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

Janett Göhring,Enrico Klotzsch,Hannes Stockinger,Lukas Schrangl,Gerhard J Schütz,Florian Kellner,Johannes B Huppa,René Platzer

doi:10.1038/s41467-021-22775-z

Janett Göhring, Enrico Klotzsch + Show 6 more

Open Access

https://doi.org/10.1038/s41467-021-22775-z

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Abstract

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.

Highlights

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
With the use of a single-molecule Förster resonance energy transfer (FRET)-based live-cell-imaging assay, we have observed synaptic unbinding between TCR and peptide/MHC complexes (pMHC) with significantly increased off-rates compared to TCR–pMHC binding in solution[9], possibly as the result of cellular motility
Dead subunits featured a tag comprised of six histidine residues each for attachment to supported lipid bilayers (SLBs) employed in our study, which contained 2% Ni-NTA-DGS for functionalization with poly-histidinetagged proteins

Summary

Introduction

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. T-cells detect the presence of even a single antigenic pMHC molecule among millions of structurally similar yet non-stimulatory pMHCs2. This is even though nominal pMHC–TCR interactions are of a rather moderate affinity, at least when measured in vitro. Tensile forces have been implicated in the discrimination of antigenic peptides from non-activating pMHCs, especially if T-cells gauge TCR-proximal signaling based on TCR–pMHC bond lifetimes as is proposed by the kinetic proofreading model[3,4,5,6]. With the use of a single-molecule Förster resonance energy transfer (FRET)-based live-cell-imaging assay, we have observed synaptic unbinding between TCR and pMHC with significantly increased off-rates compared to TCR–pMHC binding in solution[9], possibly as the result of cellular motility. The results of a more recent study suggest that, single

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