Mechanisms underlying T helper suppression by regulatory T cells

描述（由申请人提供）：本项目的总体目标是阐明T辅助细胞（Th）受调节性T细胞（TcR）抑制时发生的变化，并确定Treg介导的效应T细胞功能失活的分子机制。该项目的具体目标包括：（1）表征活化T细胞核因子（NFAT）在抑制T细胞中的作用，识别活化的分子机制，转录伴侣以及与下游靶点的相互作用;（2）确定Treg功能是否由白细胞介素（IL-）2基因的沉默控制。为了实现第一个目标，免疫荧光将揭示NFAT是否保留在细胞质中或跨膜易位，在那里它是有活性的，并且可以诱导已知抑制T细胞活化的蛋白质的表达。电泳迁移率变动分析将用于确定在AP-1不存在的情况下，活性NFAT蛋白是否可以结合受抑制的Th细胞的细胞核中的DNA。随后，将对抑制的Th 1细胞进行染色质免疫沉淀（ChIP），以确定NFAT的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。通过完成我们在目标一中的目标，我们打算揭示受抑制的T细胞内NFAT激活的转录调控，并阐明诱导和维持无反应状态所必需的基因表达模式。对于第二个目标，染色质免疫沉淀分析将确定表观遗传修饰是否调节IL-2在刺激的Th细胞和抑制的Th细胞中的表达。为了研究组蛋白去乙酰化在TCR 4的抑制活性和功能中的作用，将从BALB/c小鼠分离的TCR 4与TCR转基因D011.10小鼠共培养，并在存在或不存在组蛋白去乙酰化酶抑制剂曲古抑菌素-A的情况下刺激。我们的总体目标是在目标二是确定的作用，染色质重塑发挥在诱导T细胞耐受性和研究的机制，调节细胞因子的表达，在Treg和Treg介导的抑制Th细胞。清楚地了解IL-2位点在Treg和受抑制细胞中的调节方式，将证明对阐明Treg的行为和功能非常重要。通过揭示Treg介导的效应T细胞功能抑制的机制，可以开发针对许多自身免疫性疾病和癌症的诱导免疫耐受的新疗法。该项目与公众有关，因为这种细胞（Treg）在维持健康的免疫系统中发挥着重要作用。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.