DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation

Abstract

Significance It has been proposed that the spatial arrangement of ligands plays a key role in regulating downstream intracellular signals. Because of methodological limitations in precise ligand patterning, however, the relationship between spatial configuration of clusters and signaling dynamics remains poorly understood. By developing a DNA-based molecular “pegboard” for ligand patterning, we demonstrated that the nanometer arrangement of ligands plays significant roles in modulating signal transduction in T cells. Ligand clustering not only affects the triggering sensitivity but also determines the temporal dynamics of the intracellular signaling response. Our approach is highly translatable for studying various signaling pathways, and our results provide insights into biomolecular engineering for therapeutic uses. Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami–based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.