Mechano-ID for tagging immune cells

用于标记免疫细胞的 Mechano-ID

基本信息

批准号：
10664365
负责人：
Khalid S Salaita
金额：
$ 19.01万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-16 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10664365
关键词：
Adaptive Immune System Address Adult Affinity Amino Acids Antigens Area Autoimmune Diseases Beds Binding Sites Biological Assay Biomechanics Biotin Cancerous Cell surface Cells Cellular biology Clone Cells Complex DNA DNA Probes Data Eligibility Determination Enzymes Event Flow Cytometry Genotype Geometry Goals Human body Immobilization Immune Immune response Immunology Intuition Label Ligands Ligase Link Major Histocompatibility Complex Malignant Neoplasms Measures Mechanics Methods Modeling Molecular Mutate Nucleic Acid Probes Nucleic Acids Oligonucleotides Outcome Ovalbumin Pathway interactions Peptide Fragments Peptide Library Peptides Peroxidases Phenotype Population Process Proteins Proteomics Radial Reaction Reagent Receptor Activation Receptor Signaling Research Scanning Screening procedure Signal Transduction Specificity Spectrum Analysis Surface T-Cell Activation T-Cell Receptor T-Lymphocyte Techniques Technology Testing Transcend Transgenic Organisms Virus Work antigen-specific T cells base cancer cell cancer immunotherapy chimeric antigen receptor chimeric antigen receptor T cells design experience experimental study fighting high throughput screening immunoengineering improved innovation interest mechanical force mechanotransduction molecular mechanics mutant nanometer neoantigens nucleic acid stability pathogen phenoxy radical protein aminoacid sequence protein complex protein protein interaction public health relevance receptor receptor binding recruit screening single molecule small molecule tool transmission process virtual

项目摘要

Project Summary/Abstract The long-term goal of this proposal is to develop new tools to study how T cells defend against pathogens and eradicate cancerous cells within our bodies. ~106-108 unique T cell clones circulate in the adult human body seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, it initiates activation mechanisms that unleash a potent immune response. The very first step in T cell activation involves recognition between the T cell receptor (TCR) and the short peptides (8-11 amino acids) presented by the major histocompatibility complex (pMHC) protein. Because T cells are highly migratory and antigen recognition occurs when the T cell physically contacts a target cell, it is no surprise that the TCR-pMHC complex experiences molecular forces that influence antigen potency. Indeed, our lab and others have shown that TCR activation relies on pN force transmission to its cognate pMHC. Current screening technologies to identify antigenic peptides measure affinity without regard to mechanical force and are thus poor predictors of antigen potency. To address this problem, we will develop mechano-ID to specifically tag T cells based on the magnitude of mechanical forces transmitted through the TCR-pMHC complex. Mechano- ID integrates advances in proximity tagging with molecular tension probes pioneered by the PI. The fundamental principle behind this proposal is the concept that a well-defined TCR force unfolds a DNA hairpin, exposing a cryptic binding site that recruits enzymes. These enzymes, in turn, will generate reactive species, such phenoxy radicals that covalently tag proteins within a ~20 nm radius. Thus, mechano-ID detects energized biomechanical forces and nearby interactome rather than static protein-protein interactions. Preliminary data shows the feasibility of mechano-ID applied to primary T cells. In Aim 1, we will optimize mechano-ID to enhance its yield and specificity for force-induced tagging of TCR-pMHC mechanical events. Parameters such as reagent concentrations, timing, and the mechanical stability of the nucleic acid probes will be investigated. The assay will be integrated into a spherical bead platform to boost yield. In Aim 2, we will demonstrate mechano-ID for screening of T cell-antigen specific interactions. The altered peptide library of the SIINFEKL pMHC antigen will be tested to establish a direct correlation between antigen potency and mechano-ID signal. Chimeric antigen receptor (CAR) T cells will tested to further demonstrate mechano-ID in the area of cancer immunotherapy. The outcome of this proposal is an innovative method that allows one to tag cells based on mechanical phenotypes –thus opening the door to linking genotype to the mechanotype and enabling the field “mechanomics”. Finally, we note that mechano-ID transcends T cell biology and will be broadly useful in the study of virtually any mechanotransduction process.

项目概要/摘要该提案的长期目标是开发新工具来研究 T 细胞如何防御病原体并根除成人体内约 106-108 个独特的 T 细胞克隆。一旦 T 细胞遇到目标，就寻找其他细胞表面存在外源肽片段的证据。当细胞含有外来或突变肽时，它会启动激活机制，释放有效的免疫反应。 T 细胞激活的第一步涉及 T 细胞受体 (TCR) 和短链之间的识别。由主要组织相容性复合物 (pMHC) 蛋白呈递的肽（8-11 个氨基酸），因为 T 细胞。具有高度迁移性，并且当 T 细胞物理接触靶细胞时会发生抗原识别，因此不存在令人惊讶的是，TCR-pMHC 复合物经历了影响抗原效力的分子力。实验室和其他人已经表明，TCR 激活依赖于 pN 力传递到其同源 pMHC 电流。识别抗原肽的筛选技术可测量亲和力，而不考虑机械力和因此，它们对抗原效力的预测效果很差。为了解决这个问题，我们将专门开发机械 ID。根据通过 TCR-pMHC 复合物传递的机械力的大小来标记 T 细胞。 ID 将邻近标记技术的进步与 PI 首创的分子张力探针相结合。这一提议背后的概念是，一个原则明确的 TCR 力展开 DNA 发夹，暴露出招募酶的神秘结合位点，这些酶反过来会产生反应性物质，例如苯氧基。自由基在约 20 nm 半径内共价标记蛋白质，因此，mechano-ID 可以检测通电的生物力学。初步数据显示了力和附近的相互作用组，而不是静态的蛋白质-蛋白质相互作用。 mechano-ID 应用于原代 T 细胞的可行性在目标 1 中，我们将优化 mechano-ID 以提高其产量。以及 TCR-pMHC 机械事件参数（例如试剂）的力诱导标记的特异性。将研究核酸探针的浓度、时间和机械稳定性。将被集成到球形珠平台中以提高产量。在目标 2 中，我们将演示机械 ID。筛选 T 细胞-抗原特异性相互作用。建立测试抗原效力和机械ID信号之间的直接相关性。受体（CAR）T细胞将进行测试，以进一步证明机械ID在癌症免疫治疗领域的应用。该提案的成果是一种创新方法，允许人们根据机械表型来标记细胞 –从而打开了将基因型与机械型联系起来并实现“机械组学”领域的大门。我们注意到，mechano-ID 超越了 T 细胞生物学，并且将广泛用于几乎任何领域的研究力传导过程。