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

用 DNA 折纸定义配体空间组织在 T 细胞信号传导中的作用

• 批准号: 10680089
• 负责人: Konlin Shen
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10680089
• 关键词: 3-Dimensional; Address; Adoptive Cell Transfers; Affect; Antibodies; Antigen Presentation; Antigen-Presenting Cells; Autoimmune Diseases; Binding; Biochemistry; Biological Process; Cell Signaling Process; Cell Therapy; Cell model; Cell surface; Cells; Cellular biology; Cues; DNA; Dependence; Development; Diabetes Mellitus; Disease; Engineering; Exclusion; Foundations; Goals; Height; Human; Immune signaling; Immunotherapy; Interleukin-2; Knowledge; Lateral; Learning; Ligands; Linker DNA; Lupus; Malignant Neoplasms; Mediating; Methods; Nanotechnology; Nucleotides; Pathway interactions; Patients; Pattern; Peptide/MHC Complex; Phosphotransferases; Positioning Attribute; Process; Production; Public Health; Reagent; Research; Resolution; Resources; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Single-Stranded DNA; Structure; Surface; T cell regulation; T cell therapy; T-Cell Activation; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Techniques; Therapeutic; Work; cancer immunotherapy; cancer therapy; cell behavior; design; electron beam lithography; engineered T cells; extracellular; feasibility testing; immunological synapse; immunological synapse formation; innovation; insight; interest; light microscopy; manufacture; migration; nanometer resolution; nanopattern; nanoscale; novel; programmed cell death protein 1; receptor; scaffold; stoichiometry; success; tool

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.

项目摘要 理解和操纵细胞信号传导过程对于适应性细胞疗法（ACT）至关重要， 在治疗癌症和糖尿病等疾病方面表现出巨大的希望。当前许多挑战 制造这些治疗剂与我们缺乏对离体T细胞激活的控制有关。 在理解细胞外信号线索如何影响细胞内方面已经取得了巨大进步 州，我们对管理这些过程的详细机制的理解是不完整的。越来越多的证据 表明细胞信号传导受信号结构在表面的物理排列调节 细胞。但是，确定信号结构的空间排列如何指导细胞行为非常 由于这些结构的纳米级尺寸，难以低于传统光的分辨率极限 显微镜。 这项研究将提供至关重要的信息，以阐明空间组织在T细胞调节中的作用， 除了测试我旨在研究和操纵纳米级结构的新型工具的可行性。 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，我将将配体安排到纳米级3D图案中，然后将这些图案的配体呈现给 T细胞并表征信号传导动力学以及T细胞制造参数，例如T细胞 增殖率和IL-2分泌。这项工作的理由是，通过定义配体之间的关系 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如何在 细胞表面调节细胞内途径，这将指导优化试剂的开发以提高 离体T细胞激活。我将使用纳米技术的集成方法来实现此目标， 生物化学和细胞生物学。该项目将执行三个具体目标：1）确定关系 在细胞外受体激酶动力学和3D刺激配体排列之间，2）确定空间 抑制受体对T细胞激活的依赖性，3）创建图案化的T细胞信号试剂，这些试剂 可以触发离体初级T细胞激活。我的项目具有很高的创新性，因为它将使用DNA折纸作为一个 用单纳米分辨率进行配体模式配体的方法，仅由先进 洁净室技术，例如电子束岩性摄影。该项目的意义在于定义 信号分子和细胞内途径的空间组织之间的关系 建立纳米图案的免疫疗法试剂基础。这些知识将使我们能够更多 深入了解T细胞激活和分化的基础机制，从而有效和 有效生产用于癌症，糖尿病和其他疾病的细胞疗法。