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细胞激活涉及几个信号的整合。除了通过T细胞受体（TCR）（TCR）和通过CD28等分子的共刺激信号传导以及通过其他分子（包括跨膜受体缺陷）的信号传导在T细胞激活和发育的许多阶段至关重要。为了鉴定调节T细胞活化的新型分子，我们使用遗传学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T细胞活化缺陷。在我们的初始屏幕中，我们发现NKAP的逆转录病毒表达补充了一个Jurkat突变细胞系中的缺陷，并恢复了T细胞激活。我们已经证明，NKAP是与CIR相关联的Notch信号的新型负调节剂，这是Notch共抑制剂复合物的一部分。为了确定NKAP在体内的功能，我们用Floxed NKAP等位基因生成小鼠。 LCK-CRE NKAP有条件敲除小鼠在DN3/2选择检查点的细胞发育中具有严重的阻滞，尽管开发通常会进行。有趣的是，在NCOR或MSIN3A缺乏的小鼠（转录核心构件的两个通用成分）中，在T细胞发育过程中也具有DN3块，表明当细胞通过？选择检查点成为DP T细胞时，需要表观遗传变化。如预测的那样，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是tovo in vivo的负调控器，并且需要进行TT细胞的发育。 CD4-CRE NKAP CKO小鼠中T细胞发育的分析表明，在SP胸腺细胞中的块从半个半月发展到成熟。此外，CD4-CRE NKAP CKO小鼠的外围的T细胞数量减少，而这些幼稚的T细胞表达与最近胸腺移民一致的标志物，表明NKAP在T细胞成熟中也起着至关重要的作用。该建议将集中于理解T细胞发育和成熟过程中NKAP的功能，以发现在没有NKAP的情况下改变基因调节是如何改变T细胞的发展，并定义NKAP导致Notch靶基因上调的机制。特定目的＃1：通过NKAP特定目的2对T细胞发育和成熟的调节：在没有NKAP的情况下，基因表达改变的机制 公共卫生相关性：T细胞对于适当产生免疫反应至关重要，因为未产生T细胞会导致严重的免疫缺陷。缺乏蛋白质NKAP的小鼠在T细胞的产生中具有严重的阻滞。该建议将集中于了解NKAP如何调节T细胞的发育和成熟。