Differential Roles of Kinetic On- and Off-Rates in T-Cell Receptor Signal Integration Revealed with a Modified Fab'-DNA Ligand

Abstract

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly-binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide-MHC (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed -TCR{beta} H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rate of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells (LAT), which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from synthetic ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient. STATEMENT OF SIGNIFICANCET cells read kinetic information from ligands binding to T-cell receptors (TCRs) to make cell fate decisions. Unique kinetic features of a modified Fab-DNA ligand enable direct visualization multiple TCR signal coordination through a nascent LAT condensation event. We further observed positive feedback through a kinetic on-rate enhancement in the growing LAT condensate. These observations help unify several seemingly disparate aspects of TCR signaling that have been debated in the literature. Furthermore, calibration of the Fab-DNA ligand against native agonist pMHC establishes a basis for quantitative analysis of TCR signaling in polyclonal T cell populations.