Regulation of T cell development and maturation by NKAP

说明书(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)的抗原特异性信号和通过CD28等分子的共刺激信号外，通过包括跨膜受体Notch在内的其他分子的信号在T细胞激活和发育的许多阶段都是至关重要的。为了确定调节T细胞激活的新分子，我们使用了一种遗传方法，通过逆转录病毒表达白细胞库来挽救Jurkat突变细胞系中的T细胞激活缺陷。在我们的初步筛选中，我们发现逆转录病毒表达的NKAP弥补了一个Jurkat突变细胞系的缺陷，并恢复了T细胞的激活。我们已经证明，NKAP是一种新的Notch信号负调控因子，与CIR有关，CIR是Notch共抑制物复合体的一部分。为了确定NKAP在体内的功能，我们产生了一个NKAP等位基因。在DN3/2选择检查点，LCK-cre NKAP条件性基因敲除小鼠的T细胞发育严重受阻，尽管T细胞发育正常。有趣的是，缺乏NCoR或mSin3a的小鼠在T细胞发育过程中也会出现DN3阻断，这表明当细胞通过选择检查点成为DP T细胞时，表观遗传学变化是必要的。不出所料，QPCR检测了三个Notch靶基因CD25、Deltex 1和Hes1，发现NKAP lck-cre CKO DP T细胞的基因表达增加了8~20倍，证明NKAP在体内是Notch信号的负调控因子，是T细胞发育所必需的。对CD4cre NKAP CKO小鼠T细胞发育的分析表明，在SP胸腺细胞从半成熟到成熟的过程中，它们被阻断了。此外，CD4cre NKAP CKO小鼠外周血中T细胞数量减少，这些幼稚的T细胞表达的标志与胸腺新近迁出的T细胞一致，表明NKAP在T细胞成熟过程中也起着关键作用。这项建议将侧重于了解NKAP在T细胞发育和成熟过程中的功能，揭示在缺乏NKAP的情况下基因调控改变如何改变T细胞发育，并确定NKAP丢失导致Notch靶基因上调的机制。特异性目标1：NKAP对T细胞发育和成熟的调节

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