The function of membrane vesiculation in CTLA-4-mediate*

描述（由申请人提供）：T细胞耐受性对于预防自身免疫性疾病、移植物抗宿主病和移植存活至关重要。共刺激分子在经典的T细胞活化的双信号模型中提供“第二信号”，在维持T细胞耐受性中发挥作用，即在产生生产性免疫应答和防止对自身组织的致病性应答之间取得平衡。细胞毒性T淋巴细胞抗原-4（CTLA-4）是CD 28共刺激家族的成员，是T细胞活化和效应功能的关键调节因子。CTLA-4的功能是抵消活化信号并抑制或减弱T细胞活化和应答。在这个提议中，我们假设CTLA-4可以使用迄今为止未鉴定的和新的机制来调节T细胞耐受。在第一个具体目标中，我们提出测试存在CTLA-4内吞作用的替代方法的假设。此外，我们提出，这些替代方法是至关重要的，因为它们将通过与TCR信号体空间上不同的细胞内信号传导提供CTLA-4功能的未鉴定机制。我们将测试这些预测的一系列方法，使用显性负突变体和RNAi敲低候选细胞内运输途径中的关键蛋白。在具体目标2中，我们提出，CTLA-4是通过囊泡从活化的T细胞中释放出来的，并且膜结合的CTLA-4可以反式获得。此外，我们提出，这种来源的CTLA-4可以通过竞争配体被动地发挥作用，或者更有趣的是，可以被“受体”T细胞吸收并积极抑制T细胞刺激。我们将对CTLA-4的两种膜结合亚型（全长和配体非依赖性CTLA-4）进行这些预测。从所提出的研究中获得的见解将阐明CTLA-4介导的T细胞反应性、T细胞耐受性和耐受性丧失的调节的新机制基础。了解这些过程对于设计治疗自身免疫性疾病和过敏性疾病以及移植的新治疗方法至关重要。迄今为止，在增加CTLA-4功能的能力方面进展有限。所提出的CTLA-4功能模型将提供一种新的方法来增加体内CTLA-4介导的抑制。

DESCRIPTION (provided by applicant): T cell tolerance is critical for the prevention of autoimmune disease, graft versus host disease and for transplantation survival. There is a growing appreciation for the role of costimulatory molecules, which provide the 'second signal' in the classic two-signal model of T cell activation, play in maintaining T cell tolerance, that is striking the balance between generating productive immune responses and preventing pathogenic responses to self-tissues. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a member of the CD28 costimulation family that is a critical regulator of T cell activation and effector functions. CTLA-4 functions to counteract activating signals and inhibit or attenuate T cell activation and responses. In this proposal we hypothesize that CTLA-4 can function using a heretofore unidentified and novel mechanisms to regulate T cell tolerance. In the first specific aim we propose to test the hypothesis that there are alternate methods of CTLA-4 endocytosis. Further we propose that these alternative methods are critical since they will provide an unidentified mechanism of CTLA-4 function via intracellular signaling occurring spatially distinct from the TCR signalosome. We will test these predictions by series of approaches using dominant negative mutants and RNAi knock-down of key proteins in candidate intracellular trafficking pathways. In Specific Aim 2 we propose that CTLA-4 is released from activated T cells via vesicles and that membrane bound CTLA-4 can be acquired in trans. Further we propose that this source of CTLA-4 can function passively by competing for ligand or, more interestingly, can be taken up by the 'acceptor' T cells and actively inhibit T cell stimulation. We will test these predictions for both membrane bound isoforms of CTLA-4 (full-length and ligand-independent CTLA-4). The insights gained from the proposed studies will elucidate a novel mechanistic basis for CTLA-4-mediated regulation of T cell responsiveness, T cell tolerance and loss of tolerance. Understanding these processes is critical for designing novel therapeutic approaches to treat autoimmune disease and allergic diseases and in transplantation. To date there has been limited advancement on the ability to increase CTLA-4 function. The proposed model of CTLA-4 function would provide a novel means to increase CTLA-4-mediated inhibition in vivo.