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

人类伽马/德尔塔 T 细胞激活的细胞生物学和分子机制

• 批准号: 10625480
• 负责人: Kaushik Choudhuri
• 金额: $ 38.33万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-11 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10625480
• 关键词: Affect; Antigen Presentation; Antigen Receptors; Antigen Targeting; Antigens; Binding; Biochemical; Biological; Biological Assay; Biology; Biophysics; CRISPR/Cas technology; Cell surface; Cells; Cellular Immunity; Cellular Stress; Cellular biology; Cellular immunotherapy; Chemicals; Cytolysis; Cytoskeleton; Electron Microscopy; Elements; Epithelium; Eukaryota; Event; Genetic; Histocompatibility; Human; IL17 gene; Image; Immune; Immunity; Immunologic Surveillance; Infection; Interferon Type II; Investigation; Knowledge; Leukemic Cell; Lipids; Lymphoblastic Leukemia; Lymphocyte; MHC antigen; Malignant Neoplasms; Mediating; Medical; Membrane; Memory; Metabolic; Methods; Microbe; Modeling; Molecular; Mutagenesis; Mutation; Neutrophil Infiltration; Pathway interactions; Peptide/MHC Complex; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Population; Predisposition; Production; Protein Family; Proteins; Public Health; RNA Interference; Rationalization; Research; Role; Signal Induction; Signal Transduction; Stress; Structure; Surface; Synapses; T cell response; T-Cell Activation; T-Lymphocyte; Testing; Therapeutic; Virus; ZAP-70 Gene; adhesion receptor; butyrophilin; cancer cell; cancer type; cytokine; design; effector T cell; fluorescence imaging; genome editing; high resolution imaging; imaging modality; immune function; immunological synapse; improved; innovation; interdisciplinary approach; mevalonate; mucosal site; novel; pathogen; peripheral blood; receptor; recruit; response; segregation; spatiotemporal; stoichiometry; superresolution imaging; synaptic function; tomography; tumor; ultra high resolution; γδ T cells

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.

项目摘要/摘要 γ/delta（γδ）T细胞是一种先天样淋巴细胞，表达非常规抗原受体（γδTCR） 在他们的表面。与常规的αβT细胞不同，它们不识别肽-MHC抗原，而是 被不变应激诱导的受体和宿主/病原体相关的代谢产物激活。大多数γδT细胞 人外围血表达Vγ9Vδ2TCR，并被磷酸异源性（PIP）代谢物激活 由医学上重要的微生物，病毒和癌细胞产生。 Vγ9VΔ2T细胞通过PIPS激活为TCR- 依赖，需要与表达B7元素蛋白丁醇的PIP产生靶细胞接触 3A1（BTN3A1）。但是，PIPS和BTN3A1启动信号并激活的分子机制 Vγ9VΔ2T细胞仍未解决。我们在此提案中的总体目标是确定细胞生物学和 PIP依赖性Vγ9Vδ2T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细胞与抗原靶细胞之间的细胞细胞接触界面，称为免疫学 突触。我们建议采用包括超分辨率荧光成像的尖端方法， 电子显微镜/断层扫描（EM），基于CRISPR/CAS9的基因组编辑和基于接近的化学 标记，测试我们的中心假设：PIP介导的Vγ9Vδ2T细胞激活发生通过独特 配置的免疫突触，其中TCR，辅助受体（包括BTN3A1），细胞骨架 元素和脂质结构域在顺式和突触中作用，以启动信号传导。我们的实质性初步 调查确定了我们的方法的可行性，并指出需要“亲密接触”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 在靶细胞和T细胞中都需要BTN3A1的表达，这表明未经证实的可能性 BTN3A1的反式突触相互作用。为了检验我们的中心假设，我们提出了两个具体目标：1。）使用 超分辨率成像和EM结合生化，信号传导和功能测定 Vγ9Vδ2TT的PIP依赖分子组织，膜地形和关键信号事件 细胞突触；和2.）使用基因组编辑和RNAi方法，结合BTN3A1结构/功能 诱变，确定了PIP诱导的BTN3A1细胞表面组织变化在Vγ9Vδ2T中的作用 细胞激活。我们的方法具有创新性，因为它使用尖端的成像和生化方法来测试 PIP介导的Vγ9Vδ2T细胞活化的新型模型。拟议的研究很重要，因为它将确定 Vγ9Vδ2T细胞激活的基本生化，生物物理和细胞生物学需求， 提供基于机械的框架来利用细胞免疫疗法的γδT细胞免疫疗法。