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M. Axmann, G. Schütz, J. Huppa:
"Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay";
Journal of Visualized Experiments, 104 (2015), 531571 - 5315717.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the
surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (μM range)
to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a
microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live
T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting
Cell (APC) 2. Measurements are based on Förster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached
to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ
FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET
events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding.
Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Bioengineering, Issue 104, T-cell antigen recognition, receptor-ligand interaction kinetics, immunological synapse, Calcium flux measurement, single molecule microscopy, Förster Resonance Energy Transfer