Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.


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Research sample Sample sizes were chosen in agreement with the observed variabilities in the samples and previous experience. No statistical method was used to predetermine sample size.
Single-molecule FRET data was filtered as described in the method section of the manuscript. Partially bleached ensemble FRET data was excluded and are provided in the Source Data File.
Unless explicitly stated, all data shown were obtained from at least 3 biological independent experiments. For representative images, each experiment was successfully repeated at least three times under similar conditions.
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