Controllable Rigidity Surfaces for T Cell Mechanobiology

 描述：机械力在引导T细胞激活和随后的功能方面发挥着越来越重要的作用。我们最近发现，T细胞对呈现CD3和CD28激活配体的底物的刚性很敏感。具体地说，随着材料硬度的增加，小鼠原代CD4+T细胞在呈现CD3和CD28抗体的表面上表现出更高的活性。虽然在其他细胞系统中已经建立，主要是在整合素-ECM和钙粘附素-钙粘附素相互作用的背景下，但T细胞通过CD3/TCR和CD28的机制尚不清楚。解决这一知识差距将提供一个新的系统，在此系统上可以测试其他系统中开发的机械传感概念，并为T细胞生理学和免疫治疗的先进工具提供新的见解。这项拟议的研究试图解决这样一个问题，即T细胞与底物的相互作用像其他细胞一样，发生在多个阶段。在每个阶段控制底物刚性的能力将揭示每个阶段在机械传感中所起的作用，并将后续研究集中在负责这种能力的分子途径上。这种知识可以为复制或改进使用化学或生物制剂的软基质的益处提供新的目标。为了实现这一目标，我们提出了一种磁驱动系统，它可以按需、可逆和可重复地控制附着细胞的机械刚度。我们首先专注于这一新系统的开发和验证，它是对用于牵引力显微镜的弹性柱阵列技术的改编。该系统随后被用来独立评估T细胞在早期细胞扩散和持续收缩期间的机械感觉。在一个互补的方向上，我们还将评估CD3/TCR信号作为底物刚性的函数的动力学，底物刚性是T细胞机械传感发展模型的核心概念。这项拟议研究的成功完成将是T细胞机械生物学这一新兴跨学科领域的一项关键进展。此外，预计拟议的系统将立即和直接适用于其他蜂窝系统。

 DESCRIPTION: Mechanical forces play increasingly recognized roles in directing T cell activation and subsequent function. We recently discovered that T cells are sensitive to the rigidity of an underlying substrate presenting activating ligands to CD3 and CD28. Specifically, primary mouse CD4+ T cells exhibited increased activation on surfaces presenting antibodies to CD3 and CD28 as material rigidity increased. While established in other cell systems, predominantly in the context of integrin-ECM and cadherin-cadherin interactions, mechanosensing by T cells through the CD3/TCR and CD28 is not well understood. Addressing this gap in knowledge would provide a new system upon which the mechanosensing concepts developed in other systems could be tested, as well as provide new insight into T cell physiology and advanced tools for immunotherapy. The proposed study seeks to address the issue that T cell-substrate interaction, like other cells, occurs in multiple stages. The ability t control the rigidity of the substrate during each stage would reveal the role each one has in mechanosensing, focusing subsequent studies into the molecular pathways responsible for this ability. Such knowledge could provide new targets for replicating or improving upon the benefits of soft substrates, but using chemical or biological agents. Towards this goal, we propose a magnetically actuated system that provides on-demand, reversible, and repeatable control over the mechanical stiffness presented to an adherent cell. We first focus on development and validation of this new system, which is an adaptation of the elastomer pillar array technology used for traction force microscopy. This system is then used to independently assess T cell mechanosensing during early cell spreading and sustained contraction. In a complementary direction, we will also assess dynamics of CD3/TCR signaling as a function of substrate rigidity, a core concept in a developing model of T cell mechanosensing. Successful completion of the proposed study will be a key advance in the emerging interdisciplinary field of T cell mechanobiology. In addition, it is expected that the proposed system will be immediately and directly applicable towards other cellular systems.