Mechanisms of CD8+ T Cell Apoptosis During Acute and Chronic Viral Infections

项目总结：我们的目标是确定控制抗原特异性收缩的分子机制 急性和慢性病毒感染后的CD8+T细胞反应。在响应的高峰期， 效应性CD8+T细胞低水平表达Bcl2，对Bim介导的细胞凋亡敏感。已激活 CD8+T细胞也易通过Fas、肿瘤坏死因子等死亡受体发生凋亡 受体I(TNFRI)或肿瘤坏死因子相关的凋亡配体受体(TRAIL-R)。基因的单一突变 Bim或任何一种死亡受体都会导致抗原特异性CD8+T细胞延迟收缩 急性淋巴细胞性脉络膜脑膜炎病毒(LCMV)感染，但细胞数量最终下降。然而，损失 Bim和Fas，一种死亡受体，在急性发作后导致淋巴结特异性收缩阻断。 巨细胞病毒感染。这一提议中所涉及的具体假设是， 抗原特异性CD8+T细胞受外源性和内源性两种死亡途径控制。具体目标1将 确定Fas辅助Bim收缩抗原特异性CD8+T细胞的机制 急性巨细胞病毒感染后的淋巴转移。首先，我们将确定淋巴结收缩的缺陷是否 通过创建Bim-/-Faslpr/LPR P14 TCR转基因小鼠实现T细胞自主。这些小鼠的T细胞将会是 过继转移到野生型小鼠体内，将测量收缩。表达Fas配体的细胞 用流式细胞仪和免疫组织化学方法检测收缩过程中淋巴结的变化。这个 通过将突变的P14转基因T细胞过继转移到小鼠体内，将确定表达的重要性 选择性地表达功能性Fas配体。在具体目标2中，我们将确定死亡的贡献 受体和Bim介导的细胞凋亡在调节收缩中的作用这将通过创建双缺项来实现 诱导表达一种主要干扰FADDdd蛋白的小鼠，该蛋白可阻断死亡受体 信号，在他们的T细胞中。外源性和内源性脱氢酶对细胞凋亡的敏感性 从Bim-/-RTTA-FADDdd的NA和激活的CD8+T细胞将在体外测量刺激。至 确定所有死亡受体的丢失和BIM诱导的细胞凋亡对其他 除了淋巴结外的器官，双突变小鼠将感染引起急性 并将在体内测量抗原特异性CD8+T细胞的慢性感染和收缩。

Project Summary: Our goal is to determine the molecular mechanisms that control contraction of antigenspecific CD8+ T cell responses following acute and chronic viral infections. At the peak of the response, effector CD8+ T cells contain low levels of Bcl-2 and are susceptible to Bim-mediated apoptosis. Activated CD8+ T cells are also susceptible to apoptosis through death receptors such as Fas, Tumor Necrosis Factor Receptor I (TNFRI) or Tumor necrosis factor Related Apoptosis Ligand-Receptor (TRAIL-R). Single mutation of either Bim or any one death receptor results in delayed contraction of antigen-specific CD8+ T cells following acute lymphocytic choriomeningitis virus (LCMV) infection but cell numbers eventually decline. However, loss of both Bim and Fas, a death receptor, results in a lymph node-specific block in contraction following acute LCMV infection. The specific hypothesis that is being addressed in this proposal is that the contraction of antigen-specific CD8+ T cells is controlled by both extrinsic and intrinsic death pathways. Specific Aim 1 will determine the mechanism by which Fas aids Bim in the contraction of antigen-specific CD8+ T cells in the lymph nodes following acute LCMV infection. First, we will determine if the defects in lymph node contraction are T cell autonomous by creating Bim-/-Faslpr/lpr P14 TCR transgenic mice. T cells from these mice will be adoptively transferred into wildtype mice and contraction will be measured. The cells that express Fas Ligand in the lymph node during contraction will be determined by flow cytometry and immunohistochemistry. The importance of expression will be determined by adoptive transfer of mutant P14 transgenic T cells into mice which selectively express functional Fas Ligand. In Specific Aim 2, we will determine the contribution of death receptor and Bim-mediated apoptosis in regulating contraction. This will be accomplished by creating Bimdeficient mice that inducibly express a dominantly interfering FADDdd protein, which blocks death receptor signaling, in their T cells. The sensitivity to apoptosis following exposure to extrinsic and intrinsic deathinducing stimuli will be measured in na¿ve and activated CD8+ T cells from Bim-/-rtTA-FADDdd in vitro. To determine the extent to which loss of all death receptor and Bim-induced apoptosis affects responses in other organs besides the lymph nodes, double mutant mice will be infected with strains of LCMV that induce acute and chronic infection and contraction of antigen-specific CD8+ T cells will be measured in vivo.