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

项目概述：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CD 8 + T细胞反应。在响应的峰值， 效应CD 8 + T细胞含有低水平的Bcl-2，并且对Bim介导的细胞凋亡敏感。激活 CD 8 + T细胞也易于通过死亡受体如Fas、肿瘤坏死因子（TNF）、CD 8 + T细胞受体（CD 8 + T细胞受体）凋亡。 受体I（TNFRI）或肿瘤坏死因子相关凋亡配体-受体（TRAIL-R）。单突变 Bim或任何一种死亡受体导致抗原特异性CD 8 + T细胞在 急性淋巴细胞性脉络丛脑膜炎病毒（LCMV）感染，但细胞数量最终下降。然而，损失 Bim和Fas这两种死亡受体的结合导致淋巴结特异性阻断急性心肌梗死后的收缩。 LCMV感染。本提案所涉及的具体假设是， 抗原特异性CD 8 + T细胞受外在和内在死亡途径控制。具体目标1将 确定Fas辅助Bim收缩抗原特异性CD 8 + T细胞的机制。 急性LCMV感染后的淋巴结。首先，我们将确定淋巴结收缩的缺陷是否 通过建立Bim-/-Faslpr/lpr P14 TCR转基因小鼠，这些小鼠的T细胞将被 过继转移到野生型小鼠中并测量收缩。表达Fas配体的细胞 将通过流式细胞术和免疫组织化学来确定收缩期间淋巴结中的细胞因子。的 表达的重要性将通过将突变P14转基因T细胞过继转移到小鼠中来确定 其选择性表达功能性Fas配体。在具体目标2中，我们将确定死亡的贡献 受体和Bim介导的细胞凋亡调节收缩。这将通过创建Bimdeficient来实现 诱导表达显性干扰FADDdd蛋白的小鼠，该蛋白阻断死亡受体 在他们的T细胞中。外源性和内源性死亡诱导剂对细胞凋亡的敏感性 将在来自Bim-/-rtTA-FADDdd的初始和活化的CD 8 + T细胞中体外测量刺激。到 确定所有死亡受体的丧失和Bim诱导的细胞凋亡影响其他细胞应答的程度。 在淋巴结以外的器官中，双突变小鼠将感染LCMV株， 并且将在体内测量抗原特异性CD 8 + 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.