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

项目总结/摘要 γ/δ（γδ）T细胞是表达非常规抗原受体（γδ TCR）的天然样淋巴细胞 在他们的表面。与传统的αβ T细胞不同，它们不识别肽-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γδ T细胞在 人外周血表达Vγ9Vδ2 TCR，并被磷酸类异戊二烯（PiP）代谢物激活 由医学上重要的微生物、病毒和癌细胞产生。通过PiP的Vγ 9VS 2 T细胞活化是TCR-γ 9VS 2 T细胞活化。 依赖，并需要与表达B7家族蛋白亲酪蛋白的产生PIP的靶细胞接触 3A1（BTN3A1）。然而，PiPs和BTN 3A 1启动信号传导和激活的分子机制是不确定的。 Vγ 9VS 2 T细胞仍然未分辨。我们在这个提案中的总体目标是确定细胞生物学和 PiP依赖性Vγ 9VS 2 T细胞活化的分子基础。在αβ T细胞中，抗原诱导的信号传导发生在 T细胞和携带抗原的靶细胞之间的细胞-细胞接触界面，称为免疫接触界面。 突触我们建议采用尖端的方法，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性的化学 标记，以检验我们的中心假设：PIP介导的Vγ 9VS 2 T细胞活化通过独特的 配置的免疫突触，其中TCR，辅助受体（包括BTN 3A 1），细胞骨架 元件和脂质结构域顺式作用并穿过突触以启动信号传导。我们的初步调查 调查确定了我们的方法的可行性，并指出需要“密切接触”和突触 TCR/磷酸酶分离用于有效的PiP依赖性T细胞活化。令人惊讶的是，我们发现， BTN 3A 1的表达在靶细胞和T细胞中都是必需的，这表明BTN 3A 1的表达可能是一种未被怀疑的免疫应答。 BTN 3A 1的跨突触相互作用。为了验证我们的核心假设，我们提出了两个具体目标：1。使用 超分辨率成像和EM，结合生物化学、信号传导和功能测定， 在Vγ9Vδ2 T上的PIP依赖性分子组织、膜形貌和关键信号事件 细胞突触;和2.）使用基因组编辑和RNAi方法，结合BTN 3A 1结构/功能 突变，确定PiP诱导的BTN 3A 1在Vγ9Vδ2 T细胞表面组织中的作用 细胞激活我们的方法是创新的，因为它使用尖端的成像和生物化学方法来测试一个 PIP介导的Vγ 9VS 2 T细胞活化的新模型。拟议的研究是重要的，因为它将确定 Vγ 9VS 2 T细胞活化的基本生物化学、生物物理学和细胞生物学要求，以及 提供了一个基于机制的框架，以利用γδ T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.