Mechanistic Insights into T-Cell Antigen Recognition Through Single Molecule Microscopy

T-cell antigen recognition requires the formation of an immunological synapse, a transient interface between a T-lymphocyte and an Antigen Presenting Cell (APC). It is within this complex and dynamic bi-membranous contact where T-cell antigen receptors (TCRs) on the T-cell bind their nominal ligands, i.e., antigenic peptides embedded within major histocompatibility complex molecules (pMHC), on the APC. Productive TCR-engagement is key to initiating signaling pathways required for the activation of T-cell effector mechanisms, and hence much attention has been focused on the structural and biophysical properties of the binding partners involved. However, one central question remains unanswered: how can one explain the exquisite sensitivity and selectivity of T-cells towards antigen, which is ultimately based on short-lived TCR-pMHC interactions with moderate affinities? To approach this problem in a comprehensive manner it seems logical to study TCR-pMHC binding within the immunological synapse. Here receptors and ligands unfold their function under conditions which deviate considerably from the defined settings employed in in vitro measurements: molecules are pre-oriented, crowded and invariably subjected to mechanical forces which result from cytoskeletal restructuring and cellular motility. The last two decades have seen a surge in the use of live-cell approaches involving advanced molecular imaging and other biophysical methodologies with single molecule resolution. Here we review how such modalities have changed our perspectives on molecular interactions in situ and the way we look at T-cell antigen recognition.