Imaging and Function of the Immunological Synapse

描述(申请人提供)：T淋巴细胞将抗原识别为与抗原提呈细胞(APC)表面的MHC分子结合的小肽。除了T细胞抗原受体和载肽的MHC外，许多其他细胞表面分子在决定T细胞/APC相互作用的结果中起着必要的或调节的作用。抗原识别伴随着这些分子大规模的、依赖于细胞骨架的重排，在T细胞和APC之间形成一个有组织的接触界面，称为免疫突触。许多实验室正在研究突触如何提供或调节充分激活T细胞所需的信号。在这项赠款申请中，重点是免疫突触在将效应功能从CD4T细胞传递到APC中的作用，使用FasL(CD178)和CD40L(CD154)作为膜结合效应细胞因子的原型。由于体内大多数相互作用可能太短，不能从头合成效应细胞因子，建议的实验研究了预先形成的细胞内FasL和CD40L存储在T细胞/APC相互作用中发挥重要作用的可能性。CD40L最初被认为是细胞接触依赖性的CD4T细胞帮助B细胞的介质，此后被证明是一种有效的巨噬细胞激活剂，可以杀死树突状细胞的胞内病原体，从而启动CD4和CD8T细胞的效应功能和记忆。FasL是CD4和CD8T细胞接触依赖性杀伤的主要介质，在活化诱导的细胞死亡过程中通过T细胞自杀或自杀性杀伤来调节T细胞的反应。 利用最先进的显微镜和体外和体内功能分析相结合的方法，这一建议将检验以下假设：在大多数启动的CD4效应和记忆T细胞中，预先形成的CD40L和FasL存储在分泌溶酶体中，在瞬时抗原特异性相互作用中通过免疫突触快速输送到B细胞和其他APC，并具有不同于新合成的CD40L和FasL的重要功能后果。它还将研究1型和2型辅助T细胞形成的突触之间的结构差异，并测试由CD4T细胞形成的成熟免疫突触中黏附分子环的功能是确保抗原特异性的效应分子递送到APC并防止递送到周围旁观者细胞的假设。 与公共卫生相关：T淋巴细胞只识别其他细胞表面的抗原，称为抗原提呈细胞(APC)，并通过分泌强大的细胞因子和其他效应分子来发挥其作用。这项应用的目标是了解各种类型的CD4T细胞如何向APC递送效应性细胞因子。其应用主要集中在FasL(CD178)和CD40L(CD154)这两种关键的膜结合细胞因子上，它们控制着健康和疾病中的适应性免疫反应。拟议的实验将有助于基本了解CD4T细胞效应器的调节功能，这反过来将在移植、自身免疫、过敏、癌症免疫治疗和传染病控制方面具有公共卫生相关的应用。

DESCRIPTION (provided by applicant): T lymphocytes recognize antigen as small peptides bound to MHC molecules on the surface of antigen presenting cells (APC). In addition to the T cell antigen receptor and peptide-loaded MHC, dozens of other cell surface molecules play necessary or modulating roles in determining the outcome of the T cell/APC interaction. Antigen recognition is accompanied by large-scale, cytoskeleton-dependent rearrangements of these molecules to form an organized contact interface between the T cell and the APC termed the "immunological synapse". Many laboratories are investigating the ways in which the synapse may provide or regulate the signals necessary for full T cell activation. In this grant application, the focus is on the role of the immunological synapse in the delivery of effector functions from CD4 T cells to APC, using FasL (CD178) and CD40L (CD154) as prototypes of membrane-bound effector cytokines. Since most interactions in vivo may be too short to allow for de novo synthesis of effector cytokines, the proposed experiments examine the possibility that preformed, intracellular stores of FasL and CD40L play important roles in T cell/APC interactions. CD40L was first identified as the mediator of cell contact-dependent CD4 T cell help for B cells, and has since been shown to be a potent activator of macrophages for killing of intracellular pathogens of dendritic cells for priming effector functions and memory in CD4 and CD8 T cells. FasL is a principal mediator of contact-dependent killing by CD4 and CD8 T cells, and regulates T cell responses through T cell suicide or fratricide during activation-induced cell death. Using a combination of state-of-the-art microscopy and functional assays in vitro and in vivo, this proposal will test the hypothesis that pre-formed CD40L and FasL are stored in secretory lysosomes in most primed CD4 effector and memory T cells, are delivered rapidly through the immunological synapse to B cells and other APC in transient antigen-specific interactions, and have important functional consequences distinct from those of newly synthesized CD40L and FasL. It will also investigate structural difference between synapses formed by type 1 and type 2 helper T cells, and test the hypothesis that the function of the ring of adhesion molecules in the mature immunological synapse formed by CD4 T cells is to ensure antigen-specific delivery of effector molecules to the APC and prevent delivery to surrounding bystander cells. PUBLIC HEALTH RELEVANCE: T lymphocytes recognize antigen only on the surface of other cells, called antigen presenting cells (APC), and exert their effects by secreting potent cytokines and other effector molecules. The goal of this application is to understand how various kinds of CD4 T cells deliver effector cytokines to APC. The application is focused on FasL (CD178) and CD40L (CD154), key membrane-bound cytokines which control the adaptive immune response in health and disease. The proposed experiments will contribute to a basic understanding of the regulation CD4 T cell effector functions, which in turn will have public health-related applications in transplantation, autoimmunity, allergy, cancer immunotherapy, and control of infectious diseases.