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.

Project Narrative Adoptive T cell therapy has demonstrated tremendous potential for cancer treatment, but an incomplete understanding of T cell signaling makes manufacturing of these therapies expensive and inefficient. Because T cell signaling has been shown to be regulated by the nanoscale spatial organization of signaling structures, I propose building a DNA origami interface to present 3D patterns of signaling molecules to T cells with nanometer- scale precision. This project is relevant to public health as it will provide new insights into how T cell signaling is regulated by spatial arrangement and provide both knowledge and a platform for efficient manufacturing of therapeutic cell products for cancer immunotherapy.