Mechanoregulation of cytotoxic lymphocyte function

摘要 免疫细胞通过称为免疫突触的动态细胞-细胞连接进行交流。尽管 这些突触的生化特性已经被广泛研究，但我们对它们的机制知之甚少。 以及这些活动对免疫功能的贡献。我们使用细胞毒性淋巴细胞作为模型 研究突触力量的来源和目的的系统。细胞毒性淋巴细胞与病原体和 通过与受感染或转化的靶细胞形成免疫突触，然后分泌有毒物质而引发癌症 颗粒酶和穿孔蛋白进入细胞间隙。我们的实验室和其他人的工作 提示机械力在触发淋巴细胞激活和增强 杀戮反应的效率。我们建议的研究分为两个具体目标，将针对 在这些背景下，突触力量的功能相关性和分子基础。目标1建立在初步数据的基础上 表明细胞毒性T淋巴细胞(CTL)和自然杀伤(NK)细胞检测到 靶细胞，并使用这种机械传感能力来识别和摧毁侵袭转移的癌细胞 利基市场。为了确定这种机械形式的免疫监控是否以及如何 机械监视，塑造体内抗肿瘤免疫，我们将应用多种小鼠转移模型， 原子力显微镜，以及临床免疫治疗试验的分析。目标2是以先前工作为前提的 提示CTL利用机械力增强分泌的穿孔素的成孔活性。这一种 物理化学协同作用需要机械输出和分泌输出之间的高度协调 在突触内，但淋巴细胞如何实现这种耦合仍不清楚。我们已经开发出一种 基于成像的生物物理方法来解决这个问题，这将使我们能够建立 细胞毒性的机械力化学编排具有前所未有的精确度。我们建议的研究将 采用技术创新的方法，包括对淋巴细胞力施加于 微米级的生物物理探测器。他们还将介绍一些创新的概念，包括 细胞的刚性可以触发免疫监视，以及淋巴细胞亚群采用不同的 用于指定其效应器响应的机械签名。我们的工作还将解决一个简单但技术上 这个令人困扰的问题已经制约了这个领域一段时间，即机械生物学原理 实际上会影响体内的免疫力。理解免疫突触功能的生物物理维度 可能会为临床上调节和评估淋巴细胞活性提供新的策略。 因此，这里描述的研究与美国国立卫生研究院的任务高度相关，因为它们将有助于 能够改善人类健康的知识进步。

Summary Immune cells communicate through dynamic cell-cell junctions known as immune synapses. Although the biochemical properties of these synapses have been studied extensively, we know little about their mechanical activities and how these activities contribute to immune function. We use cytotoxic lymphocytes as a model system to investigate the origins and purposes of synaptic force. Cytotoxic lymphocytes fight pathogens and cancer by forming an immune synapse with an infected or transformed target cell and then secreting toxic granzymes and the pore forming protein perforin into the intercellular space. Work from our lab and others suggests critical roles for mechanical forces both in triggering lymphocyte activation and in enhancing the efficiency of killing responses. Our proposed studies, which are divided into two Specific Aims, will address the functional relevance and molecular bases of synaptic force in these contexts. Aim 1 builds on preliminary data indicating that cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells detect the physical stiffening of target cells and use this mechanosensing capacity to identify and destroy cancer cells invading the metastatic niche. To determine if and how this mechanical form of immunosurveillance, which we call mechanosurveillance, shapes anti-tumor immunity in vivo, we will apply multiple murine metastasis models, atomic force microscopy, and analysis of clinical immunotherapy trials. Aim 2 is premised on prior work indicating that CTLs use mechanical force to potentiate the pore forming activity of secreted perforin. This sort of physicochemical synergy demands a high degree of coordination between mechanical and secretory output within the synapse, but how lymphocytes achieve this coupling remains unknown. We have developed an imaging-based biophysical approach to address this problem, which will enable us to establish the mechanochemical choreography of cytotoxicity with unprecedented precision. Our proposed studies will employ technically innovative methods, including super-resolution imaging of lymphocyte force exertion against micron-scale biophysical probes. They will also introduce a number of innovative concepts, including the idea that cellular rigidity can trigger immunosurveillance and the idea that lymphocyte subsets employ distinct mechanical signatures to specify their effector responses. Our work will also address a simple but technically vexing issue that has constrained the field for some time, namely whether mechanobiological principles actually influence immunity in vivo. Understanding the biophysical dimensions of immune synapse function could potentially reveal new strategies for the modulation and assessment of lymphocyte activity in the clinic. As such, the studies described herein are highly relevant to the NIH mission in that they will contribute to the advancement of knowledge that could improve human health.