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: 10311048
• 负责人: Anna Kellner
• 金额: $ 4.68万
• 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/10311048
• 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.

项目摘要/摘要 T细胞通过物理方式探测自身环境，从而调查身体是否存在潜在威胁，包括肿瘤细胞。 互动。在这些物理相互作用中，T细胞受体(TCR)产生的分子作用力“拉” 通过加强和延长TCR有助于区分抗原组织和自身组织的潜在威胁 结合，从而导致增强的T细胞激活信号。这是一个耐人寻味的概念，因为拉力 分子键被认为会导致键的快速解离和信号的丢失。就像这台机械 力对T细胞的激活和功能至关重要，免疫系统内的分子力的作用应该 在其他TCR共受体相互作用中也被探索。这包括编程之间的交互 T细胞上的死亡受体1和肿瘤细胞上的程序性细胞死亡配体1/2；相互作用 这是癌症治疗的革命性进展，但目前人们对此仍知之甚少。具体地说，已经观察到 当活跃的T细胞在身体上爬行并与表达PDL1或PDL2的肿瘤细胞接触时，T细胞 由于PD1信号而关闭并失去其效应器功能，但机制未知。AS PD1 与TCR密切相关，我们假设PD1受体也是受力调节的，并且 分子水平的机械力对PD1信号也是至关重要的。我们研究的目标是确定 机械力在PD1信令中的机械作用，我们将通过以下三个方面来完成 目标。目的1.建立和鉴定重组PDL1/PDL2力传感器以定量细胞毒中的PD1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PDL1/2力传感器， 利用荧光团-猝灭剂对功能化的杂交DNA双链。在PD1强制施加时 在受体的作用下，DNA双链被机械地展开，导致荧光团和猝灭剂分离。这 荧光的增加可以使用传统的荧光显微镜来绘制和量化，因为 DNA双链可以被调节以显示受体施加的特异力。这项研究的结果将允许 美国首次获得T细胞中PD1分子作用力的图谱。目标2.确定影响人体健康的氨基酸 对PD1-PDL1/2复合体的机械稳定性的影响。我们将使用定点突变来产生一个 PDL1和PDL2的突变文库，以确定哪些氨基酸对分子作用力有贡献。目标3. 确定PD1分子作用力在T细胞信号传导中的作用。我们将机械地限制PD1的力量 受体可以传递这一信息，并将其与细胞内信号联系起来。这些研究的结果将 建立PD1机制在T细胞信号转导中的作用，并将提供对发生的分子事件的洞察 在肉体的“死亡之吻”中。理解T细胞生物学和肿瘤中的机械力范式 认知将极大地扩展我们对肿瘤-免疫系统相互作用的知识，并将打开新的 癌症免疫疗法的途径。

项目成果

Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.

• DOI: 10.1038/s41592-020-0929-2
• 发表时间: 2020-10
• 期刊: Nature methods
• 影响因子: 48
• 作者: Brockman JM;Su H;Blanchard AT;Duan Y;Meyer T;Quach ME;Glazier R;Bazrafshan A;Bender RL;Kellner AV;Ogasawara H;Ma R;Schueder F;Petrich BG;Jungmann R;Li R;Mattheyses AL;Ke Y;Salaita K
• 通讯作者: Salaita K

Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope.

• DOI: 10.1038/s41467-021-24602-x
• 发表时间: 2021-08-03
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Blanchard A;Combs JD;Brockman JM;Kellner AV;Glazier R;Su H;Bender RL;Bazrafshan AS;Chen W;Quach ME;Li R;Mattheyses AL;Salaita K
• 通讯作者: Salaita K

The magnitude of LFA-1/ICAM-1 forces fine-tune TCR-triggered T cell activation.

• DOI: 10.1126/sciadv.abg4485
• 发表时间: 2022-02-25
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Ma VP;Hu Y;Kellner AV;Brockman JM;Velusamy A;Blanchard AT;Evavold BD;Alon R;Salaita K
• 通讯作者: Salaita K

Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency.

• DOI: 10.1021/acs.biomac.0c00264
• 发表时间: 2020-07-13
• 期刊: Biomacromolecules
• 影响因子: 6.2
• 作者: Perdue LA;Do P;David C;Chyong A;Kellner AV;Ruggieri A;Kim HR;Salaita K;Lesinski GB;Porter CC;Dreaden EC
• 通讯作者: Dreaden EC