Defining the role of ligand spatial organization in T cell signaling with DNA origami

项目摘要 了解和操纵细胞信号传递过程对于过继细胞疗法(ACT)至关重要，ACT 在治疗癌症和糖尿病等疾病方面显示出巨大的希望。中国当前面临的许多挑战 制造这些治疗药物与我们缺乏对体外T细胞激活的控制有关。尽管 在理解细胞外信号信号如何影响细胞内方面已经取得了巨大的进展 但是，我们对管理这些进程的详细机制的理解是不完整的。越来越多的证据 提示细胞信号是由细胞表面信号结构的物理排列来调节的 细胞。然而，确定信号结构的空间排列如何指导细胞行为是非常重要的 由于这些结构的纳米级尺寸低于传统光的分辨率限制，因此很难实现 显微镜。 这项研究将为阐明空间组织在T细胞调节中的作用提供关键信息。 以及测试我设计的新工具的可行性，以研究和操纵纳米级的结构。 这项研究的目的是确定信号分子的3D空间排列如何影响T细胞 行为。为此，我将把配体排列成纳米级的3D图案，然后将这些图案化的配体呈现给 T细胞和表征信号动力学，以及T细胞制造参数 细胞增殖率和IL-2分泌。这项工作的基本原理是通过定义配体之间的关系 排列和T细胞信号，我们将更好地理解信号分子是如何组织在 细胞表面调节细胞内途径，这将指导开发高效的优化试剂 体外T细胞活化。我将利用从纳米技术中汲取的综合方法来实现这一目标， 生物化学和细胞生物学。该项目将进行三个具体目标：1)确定关系 细胞外受体激酶动力学和3D刺激配体排列之间的关系，2)决定空间 抑制性受体对T细胞激活的依赖性，以及3)创建图案化的T细胞信号试剂 可以触发体外初级T细胞的激活。我的项目很有创新性，因为它将使用DNA折纸作为一种 一种以单纳米分辨率构图配体的方法，其精度只有先进的 纯净室技术，如电子束光刻。这个项目的意义在于定义了 信号分子的空间组织与细胞内通路的关系 为纳米免疫治疗试剂的研究奠定基础。这一知识将使我们能够更多地 深入了解T细胞活化和分化的机制，使高效和 有效制造治疗癌症、糖尿病和其他疾病的细胞疗法。

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 I have designed to study and manipulate structures on the nanoscale. The objective of this study is to determine how 3D spatial arrangements of signaling molecules affect T cell behavior. To do this, I will arrange ligands into nanoscale 3D patterns, then present these patterned ligands to T cells and characterize signaling dynamics, as well as T cell manufacturing parameters such as T cell proliferation rate and IL-2 secretion. The rationale for this work 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. I will accomplish this goal using an integrated approach drawing from nanotechnology, biochemistry, and cell biology. This project will conduct 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. My project is highly innovative because it will use DNA origami as a method to pattern ligands with single nanometer-resolution, a degree of precision rivaled only by advanced cleanroom techniques such as electron beam lithography. This project’s significance lies in defining 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.