FAS REGULATES T CELL DEVELOPMENT/FUNCTION IN LPR MICE

FAS 调节 LPR 小鼠 T 细胞发育/功能

• 批准号: 6650796
• 负责人: Ralph C Budd
• 金额: $ 33.98万
• 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 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6650796
• 关键词: CD95 molecule; DNA binding protein; T cell receptor; T lymphocyte; apoptosis; biological signal transduction; cysteine endopeptidases; genetically modified animals; human subject; laboratory mouse; lymphocyte proliferation; mitogen activated protein kinase; nuclear factor kappa beta; tissue /cell culture; transfection

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.

描述（改编自研究者摘要）：本申请 探讨Fas天然抑制剂对免疫功能的影响， 半胱天冬酶-8（FLICE），称为FLIP（FLICE抑制蛋白）。它还提请 过表达FLIP的小鼠和Fas缺陷的lpr小鼠之间的相似之处。的 初步研究结果表明，可溶性Fas配体（FasL）可以共刺激 抗CD 3增殖和IL-2产生的原代T细胞在一个 半胱天冬酶依赖型。T细胞的Fas共刺激也上调ERK 和NF-κ B活性。这可能是通过FLIP介导的物理 FLIP与Raf-1（ERK通路的上游调节因子）的关联，以及 TRAF-1和RIP（与NF-κ B通路连接）。因此，FLIP具有 双重功能，不仅抑制Fas介导的细胞死亡， 在与胱天蛋白酶-8竞争的情况下，它也正性地发出信号以增强TCR信号。 目的1检查是否FLIP裂解的胱天蛋白酶-8实际上是需要的 增加ERK和NF-κ B活性。FLIP含有两种潜在的半胱天冬酶 裂解位点这些已经被突变，突变的FLIP将稳定地 转染到T-和B-细胞系中以及逆转录病毒转染到 原代T细胞不可裂解的FLIP应与内源性FLIP竞争， 在DISC（死亡诱导物信号复合物）处与胱天蛋白酶-8或FADD结合 降低ERK和NF-κ B的激活。目标2研究了 FLIP转基因（Tg）小鼠中CD 8+细胞的耗竭，以及这是否会导致 从FLIP诱导的TCR信号传导增加和选择性过早细胞死亡 CD 8 + T细胞在缺乏Fas的lpr小鼠中则相反 表情将TCR-Tg小鼠OT-1培育成FLIP-Tg和lpr小鼠， 卵清蛋白肽（OVAp）将用于监测ERK和NF-κ B活化， 增殖、体内细胞周期和死亡。目标3检查FLIP是否 过度表达或Fas缺乏导致“误选“保留 胸腺细胞，即在阳性选择中存活但随后不遇到 任何适当的肽/MHC组合在外周，并通常是 通过凋亡消除，但保留在过表达FLIP或 缺少Fas。这种“错误选择的”CD 8 + T细胞可能是免疫缺陷的来源。 在lpr小鼠中积累CD 4 -8-T细胞。目的4研究FLIP水平是否 表达决定哪些T细胞成为记忆T细胞。我们探索一个模型， 该FLIP水平与电池循环的强度成比例地降低， 使快速循环的T细胞对Fas诱导的死亡敏感。但这些 周期较不强烈的T细胞将维持FLIP水平并存活至 变成记忆T细胞