Differential Analysis Of T-Cell Activation By A Library Of Membrane-Tethered, Monovalent, Fab'-Based Synthetic T-Cell Receptor Agonists

T cells mediate adaptive immunity by identifying pathogenic peptide-major histocompatibility complex (pMHC) molecules presented on the surface of antigen presenting cells (APCs). The T cell-APC junction, where T-cell receptors (TCRs) bind pMHC, is necessary for productive TCR triggering; T cells activate in response to single-molecule densities of membrane-bound agonist pMHC, but they do not respond to pMHC in solution. Crosslinked pMHC or multivalent antibodies can be used to activate T cells from solution, but these widely used strategies produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a library of strictly monovalent Fab’ fragments that bind three distinct epitopes on the TCR complex and activate T cells when coupled to a supported lipid bilayer via DNA complementation. Like pMHC, these synthetic Fab’-based ligands are inactive from solution. I will discuss the differential activation profiles of T cells in response to the library of ligands and the insights these data provide about the physiochemical and mechanical requirements for productive TCR triggering. The intrinsically modular nature of the hybrid Fab’-DNA ligands facilitates control of numerous physical aspects of the TCR triggering process to explore T-cell activation by synthetic ligands. Notably, the Fab's used can activate polyclonal populations of T cells, and so they can be used in studies for which T-cell activation by pMHC is not possible. These studies additionally have potential to inform the design of cancer immunotherapies such as bispecific T cell engagers (BiTEs), which are structurally similar to the synthetic Fab’ ligands.