Role of GRAIL in CD25+ T Regulatory Cells

描述（由申请人提供）：最近对CD 25 + T调节（CD 25 Treg）细胞的表征强调了这些细胞在调节广泛疾病状态下免疫应答的重要性。CD 25 Treg细胞是具有无反应性表型的调节性T细胞的亚群，其通过知之甚少的机制以抗原特异性方式抑制免疫应答。迄今为止，CD 25 Treg细胞中唯一独特的遗传标记是转录因子Foxp 3。我们实验室的研究已经证明了GRAIL（一种E3泛素连接酶，对于体内CD 4 T细胞无反应性的发展是必要的）和CD 25 Treg细胞之间的联系。我们假设GRAIL对天然存在和诱导的CD 25 Treg细胞的发育和功能很重要。几条证据支持这一假设：1）GRAIL在天然存在的CD 25 Treg细胞中以比CD 25-CD 4 T细胞高10倍的水平表达，2）体内耐受性免疫导致诱导表达抗原特异性耐受化T细胞的长寿命CD 25。这些细胞的基因表达分析揭示GRAIL mRNA上调（相对于CD 25耐受化的700倍增加;相对于天然存在的CD 25 Treg细胞100倍增加）。此外，GRAIL表达与Foxp 3表达相关联，强烈提示在该诱导耐受化群体中的抑制表型。作为理解GRAIL在CD 25 Treg细胞抑制功能中的作用的第一步，我们证明了GRAIL在抗原特异性T细胞系中的强制表达足以在体外传递抑制表型。这项研究建议的具体目标是：1）通过产生表达GRAIL（一种显性阴性形式）的骨髓嵌合小鼠来确定GRAIL在天然存在的CD 25 Treg细胞发育和功能中的作用，2）为了通过功能性表征诱导的CD 25 + T细胞或GRAIL RNAi构建体来确定GRAIL是否在活化的抑制细胞中上调，表达GRAIL的耐受化细胞和建立激活的抑制细胞中GRAIL表达的动力学。

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.