Probing mesoscale receptor organization in T cell signaling with DNA origami

项目摘要 理解和操纵细胞信号传导过程对于过继细胞疗法（ACT）至关重要， 在治疗癌症和糖尿病等疾病方面显示出巨大的前景。目前的许多挑战， 制造这些治疗剂与我们缺乏对离体T细胞活化的控制有关。虽然 在理解细胞外信号如何影响细胞内信号方面已经取得了巨大的进展， 国家，我们对这些过程的详细机制的理解是不完整的。安装 有证据表明，细胞信号传导是由信号传导结构的物理排列调节的。 细胞的表面。然而，确定信号结构的空间排列如何指导细胞行为， 由于这些结构的纳米级尺寸，这是非常困难的，这是低于传统的分辨率限制 光镜 本研究将为阐明T细胞空间结构的作用提供重要信息。 监管，以及测试新工具的可行性，以研究和操纵纳米结构。 我们的目标是确定信号分子的3D空间排列如何影响T细胞行为。做 因此，我们将使用DNA折纸纳米结构将配体排列成纳米级3D图案，然后呈现这些 模式化的T细胞配体和表征信号动力学，我们还将评估ACT相关参数 如T细胞增殖率和IL-2分泌。我们的基本原理是，通过定义 配体排列和T细胞信号传导，我们将更好地了解信号分子的组织 在细胞表面调节细胞内途径，这将指导优化试剂的开发， 有效的离体T细胞活化。该项目将利用纳米技术，生物化学和细胞， 实现三个具体目的：1）确定细胞外受体激酶动力学之间的关系 和三维刺激配体排列，2）确定T细胞上抑制性受体的空间依赖性 活化，和3）产生可触发离体原代T细胞活化的图案化T细胞信号传导试剂。 我们将定义信号分子的空间组织和细胞内的 途径，并建立纳米图案化免疫治疗试剂的基础。这些知识将 使我们能够更深入地了解T细胞活化和分化的机制， 用于癌症、糖尿病和其他疾病的细胞疗法的高效和有效的制造。

PROJECT SUMMARY Understanding and manipulating cell signaling processes is crucial for adoptive cell therapies (ACT), which show significant promise in treating diseases such as cancer and diabetes. Many of the current challenges in manufacturing these therapeutics are related to our lack of control over ex vivo T cell activation. Though tremendous progress has been made in understanding how extracellular signaling cues influence intracellular states, our understanding of detailed mechanisms governing these processes is incomplete. Mounting evidence suggests that cell signaling is regulated by the physical arrangement of signaling structures at the surface of cells. However, determining how the spatial arrangement of signaling structures guides cell behavior is very difficult due to the nanoscale size of these structures, which is below the resolution limit of traditional light microscopy. This study will provide crucial information towards elucidating the role of spatial organization in T cell regulation, as well as test the feasibility of novel tools to study and manipulate structures on the nanoscale. Our objective is to determine how 3D spatial arrangements of signaling molecules affect T cell behavior. To do so, we will use DNA origami nanostructures to arrange ligands into nanoscale 3D patterns, then present these patterned ligands to T cells and characterize signaling dynamics, we will also assess ACT-relevant parameters such as T cell proliferation rate and IL-2 secretion. Our rationale is that by defining the relationship between ligand arrangement and T cell signaling, we will better understand how the organization of signaling molecules at the cell surface regulates intracellular pathways, which will guide the development of optimized reagents for efficient ex vivo T cell activation. This project will leverage nanotechnology, biochemistry, and cell to accomplish three Specific Aims: 1) determine the relationship between extracellular receptor kinase dynamics and 3D stimulatory ligand arrangement, 2) determine the spatial dependence of inhibitory receptors on T cell activation, and 3) create patterned T cell signaling reagents that can trigger ex vivo primary T cell activation. We will define the relationship between the spatial organization of signaling molecules and intracellular pathways, and in establishing the foundation for nanopatterned immunotherapy reagents. This knowledge will allow us to more deeply understand the mechanisms underlying T cell activation and differentiation, enabling efficient and efficacious manufacturing of cell therapies for cancer, diabetes, and other diseases.