Regulation of T cell development and maturation by NKAP

描述（由申请人提供）：T细胞激活涉及多个信号的整合。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传导在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，我们使用遗传方法通过白细胞文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。在我们最初的筛选中，我们发现NKAP的逆转录病毒表达补充了一个Jurkat突变细胞系的缺陷，并恢复了T细胞的激活。我们已经证明NKAP是Notch信号的一种新的负调节因子，与CIR相关，后者是Notch共抑制因子复合物的一部分。为了确定NKAP在体内的功能，我们制造了带有固定NKAP等位基因的小鼠。Lck-cre NKAP条件敲除小鼠在？？T细胞在dn3 /2选择检查点发育，尽管？？T细胞的发育正常进行。有趣的是，缺乏nor或mSin3a（两种转录辅助抑制因子复合物的通用成分）的小鼠在T细胞发育过程中也有DN3阻滞，这表明细胞通过？-选择检查点变成DP T细胞。正如预测的那样，通过QPCR检测三个Notch靶基因CD25、Deltex1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在体内作为Notch信号的负调控因子，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胸腺细胞在从半成熟到成熟的过程中存在阻滞。此外，CD4-cre NKAP cKO小鼠外周T细胞数量减少，这些幼稚T细胞表达与新近胸腺迁移一致的标记物，表明NKAP在T细胞成熟中也起着关键作用。本研究将重点了解NKAP在T细胞发育和成熟过程中的功能，揭示NKAP缺失时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。特异性目标#1:NKAP对T细胞发育和成熟的调控特异性目标#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 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.