Mechano-ID for tagging immune cells

用于标记免疫细胞的 Mechano-ID

• 批准号: 10608815
• 负责人: Khalid S Salaita
• 金额: $ 25.45万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608815
• 关键词: Adaptive Immune System; Address; Adult; Affinity; Amino Acids; Antigens; Area; Autoimmune Diseases; Beds; Binding; Binding Sites; Biological Assay; Biomechanics; Biotin; Cancerous; Cell surface; Cells; Cellular biology; Clone Cells; Complex; DNA; DNA Probes; Data; Eligibility Determination; Enzymes; Event; FDA approved; Flow Cytometry; Genotype; Geometry; Goals; Human; Human body; Immobilization; Immune; Immune response; Immunology; Label; Ligands; Ligase; Link; Major Histocompatibility Complex; Malignant Neoplasms; Maps; 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; 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; meter; molecular mechanics; mutant; nanometer; neoantigens; nucleic acid stability; particle; 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

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.

项目总结/摘要 这项提案的长期目标是开发新的工具来研究T细胞如何防御病原体 根除我们体内的癌细胞~106-108个独特的T细胞克隆在成人体内循环 寻找在其他细胞表面存在外源肽片段的证据。一旦T细胞遇到目标 细胞与外来或突变肽，它启动激活机制，释放强大的免疫反应。 T细胞活化的第一步涉及T细胞受体（TCR）和短链间的识别。 由主要组织相容性复合体（pMHC）蛋白呈递的肽（8-11个氨基酸）。因为T细胞 是高度迁移的，当T细胞物理接触靶细胞时发生抗原识别， 令人惊讶的是，TCR-pMHC复合物经历影响抗原效力的分子力。的确，我们的 实验室和其他人已经表明TCR活化依赖于pN力传递到其同源pMHC。电流 鉴定抗原肽的筛选技术测量亲和力而不考虑机械力， 因此不能很好地预测抗原效力。为了解决这个问题，我们将专门开发mechano-ID， 基于通过TCR-pMHC复合物传递的机械力的大小来标记T细胞。机械- ID集成了PI开创的邻近标记与分子张力探针的进展。根本 这一建议背后的原理是这样一个概念，即一个明确的TCR力展开一个DNA发夹，暴露出一个DNA发夹。 募集酶的隐蔽结合位点。这些酶，反过来，将产生反应性物种，如苯氧基 在~20 nm半径内共价标记蛋白质的自由基。因此，机械ID检测通电的生物力学 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。初步数据显示， 机械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 和对TCR-pMHC机械事件的力诱导标记的特异性。试剂等参数 将研究核酸探针的浓度、时间和机械稳定性。测定 将被集成到一个球形珠平台，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。SIINFEKL pMHC抗原的改变的肽文库将 以建立抗原效力和机械ID信号之间的直接相关性。嵌合抗原 将测试CAR受体T细胞，以进一步证明癌症免疫治疗领域的机械ID。的 这一提议的结果是一种创新的方法，允许人们基于机械表型标记细胞 - 从而打开了将基因型与机械型联系起来的大门，并使“机械组学”领域成为可能。最后， 我们注意到，mechano-ID超越了T细胞生物学，将广泛用于几乎任何 机械传导过程