DNA mechanotechnology to investigate the role of PD1 biophysics in T cell signaling

DNA 机械技术研究 PD1 生物物理学在 T 细胞信号传导中的作用

• 批准号: 9911685
• 负责人: Anna Kellner
• 金额: $ 4.55万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-02 至 2022-12-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911685
• 关键词: Address; Amino Acids; Antigens; Apoptosis; Area; Binding; Biophysical Process; Biophysics; Cell Communication; Cell physiology; Cells; Cellular biology; Cessation of life; Chemicals; Complex; Cytotoxic T-Lymphocytes; DNA; DNA Probes; Data; Dissociation; Engineering; Environment; Event; Fluorescence; Fluorescence Microscopy; Genes; Goals; Hybrids; Hydrophobicity; Immobilization; Immune; Immune checkpoint inhibitor; Immune response; Immune system; Immunofluorescence Immunologic; Knowledge; Laboratories; Libraries; Ligands; Maps; Measures; Mechanics; Mediating; Medicine; Methods; Molecular; Molecular Probes; Nature; Nobel Prize; Optics; Physiology; Play; Positioning Attribute; Proteins; Recombinants; Regulation; Research; Rest; Role; Rupture; Scanning; Signal Transduction; Site-Directed Mutagenesis; Surface; Surveys; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Tissues; adaptive immunity; base; cancer cell; cancer immunotherapy; cancer therapy; career; checkpoint receptors; design; experience; experimental study; fluorophore; force sensor; immune checkpoint; immunoengineering; insight; mechanical force; molecular mechanics; mutant; neoplastic cell; novel; pathogen; prevent; programmed cell death ligand 1; receptor; skills; therapy design; tool; tumor; tumor-immune system interactions

PROJECT SUMMARY/ABSTRACT T cells survey the body for potential threats, including tumor cells, by probing their environment through physical interactions. During these physical interactions, molecular forces generated by the T cell receptor (TCR) “pulling” on potential threats help discriminate antigenic tissue from self-tissue by strengthening and prolonging TCR bonds, thus causing an enhanced T cell activation signal. This is an intriguing concept, as a force pulling on a molecular bond would be thought to cause rapid bond dissociation and loss of signaling. As this mechanical force is critical for T cell activation and function, the role of molecular forces within the immune system should be explored in other TCR co-receptor interactions as well. This includes the interaction between programmed death receptor 1 (PD1) on T cells and programmed cell death ligand 1/2 (PDL1/2) on tumor cells; an interaction that is revolutionizing cancer treatment but is currently still poorly understood. Specifically, it has been observed that when an active T cell physically crawls on and engages a tumor cell expressing PDL1 or PDL2, the T cell shuts down and loses its effector functions due to PD1 signaling, but through an unknown mechanism. As PD1 closely associates with the TCR, we hypothesize that the PD1 receptor is also force-regulated, and that molecular-level mechanical forces are also critical to PD1 signaling. The goal of our research is to determine the mechanistic role of mechanical forces in PD1 signaling, which we will accomplish through the following three aims. Aim 1. Create and characterize recombinant PDL1/PDL2 force sensors to quantify PD1 forces in cytotoxic T cells. Using molecular probes previously developed in our lab, we will design PDL1/2 force sensors that consist of a hybridized DNA duplex functionalized with a fluorophore-quencher pair. Upon force application by the PD1 receptor, the DNA duplex is mechanically unfolded, causing separation of the fluorophore and quencher. This increase in fluorescence can be mapped and quantified using conventional fluorescence microscopy, as the DNA duplex can be tuned to display specific forces exerted by the receptors. The results of this study will allow us to obtain the first maps of PD1 molecular forces in T cells. Aim 2. Determine the amino acids that contribute to the mechanical stability of the PD1-PDL1/2 complex. We will use site-directed mutagenesis to generate a mutant library of PDL1 and PDL2 to determine which amino acids contribute to molecular forces. Aim 3. Determine the role of PD1 molecular forces in T cell signaling. We will mechanically cap the forces the PD1 receptor can transmit and relate this information to intracellular signaling. The results of these studies will establish the role of PD1 mechanics in T cell signaling and will provide insight to the molecular events occurring during the physical “kiss of death.” Understanding the paradigms of mechanical forces in T cell biology and tumor recognition will greatly expand our knowledge of tumor-immune system interactions and will open doors to new avenues of cancer immunotherapies.

项目总结/摘要 T细胞通过物理探测它们的环境来调查身体的潜在威胁，包括肿瘤细胞， 交互.在这些物理相互作用中，T细胞受体（TCR）“拉动” 通过加强和延长TCR， 结合，从而引起增强的T细胞活化信号。这是一个有趣的概念，作为一种力量， 分子键被认为会导致键的快速解离和信号的丢失。由于这种机械 力对于T细胞活化和功能至关重要，分子力在免疫系统中的作用应该 也可以在其他TCR共受体相互作用中进行探索。这包括编程之间的相互作用 T细胞上的死亡受体1（PD 1）和肿瘤细胞上的程序性细胞死亡配体1/2（PDL 1/2）; 这是癌症治疗的革命，但目前仍然知之甚少。具体来说，据观察， 当活性T细胞在表达PDL 1或PDL 2的肿瘤细胞上物理爬行并接合时， 由于PD 1信号传导，但通过未知的机制关闭并失去其效应器功能。如pd 1 与TCR密切相关，我们假设PD 1受体也是力调节的， 分子水平的机械力对于PD 1信号传导也是至关重要的。我们研究的目的是确定 机械力在PD 1信号传导中的机械作用，我们将通过以下三个方面来实现 目标。目标1。创建并表征重组PDL 1/PDL 2力传感器，以量化细胞毒性中的PD 1力 T细胞。使用我们实验室以前开发的分子探针，我们将设计PDL 1/2力传感器， 用荧光团-猝灭剂对功能化的杂交DNA双链体。PD 1施力后 当DNA双链体与受体结合时，DNA双链体被机械解折叠，导致荧光团和猝灭剂分离。这 荧光的增加可以使用常规荧光显微镜进行绘图和定量，因为 DNA双链体可以被调整以显示由受体施加的特定力。这项研究的结果将使 我们获得了T细胞中PD 1分子力的第一张图。目标2.确定氨基酸， PD 1-PDL 1/2复合物的机械稳定性。我们将使用定点突变来产生一个 PDL 1和PDL 2的突变体库，以确定哪些氨基酸对分子力有贡献。目标3。 确定PD 1分子力在T细胞信号传导中的作用。我们将机械地限制PD 1 受体可以传递该信息并将其与细胞内信号传导联系起来。这些研究的结果将 建立PD 1机制在T细胞信号传导中的作用，并将为发生的分子事件提供见解 在肉体的“死亡之吻”理解T细胞生物学和肿瘤中的机械力范式 认识到这一点将极大地扩展我们对肿瘤-免疫系统相互作用的认识，并将为新的免疫系统打开大门。 癌症免疫疗法的途径。