Mechanisms underlying T helper suppression by regulatory T cells

描述(由申请人提供)：这个项目的总体目标是阐明调节性T细胞(Treg)抑制T辅助细胞(Th)内发生的变化，并确定Treg介导的效应器T细胞功能失活的分子机制。该项目的具体目标包括(1)确定活化T细胞核因子(NFAT)在受抑制T细胞中的作用，确定激活的分子机制、转录伙伴以及与下游靶点的相互作用；以及(2)确定Treg功能是否受白介素2(IL-2)基因沉默的控制。为了接近第一个目标，免疫荧光将揭示NFAT是保留在细胞质中还是跨膜转运，在那里它是活跃的，并可以诱导已知抑制T细胞激活的蛋白质的表达。在没有AP-1的情况下，凝胶迁移率改变分析将被用来确定活性NFAT蛋白是否能在受抑制的Th细胞的核中结合DNA。随后，将对受抑制的Th1细胞进行染色质免疫沉淀(ChIP)，以确定NFAT激活是否涉及将该蛋白直接募集到T细胞失活基因的启动子区域。通过完成我们在目标1中的目标，我们打算揭示被抑制的T细胞中NFAT激活的转录调节，并阐明诱导和维持无反应状态所必需的基因表达模式。对于第二个目的，染色质免疫沉淀分析将确定表观遗传修饰是否调节刺激的Tregs和抑制的Th细胞中IL-2的表达。为了研究组蛋白去乙酰化在Tregs抑制活性和功能中的作用，将BALB/c小鼠分离的Tregs与TCR转基因D011.10小鼠共培养，并在有或没有组蛋白去乙酰化酶抑制剂trichostatin-A的情况下进行刺激。我们在目标2中的总体目标是确定染色质重塑在T细胞耐受诱导中的作用，并研究Tregs中细胞因子表达的调节机制和Treg介导的Th细胞抑制机制。清楚地了解Tregs和受抑制的细胞中Theil-2基因是如何调控的，对于阐明Treg的行为和功能将是非常有价值的。通过揭示Treg介导的抑制效应T细胞功能的机制，可以开发出诱导免疫耐受的新疗法，以对抗一系列自身免疫性疾病和癌症。这个项目与广大公众相关，因为这个细胞(Treg)在维持健康的免疫系统方面发挥着重要作用。通过研究这种细胞类型(Treg)的行为，可以更好地了解不同人类疾病的原因，并开发新的治疗方法。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate the changes that occur within T helper (Th) cells when suppressed by regulatory T cells (Tregs) and identify the molecular mechanisms underlying Treg mediated inactivation of effector T cell functions. The specific aims of this project include (1) characterizing the role of Nuclear Factor of Activated T cells (NFAT) within suppressed T cells, identifying the molecular mechanisms of activation, transcriptional partners and the interaction with downstream targets; and (2) determining if Treg function is controlled by silencing of the interleukin (IL-)2 gene. To approach the first aim, immunofluorescence will reveal if NFAT is retained in the cytoplasm or translocated across the membrane, where it is active and can induce the expression of proteins known to suppress T cell activation. Electrophoretic mobility shift assays will be used to determine if active NFAT proteins can bind DNA in the nucleus of suppressed Th cells, in the absence of AP-1. Subsequently, chromatin immunoprecipitation (ChIP) will be performed on suppressed Th1 cells to determine if activation by NFAT involves direct recruitment of this protein to the promoter regions of T cell inactivating genes. By completing our goals in aim one we intend to uncover the transcriptional regulation of NFAT activation within suppressed T cells and elucidate gene expression patterns that are necessary for the induction and maintenance of the unresponsive state. For the second aim, chromatin immunoprecipitation assays will determine if epigenetic modifications regulate IL-2expression in stimulated Tregs and suppressed Th cells. To study the role of histone deacetylation in the suppressor activity and function of Tregs, isolated Tregs from BALB/c mice will be cocultured with TCR transgenic D011.10 mice and stimulated in the presence or absence of the histone deacetylase inhibitor, Trichostatin-A. Our overall goal in aim two is to determine the role that chromatin remodeling plays in the induction of T cell tolerance and investigate the mechanisms that regulate cytokine expression in Tregs and in Treg-mediated suppression of Th cells. A clear understanding of how theIL-2 locus is regulated in both Tregs and suppressed cells will prove invaluable in elucidating Treg behavior and function. By uncovering the mechanisms of Treg-mediated suppression of effector T cell function, novel therapies inducing immune tolerance can be developed against a host of autoimmune diseases and cancers. This project is relevant to the public at large because of the importance this cell (Treg) plays in maintaining a healthy immune system. By studying the behavior of this cell type (Treg), the causes of different human diseases can be better understood and new therapies developed.