CELL BIOLOGY AND MOLECULAR MECHANISMS OF HUMAN GAMMA/DELTA T CELL ACTIVATION

项目摘要/摘要 γ/增量(γδ)T细胞是表达非常规抗原受体(γδ)的先天类淋巴细胞。 在它们的表面。与传统的αβT细胞不同，它们不识别多肽-MHC抗原，而是 由不变的应激诱导受体和宿主/病原体相关代谢物激活。大多数γδT细胞在 人外周血表达V-γ-9V-δ-2TCR，并被类异戊二烯(PIP)代谢物激活 由医学上重要的微生物、病毒和癌细胞产生。由PIPS激活的Vγ9Vδ2 T细胞是TCR- 依赖，并需要与表达B7家族蛋白的PIP产生的靶细胞接触 3A1(BTN3A1)。然而，PIPs和BTN3A1启动信号和激活的分子机制 Vγ9Vδ2 T细胞仍未溶解。我们在这项提案中的总体目标是鉴定细胞生物学和 依赖PIP的Vγ9Vδ2 T细胞活化的分子基础在αβT细胞中，抗原诱导的信号发生在 T细胞和携带抗原的靶细胞之间的细胞-细胞接触界面，称为免疫学 Synapse。我们建议使用尖端方法，包括超分辨率荧光成像， 电子显微镜/断层扫描(EM)、基于CRISPR/Cas9的基因组编辑和基于邻近的化学 标签，来检验我们的中心假设：PIP介导的Vγ9Vδ2T细胞激活是通过一种独特的 配置免疫突触，其中TCR、辅助受体(包括BTN3A1)、细胞骨架 元件和类脂域在顺式和跨突触中作用，启动信号传递。我们实质性的初步调查 调查确定了我们方法的可行性，并指出有必要进行密切接触和突触 TCR/磷酸酶分离可有效激活依赖于PIP的T细胞。令人惊讶的是，我们发现 靶细胞和T细胞都需要BTN3A1的表达，这表明可能是意外的 BTN3A1的跨突触相互作用。为了验证我们的中心假设，我们提出了两个具体目标：1)vbl.使用 超分辨率成像和EM，结合生化、信号和功能分析，建立 Vγ9Vδ2T依赖于PiP的分子组织、膜形态和关键信号事件 细胞突触；2.)使用基因组编辑和RNAi方法，结合BTN3A1结构/功能 诱变，确定PIP诱导的Vγ9Vδ2T细胞表面结构改变的作用 细胞激活。我们的方法是创新的，因为它使用了尖端的成像和生化方法来测试 PIP介导Vγ9Vδ2T细胞活化的新模型拟议的研究具有重要意义，因为它将确定 Vγ9Vδ2 T细胞激活的基本生化、生物物理和细胞生物学要求，以及 提供一个基于机制的框架，以利用γδT细胞免疫进行细胞免疫治疗。

PROJECT SUMMARY/ABSTRACT Gamma/delta (γδ) T cells are innate-like lymphocytes that express unconventional antigen receptors (γδ TCR) on their surface. Unlike conventional αβ T cells, they do not recognize peptide-MHC antigens but are instead activated by invariant stress-inducible receptors and host/pathogen-associated metabolites. Most γδ T cells in human peripheral blood express the Vγ9Vδ2 TCR and are activated by phosphoisoprenoid (PiP) metabolites produced by medically important microbes, viruses and cancer cells. Vγ9Vδ2 T cell activation by PiPs is TCR- dependent, and requires contact with PiP-producing target cells that express the B7-family protein butyrophilin 3A1 (BTN3A1). However, the molecular mechanisms by which PiPs and BTN3A1 initiate signaling and activate Vγ9Vδ2 T cells remain unresolved. Our overall objective in this proposal is to identify the cell biological and molecular basis of PiP-dependent Vγ9Vδ2 T cell activation. In αβ T cells, antigen-induced signaling occurs at the cell-cell contact interface between T cells and antigen-bearing target cells, known as the immunological synapse. We propose to employ cutting edge approaches that include super-resolution fluorescence imaging, electron microscopy/tomography (EM), CRISPR/Cas9-based genomic editing, and proximity-based chemical tagging, to test our central hypothesis: that PiP-mediated Vγ9Vδ2 T cell activation occurs through a uniquely configured immunological synapse, in which TCR, accessory receptors (including BTN3A1), cytoskeletal elements, and lipid domains act in cis and across the synapse to initiate signaling. Our substantial preliminary investigations establish the feasibility of our approach and point to a need for `close contacts' and synaptic TCR/phosphatase segregation for effective PiP-dependent T cell activation. Surprisingly, we find that expression of BTN3A1 is required in both target cells and T cells, pointing to the possibility of an unsuspected trans-synaptic interaction of BTN3A1. To test our central hypothesis, we propose two Specific Aims: 1.) Using super-resolution imaging and EM, in conjunction with biochemical, signaling and functional assays, establish the PiP-dependent molecular organization, membrane topography, and key signaling events at the Vγ9Vδ2 T cell synapse; and 2.) Using genome-editing and RNAi approaches, combined with BTN3A1 structure/function mutagenesis, establish the role of PiP-induced changes in cell-surface organization of BTN3A1 in Vγ9Vδ2 T cell activation. Our approach is innovative as it uses cutting-edge imaging and biochemical methods to test a novel model for PiP-mediated Vγ9Vδ2T cell activation. The proposed research is significant as it will identify the fundamental biochemical, biophysical and cell biological requirements for Vγ9Vδ2 T cell activation, and provide a mechanistically-based framework to harness γδ T cell immunity for cellular immunotherapy.