Regulation of T cell development and maturation by NKAP

描述（由申请人提供）：T细胞活化涉及几种信号的整合。除了通过T细胞受体（TCR）的抗原特异性信号传导和通过分子如CD 28的共刺激信号传导之外，通过包括跨膜受体Notch的另外的分子的信号传导在T细胞活化和发育的许多阶段是关键的。为了鉴定调节T细胞活化的新分子，我们使用遗传方法通过白细胞文库的逆转录病毒表达来拯救Jurkat突变细胞系中的T细胞活化缺陷。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一个Jurkat突变细胞系中的缺陷，并恢复了T细胞活化。我们已经证明，NKAP是一种新的Notch信号负调控因子，与CIR相关，CIR是Notch辅助阻遏复合物的一部分。为了确定NKAP在体内的功能，我们产生了具有floxed NKAP等位基因的小鼠。Lck-cre NKAP条件性基因敲除小鼠在？在DN 3/2选择检查点的T细胞发育，虽然？？T细胞发育正常。有趣的是，NcoR或mSin 3a（转录辅阻遏物复合物的两种通用组分）缺陷的小鼠在T细胞发育期间也具有DN 3阻断，这表明当细胞通过？选择检查点成为DP T细胞。如所预测的，通过QPCR对三种Notch靶基因CD 25、Deltex 1和Hes 1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在体内起Notch信号传导的负调节剂的作用，并且是T细胞发育所需的。对CD 4-cre NKAP cKO小鼠中T细胞发育的分析表明，随着SP胸腺细胞从半成熟发展到成熟，SP胸腺细胞内存在阻滞。此外，在CD 4-cre NKAP cKO小鼠中，外周中T细胞数量减少，并且这些初始T细胞表达与近期胸腺移出一致的标志物，表明NKAP在T细胞成熟中也起关键作用。该提案将侧重于了解NKAP在T细胞发育和成熟过程中的功能，以揭示在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对T细胞发育和成熟的调节具体目标#2：NKAP缺失时基因表达改变的机制

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