Advanced Quantification of Receptor-Ligand Interaction Lifetimes via Single-Molecule FRET Microscopy.

Receptor-ligand interactions at cell interfaces initiate signaling cascades essential for cellular communication and effector functions. Specifically, T cell receptor (TCR) interactions with pathogen-derived peptides presented by the major histocompatibility complex (pMHC) molecules on antigen-presenting cells are crucial for T cell activation. The binding duration, or dwell time, of TCR-pMHC interactions correlates with downstream signaling efficacy, with strong agonists exhibiting longer lifetimes compared to weak agonists.

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide.

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes.

Single-molecule Förster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes.

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.

TIM-3 and CEACAM1 do not interact in cis and in trans.

TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing.

TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells.

The main function of T cells is to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data have indicated that global clustering of T cell antigen receptors (TCRs) occurs before T cell activation. Such pre-activation clustering has been interpreted as representing a potential regulatory mechanism that fine tunes the T cell response.

Monomeric TCRs drive T cell antigen recognition.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements.

A cellular platform for the evaluation of immune checkpoint molecules.

Blockade of the T cell coinhibitory molecules CTLA-4 and PD-1 has clinical utility to strengthen T cell responses. In addition to these immune checkpoints an ever-growing number of molecules has been implicated in generating coinhibitory signals in T cells. However, investigating coinhibitory molecules in primary human cells is complicated by the restricted expression and promiscuity of both coinhibitory receptors and their ligands.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

Background & Aims: Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation.