翻转水平是否 表达决定了哪些T细胞成为记忆T细胞。我们在中探索一个模型 这些翻转水平与细胞周期的强度成比例地下降， 使快速循环的T细胞对Fas诱导的死亡敏感。然而，那些 循环强度较低的T细胞将维持翻转水平，并存活到 成为记忆T细胞。

DESCRIPTION (Adapted from the Investigator's abstract): This application explores the influence on immune function by the natural inhibitor of Fas and caspase-8 (FLICE) known as FLIP (FLICE-inhibitory protein). It also draws parallels between mice over-expressing FLIP and Fas-deficient lpr mice. The preliminary findings show that soluble Fas-ligand (FasL) can co-stimulate with anti-CD3 proliferation and IL-2 production by primary T-cells in a caspase-dependent fashion. Fas co-stimulation of T-cells also up-regulates ERK and NF-kappaB activities. This is likely mediated by FLIP via the physical association of FLIP with Raf-1 (the upstream regulator of the ERK pathway), and TRAF-1 and RIP (which connect with the NF-kappaB pathway). As such FLIP has a dual function of not only inhibiting Fas-mediated cell death through competition with caspase-8, it also signals positively to augment TCR signals. Aim 1 examines whether FLIP cleavage by caspase-8 is actually required for it to augment ERK and NF-kappaB activities. FLIP contains two potential caspase cleavage sites. These have been mutated and the mutant FLIP will be stably transfected into T- and B-cell lines as well as retrovirally transfected into primary T-cells. The non-cleavable FLIP should compete with endogenous FLIP for binding to caspase-8 or to FADD at the DISC (Death-Inducer Signaling Complex) to decrease activation of ERK and NF-kappaB. Aim 2 studies the reason for the depletion of CD8+ cells in FLIP-transgenic (Tg) mice, and whether this results from FLIP-induced increased TCR signaling and premature cell death selectively by CD8+ T-cells. The opposite scenario is considered in lpr mice that lack Fas expression. The TCR-Tg mouse OT-1 has been bred to FLIP-Tg and lpr mice and ovalbumin peptide (OVAp) will be used to monitor ERK and NF-kappaB activation, proliferation, in vivo cell cycling, and death. Aim 3 examines whether FLIP over-expression or Fas deficiency cause retention of ""misselected"" thymocytes, T-cells that survived positive selection but then do not encounter any proper peptide/MHC combination in the periphery, and are normally eliminated by apoptosis, but are retained in mice over-expressing FLIP or lacking Fas. Such "misselected" CD8+ T-cells may be the source of the accumulating CD4-8- T-cells in lpr mice. Aim 4 studies whether levels of FLIP expression determine which T-cells become memory T-cells. We explore a model in which FLIP levels decrease proportional to the intensity of cell cycling, making rapidly cycling T-cells sensitive to Fas-induced death. However, those T-cells which cycle less intensely will maintain FLIP levels and survive to become memory T-cells.