A FRET-based sensor for probing forces exerted by single T cell receptors on their ligands

T cells employ T cell receptor (TCR) complexes to probe antigenic peptide presented on the surface of antigen-presenting cells (APCs). This interaction is of fundamental importance for the adaptive immune system and takes place in a highly structured interface between the cells, which is termed immunological synapse. In a remarkably sensitive and selective process, T cells are able to distinguish between peptides originating from harmful and harmless substances and trigger an appropriate immunological response. Despite its importance, the mechanisms behind the process remain elusive. Prompted by evidence suggesting that mechanical forces play a key role, we developed a FRET-based molecular force sensor to be inserted into immunological synapses. The sensor is composed of an anchor unit, a spring-like peptide derived from spider silk, and a TCR-binding ligand. The peptide, acting as a molecular spring, has donor and acceptor fluorophores attached near the opposing termini. Force-induced changes in the end-to-end distance can therefore be monitored via the FRET efficiency. As surrogate APCs, we functionalized planar lipid bilayers (PLBs) with force sensors featuring antibody-derived single chain fragments as the TCR-binding ligands. After seeding T cells onto the PLBs, forces arising in single receptor-ligand interactions were recorded employing a combined single molecule FRET and tracking approach using TIRF microscopy. On gel-phase PLBs, where the sensors were virtually immobile, we observed forces of 4-6 pN. In experiments on liquid PLBs, where the sensors could diffuse freely, only weak pulling events were found. This suggests that forces occur primarily parallel to the cell membrane.