TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity

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Abstract
The use of a novel FRET-based imaging system provides an in situ view of the kinetics of T-cell receptor (TCR) binding to peptide MHC complexes in their natural environment, the immunological synapse. Previously the mater of how containment in this environment would affect the molecular interactions that drive cell–cell interactions has been a matter of speculation. Now that they have been measured, both expected effects (enhanced association rate due to optimal orientation) and unexpected (a very active cytoskeletal component destabilizing TCR binding) are revealed. This work is of relevance to T-cell immunology and to in cell–cell interactions more generally. T lymphocytes, which are an integral part of most adaptive immune responses, recognize foreign antigens through the binding of antigenic peptide–major histocompatibility complex (pMHC) molecules on other cells to specific T-cell antigen receptors (TCRs). Using single-molecule microscopy and fluorescence resonance energy transfer, the kinetics of TCR–pMHC binding are now measured in situ, revealing accelerated kinetics and increased affinity when compared with solution measurements. The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptide–major histocompatibility complex (pMHC) molecules on other cells1. If productive, these interactions promote the formation of an immunological synapse2,3. Here we show that synaptic TCR–pMHC binding dynamics differ significantly from TCR–pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCR–pMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4–12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR–pMHC complexes, indicating that the TCR binds pMHC independently of CD4.