Mechano-ID for tagging immune cells

项目摘要/摘要 这项提议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 并根除体内的癌细胞。大约106-108个独特的T细胞克隆在成人体内循环 寻找其他细胞表面存在异源多肽碎片的证据。一旦T细胞遇到靶细胞 当细胞中含有外源或突变的多肽时，它会启动激活机制，从而释放出强大的免疫反应。 T细胞活化的第一步涉及T细胞受体(TCR)和短链T细胞之间的识别 主要组织相容性复合体(PMHC)蛋白提供的多肽(8-11个氨基酸)。因为T细胞 是高度迁移的，当T细胞与靶细胞物理接触时，会发生抗原识别，它不是 令人惊讶的是，TCR-pMHC复合体经历了影响抗原效力的分子力。的确，我们的 Lab和其他人已经证明TCR的激活依赖于PN力传递到其同源的pMHC。当前 识别抗原肽的筛选技术测量亲和力而不考虑机械力和 因此不能很好地预测抗原的效力。为了解决这个问题，我们将开发机械ID来具体 根据通过TCR-pMHC复合体传递的机械力的大小来标记T细胞。机械式- ID集成了PI首创的分子张力探头在邻近标记方面的进步。最基本的 这一提议背后的原理是这样一个概念，即一个定义明确的TCR力展开DNA发夹，暴露出一个 招募酶的神秘结合部位。这些酶反过来会产生活性物质，如苯氧基。 在~20纳米半径内共价标记蛋白质的自由基。因此，机械ID检测到了充满能量的生物力学 力和附近的相互作用体而不是静态的蛋白质-蛋白质相互作用。初步数据显示， 机械力识别技术应用于原代T细胞的可行性研究在目标1中，我们将优化机械ID以提高其产量 以及对TCR-pMHC机械事件的力诱导标记的特异性。试剂等参数 将对核酸探针的浓度、时间和机械稳定性进行调查。化验结果 将集成到球形微珠平台中，以提高产量。在目标2中，我们将演示机械ID 筛选T细胞与抗原的特异性相互作用。SIINFEKL pMHC抗原的改变的多肽文库将 进行测试，以确定抗原效力和机械ID信号之间的直接关联。嵌合抗原 受体(CAR)T细胞将进行测试，以进一步证明在癌症免疫治疗领域的机械性ID。这个 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来标记细胞 -从而打开了将基因型与机械型联系起来的大门，并使该领域能够进行“机械组学”。最后， 我们注意到，机械ID超越了T细胞生物学，并将在几乎所有 机械转导过程。

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.