Role of GRAIL in CD25+ T Regulatory Cells

描述（由申请人提供）：最近CD25+ T调节（CD25 Treg）细胞的特性强调了这些细胞在多种疾病状态下调节免疫反应的重要性。CD25 Treg细胞是调节性T细胞的一个亚群，具有无能表型，通过一种鲜为人知的机制以抗原特异性的方式抑制免疫反应。迄今为止，CD25 Treg细胞中唯一独特的遗传标记是转录因子Foxp3。我们实验室的研究已经证明了GRAIL和CD25 Treg细胞之间的联系，GRAIL是一种E3泛素连接酶，是体内CD4 T细胞能量发展所必需的。我们假设GRAIL对自然发生和诱导的CD25 Treg细胞的发育和功能很重要。有几条证据支持这一假设：1)GRAIL在自然发生的CD25 Treg细胞中的表达水平比CD25- CD4 T细胞高10倍；2)体内耐受免疫可诱导长寿命表达抗原特异性CD25的耐受T细胞。这些细胞的基因表达分析显示GRAIL mRNA上调（与CD25耐受细胞相比增加700倍；与自然发生的CD25 Treg细胞相比增加100倍）。此外，GRAIL的表达与Foxp3的表达密切相关，这表明在这种诱导耐受人群中存在抑制表型。作为了解GRAIL在CD25 Treg细胞抑制功能中的作用的第一步，我们证明了在抗原特异性T细胞系中强制表达GRAIL足以在体外传递抑制表型。本研究计划的具体目的是：1)通过生成表达GRAIL（显性阴性形式）或GRAIL RNAi构建体的骨髓嵌合小鼠，确定GRAIL在自然发生的CD25 Treg细胞发育和功能中的作用；2)通过功能表征诱导表达耐受细胞的CD25+ GRAIL，并建立激活抑制细胞中GRAIL的表达动力学，确定GRAIL是否在活化抑制细胞中上调。

DESCRIPTION (provided by applicant): The recent characterization of CD25+ T regulatory (CD25 Treg) cells has underscored the importance of these cells in modulating immune responses in a broad range of disease states. CD25 Treg cells are a subset of regulatory T cells with an anergic phenotype that suppress immune responses in an antigen specific fashion by a poorly understood mechanism. To date, the only unique genetic marker in CD25 Treg cells is the transcription factor, Foxp3. Studies from our laboratory have demonstrated a link between GRAIL, an E3 ubiquitin ligase that is necessary for the development of CD4 T cell anergy in vivo, and CD25 Treg cells. We hypothesize that GRAIL is important for the development and function of naturally occurring and induced CD25 Treg cells. Several lines of evidence support this hypothesis: 1) GRAIL is expressed in naturally occurring CD25 Treg cells at levels 10-fold greater than CD25- CD4 T cells, 2) a tolerizing immunization in vivo leads to the induction of long-lived CD25 expressing antigen-specific tolerized T cells. Gene expression analysis of these cells reveals that GRAIL mRNA is upregulated (700-fold increase vs. CD25- tolerized; 100-fold increase vs, naturally occurring CD25 Treg cells). Moreover, GRAIL expression is linked with Foxp3 expression strongly suggesting a suppressor phenotype in this induced tolerized population. As an initial step to understanding the role of GRAIL in CD25 Treg cell suppressor function, we demonstrate that enforced expression of GRAIL in an antigen-specific T cell line is sufficient to convey a suppressor phenotype in vitro. The specific aims of this research proposal are: 1) To establish the role of GRAIL in naturally occurring CD25 Treg cell development and function by generating bone marrow chimeric mice expressing GRAIL, a dominant negative form, or GRAIL RNAi construct 2) To determine if GRAIL is upregulated in activated suppressor cells by functionally characterizing the induced CD25+ GRAIL expressing tolerized cells and establishing the kinetics of GRAIL expression in activated suppressor cells.