UC Berkeley

T-cell receptors (TCRs) recognize sparse pathogenic peptides (p) displayed in major histocompatibility complex (MHC) proteins on the surface of antigen presenting cells (APCs). Wide natural diversity of TCR clonotypes within an individual enables a robust immune response but complicates efforts to study and therapeutically control T cell activation because the pMHC ligands for almost all natural TCRs are not known. Antibodies are widely used to universally activate T cells, but unlike monovalent pMHC, antibodies activate T cells from solution by inducing extensive TCR crosslinking and have high affinity for TCR. In contrast, the physical junction between the T cell and APC is required for productive T cell activation in response a few tens of monovalent, membrane-bound pMHC that bind TCR relatively weakly. These differences suggest that antibodies short-circuit native T cell signal integration processes that confer single molecule sensitivity. We have engineered a class of membrane-associated, synthetic TCR agonists that mimic biophysical properties of native pMHC in order to activate polyclonal T cells in a physiologically-relevant manner. The modular ligand design – an anti-TCR Fab’ fragment conjugated to a DNA oligonucleotide – enables experimental control over physical parameters of ligand:TCR interactions including binding epitope, ligand geometry, and affinity. We find that, when presented on a supported lipid bilayer, high-affinity Fab’-DNA ligands activate T cells with similar potency as strong pMHC agonists, regardless of which epitope they bind. However, a short intermembrane space at binding events is necessary to maintain the native mechanical TCR triggering process. Notably, like pMHC, Fab’-DNA must be tethered to an opposing surface in order to trigger TCR; it is inactive from solution. By tuning Fab’-DNA binding kinetics to match naturally low pMHC:TCR affinities, we find roles for both on- and off-rates in coordinating the T cell activation response. The results obtained from these studies can aide in defining design principles for effective T cell therapies. Moreover, the ability of Fab’-DNA ligands to trigger TCRs in polyclonal T cells opens avenues to quantitate signal integration in human T cell populations using a membrane-associated, monovalent TCR agonist.