Regulation of T cell development and maturation by NKAP

NKAP 对 T 细胞发育和成熟的调节

• 批准号: 8011439
• 负责人: Virginia Smith Shapiro
• 金额: $ 37.4万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8011439
• 关键词: Alleles; Antigens; Apoptosis; CD28 gene; Cell Cycle Progression; Cell Line; Cell Maturation; Cells; Chemosensitization; Complement; Complex; Defect; Deltex Homolog 1; Development; Emigrant; Epigenetic Process; Failure; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Generic Drugs; Genetic; IL2RA gene; Immune; Immune response; Knockout Mice; Leukocytes; Libraries; Mus; Play; Proteins; Regulation; Repression; Role; Signal Transduction; Staging; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Thymocyte Development; Transcription Repressor/Corepressor; Up-Regulation; in vivo; mutant; notch protein; novel; public health relevance; thymocyte

DESCRIPTION (provided by applicant): T cell activation involves the integration of several signals. In addition to antigen specific signaling through the T cell receptor (TCR) and costimulatory signaling through molecules such as CD28, signaling through additional molecules including the transmembrane receptor Notch are critical at many stages of T cell activation and development. To identify novel molecules that regulate T cell activation, we used a genetic approach to rescue the T cell activation defect in a Jurkat mutant cell line by retroviral expression of a leukocyte library. In our initial screen, we found that retroviral expression of NKAP complemented the defect in one Jurkat mutant cell line and restored T cell activation. We have demonstrated that NKAP is a novel negative regulator of Notch signaling that associates with CIR, which is part of the Notch co-repressor complex. To determine the function of NKAP in vivo, we generated mice with a floxed NKAP allele. Lck-cre NKAP conditional knockout mice have a severe block in ??T cell development at the DN3/2-selection checkpoint, although ??T cell development proceeds normally. Interestingly, mice deficient in either NcoR or mSin3a, two generic components of transcriptional corepressor complexes, also have a DN3 block during T cell development, indicating that epigenetic changes are required as cells pass the ?-selection checkpoint to become DP T cells. As predicted, examination of three Notch target genes, CD25, Deltex1 and Hes1 by QPCR demonstrated that NKAP lck-cre cKO DP T cells had increases in gene expression by 8- to 20-fold, proving that the NKAP functions as a negative regulator of Notch signaling in vivo, and is required for T cell development. Analysis of T cell development in CD4-cre NKAP cKO mice demonstrates a block within SP thymocytes as they progress from semimature to mature. In addition, there are decreased numbers of T cells in the periphery in CD4-cre NKAP cKO mice, and these naive T cells express markers consistent with being recent thymic emigrants, indicating that NKAP also plays a critical role in T cell maturation. This proposal will focus on understanding the function of NKAP during T cell development and maturation, to uncover how altered gene regulation in the absence of NKAP alters T cell development and to define the mechanism whereby loss of NKAP leads to upregulation of Notch target genes. Specific Aim #1: Regulation of T cell development and maturation by NKAP Specific Aim #2: Mechanism of altered gene expression in the absence of NKAP PUBLIC HEALTH RELEVANCE: T cells are critical to proper generation of the immune response, as failure to produce T cells results in severe immune deficiency. Mice deficient in the protein NKAP have a severe block in the generation of T cells. This proposal will focus on understanding how NKAP regulates T cell development and maturation.

描述（由申请人提供）：T 细胞激活涉及多种信号的整合。除了通过 T 细胞受体 (TCR) 的抗原特异性信号传导和通过 CD28 等分子的共刺激信号传导之外，通过包括跨膜受体 Notch 在内的其他分子的信号传导在 T 细胞激活和发育的许多阶段也至关重要。为了鉴定调节 T 细胞激活的新分子，我们使用遗传方法通过白细胞库的逆转录病毒表达来挽救 Jurkat 突变细胞系中的 T 细胞激活缺陷。在我们的初步筛选中，我们发现 NKAP 的逆转录病毒表达补充了 Jurkat 突变细胞系的缺陷并恢复了 T 细胞活化。我们已经证明 NKAP 是一种新型的 Notch 信号负调节因子，与 CIR 相关，而 CIR 是 Notch 共抑制复合物的一部分。为了确定 NKAP 的体内功能，我们培育了具有 floxed NKAP 等位基因的小鼠。尽管 T 细胞发育正常，但 Lck-cre NKAP 条件敲除小鼠在 DN3/2 选择检查点的 T 细胞发育受到严重阻碍。有趣的是，缺乏 NcoR 或 mSin3a（转录辅阻遏物复合物的两种通用成分）的小鼠在 T 细胞发育过程中也有 DN3 阻断，这表明当细胞通过 β-选择检查点成为 DP T 细胞时需要表观遗传变化。正如预测的那样，通过 QPCR 对三个 Notch 靶基因 CD25、Deltex1 和 Hes1 的检查表明，NKAP lck-cre cKO DP T 细胞的基因表达增加了 8 至 20 倍，证明 NKAP 作为负调节因子发挥作用。 Notch 信号传导在体内，是 T 细胞发育所必需的。对 CD4-cre NKAP cKO 小鼠 T 细胞发育的分析表明，SP 胸腺细胞从半成熟到成熟的过程中出现了阻碍。此外，CD4-cre NKAP cKO小鼠外周T细胞数量减少，这些初始T细胞表达的标记物与最近的胸腺移出物一致，表明NKAP在T细胞成熟中也发挥着关键作用。该提案将重点了解 NKAP 在 T 细胞发育和成熟过程中的功能，揭示在 NKAP 缺失的情况下基因调控的改变如何改变 T 细胞发育，并确定 NKAP 缺失导致 Notch 靶基因上调的机制。具体目标#1：NKAP 调节 T 细胞发育和成熟 具体目标#2：缺乏 NKAP 时基因表达改变的机制 公共卫生相关性：T 细胞对于免疫反应的正常产生至关重要，因为无法产生 T 细胞会导致严重的免疫缺陷。缺乏 NKAP 蛋白的小鼠 T 细胞的生成严重受阻。该提案将重点了解 NKAP 如何调节 T 细胞的发育和成熟。