Pattern and Precision: DNA-Based Mapping of Spatial Rules for T Cell Activation

The nanoscale spatial arrangement of T cell receptors (TCRs) ligands critically influences their activation potential in CD8+ T cells, yet a comprehensive understanding of the molecular landscape induced by engagement with native peptide-MHC class I (pMHC-I) remains incomplete. Using DNA origami as platform for multivalent engineering, we precisely organize pMHC-I molecules into defined spatial configurations to systematically investigate the roles of valency, inter-ligand spacing, geometric pattern, and molecular flexibility in regulating T cell function. We find that reducing inter-ligand spacing to [~]7.5 nm enhances T cell activation by up to eightfold compared to wider spacing ([~]22.5 nm), and that as few as six pMHC-I molecules are sufficient to elicit a robust response. Notably, the geometry of pMHC-I presentation emerges as a key determinant of signaling strength, with hexagonal arrangements proving most effective. In contrast, the introduction of flexible linkers into pMHC-I impairs TCR triggering. Together, these findings define spatial parameters that govern pMHC-I-TCR interactions at the T cell interface and provide design principles for engineering next-generation T cell-based immunotherapies.