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Binding of the T cell receptor complex to its ligand, the subsequent molecular rearrangement, and the concomitant cell-scale shape changes represent the very first steps of adaptive immune recognition. The first minutes of the interaction of T cells and antigen presenting cells have been extensively scrutinized; yet, gaps remain in our understanding of how the biophysical properties of the environment may impact the sequence of events. In particular, many pioneering experiments were done on immobilized ligands and gave major insights into the process of T cell activation, whereas later experiments have indicated that ligand mobility was of paramount importance, especially to enable the formation of T cell receptor clusters. Systematic experiments to compare and reconcile the two schools are still lacking. Furthermore, recent investigations using compliant substrates have elucidated other intriguing aspects of T cell mechanics. Here we review experiments on interaction of T cells with planar artificial antigen presenting cells to explore the impact of mechanics on adhesion and actin morphodynamics during the spreading process. We enumerate a sequence tracing first contact to final spread state that is consistent with current understanding. Finally, we interpret the presented experimental results in light of a mechanical model that captures all the different morphodynamic states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest., Competing Interests: Declaration of interests PSL valorisation has submitted a patent related to the J-DNA (PCT FR2018/053533) with D.K., T.S., and C.G. among the inventors., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Antibodies are key tools in biomedical research and medicine. Their binding properties are classically measured in solution and characterized by an affinity. However, in physiological conditions, antibodies can bridge an immune effector cell and an antigen-presenting cell, implying that mechanical forces may apply to the bonds. For example, in antibody-dependent cell cytotoxicity-a major mode of action of therapeutic monoclonal antibodies-the Fab domains bind the antigens on the target cell, whereas the Fc domain binds to the activating receptor CD16 (also known as FcgRIII) of an immune effector cell, in a quasi-bidimensional environment (2D). Therefore, there is a strong need to investigate antigen/antibody binding under force (2D) to better understand and predict antibody activity in vivo. We used two anti-CD16 nanobodies targeting two different epitopes and laminar flow chamber assay to measure the association and dissociation of single bonds formed between microsphere-bound CD16 antigens and surface-bound anti-CD16 nanobodies (or single-domain antibodies), simulating 2D encounters. The two nanobodies exhibit similar 2D association kinetics, characterized by a strong dependence on the molecular encounter duration. However, their 2D dissociation kinetics strongly differ as a function of applied force: one exhibits a slip bond behavior in which off rate increases with force, and the other exhibits a catch-bond behavior in which off rate decreases with force. This is the first time, to our knowledge, that catch-bond behavior was reported for antigen-antibody bond. Quantification of natural killer cells spreading on surfaces coated with the nanobodies provides a comparison between 2D and three-dimensional adhesion in a cellular context, supporting the hypothesis of natural killer cell mechanosensitivity. Our results may also have strong implications for the design of efficient bispecific antibodies for therapeutic applications., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Spreading of T cells on antigen presenting cells is a crucial initial step in immune response. Spreading occurs through rapid morphological changes concomitant with the reorganization of surface receptors and of the cytoskeleton. Ligand mobility and frictional coupling of receptors to the cytoskeleton were separately recognized as important factors but a systematic study to explore their biophysical role in spreading was hitherto missing. To explore the impact of ligand mobility, we prepared chemically identical substrates on which molecules of anti-CD3 (capable of binding and activating the T cell receptor complex), were either immobilized or able to diffuse. We quantified the T cell spreading area and cell edge dynamics using quantitative reflection interference contrast microscopy, and imaged the actin distribution. On mobile ligands, as compared to fixed ligands, the cells spread much less, the actin is centrally, rather than peripherally distributed and the edge dynamics is largely altered. Blocking myosin-II or adding molecules of ICAM1 on the substrate largely abrogates these differences. We explain these observations by building a model based on the balance of forces between activation-dependent actin polymerization and actomyosin-generated tension on one hand, and on the frictional coupling of the ligand-receptor complexes with the actin cytoskeleton, the membrane and the substrate, on the other hand. Introducing the measured edge velocities in the model, we estimate the coefficient of frictional coupling between T Cell receptors or LFA-1 and the actin cytoskeleton. Our results provide for the first time, to our knowledge, a quantitative framework bridging T cell-specific biology with concepts developed for integrin-based mechanisms of spreading., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The binding properties of biomolecules play a crucial role in many biological phenomena, especially cell adhesion. Whereas the attachment kinetics of soluble proteins is considered well known, complex behavior arises when protein molecules are bound to the cell membrane. We probe the hidden kinetics of ligand-receptor bond formation using single-molecule flow chamber assays and Brownian dynamics simulations. We show that, consistent with our recently proposed hypothesis, association requires a minimum duration of contact between the reactive species. In our experiments, ICAM-1 anchored on a flat substrate binds to anti-ICAM-1 coated onto flowing microbeads. The interaction potential between bead and substrate is measured by microinterferometry and is used as an ingredient to simulate bead movement. Our simulation calculates the duration of ligand-receptor contacts imposed by the bead movement. We quantitatively predict the reduction of adhesion probability measured for shorter tether length of the ligand or if a repulsive hyaluronan layer is added onto the surface. To account for our results, we propose that bond formation may occur in our system by crossing of a diffusive plateau in the energy landscape, on the timescale of 5 ms and an energy barrier of 5 k(B)T, before reaching the first detectable bound state. Our results show how to relate cell-scale behavior to the combined information of molecular reactivity and biomolecule submicron-scale environment., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The efficiency of many cell-surface receptors is dependent on the rate of binding soluble or surface-attached ligands. Much effort was exerted to measure association rates between soluble molecules (three-dimensional k(on)) and, more recently, between surface-attached molecules (two-dimensional [2D] k(on)). According to a generally accepted assumption, the probability of bond formation between receptors and ligands is proportional to the first power of encounter duration. Here we provide new experimental evidence and review published data demonstrating that this simple assumption is not always warranted. Using as a model system the (2D) interaction between ICAM-1-coated surfaces and flowing microspheres coated with specific anti-ICAM-1 antibodies, we show that the probability of bond formation may scale as a power of encounter duration that is significantly higher than 1. Further, we show that experimental data may be accounted for by modeling ligand-receptor interaction as a displacement along a single path of a rough energy landscape. Under a wide range of conditions, the probability that an encounter of duration t resulted in bond formation varied as erfc[(t(0)/t)(1/2)], where t(0) was on the order of 10 ms. We conclude that the minimum contact time for bond formation may be a useful parameter to describe a ligand-receptor interaction, in addition to conventional association rates.

We used a combination of laminar flow chamber and reflection interference microscopy to study the formation and rupture of single bonds formed between Fc-ICAM-1 attached to a substrate and anti-ICAM-1 carried by micrometric beads in the presence of a repulsive hyaluronan (HA) layer adsorbed onto the substrate. The absolute distance between the colloids and the surface was measured under flow with an accuracy of a few nanometers. We could verify the long-term prediction of classical lubrication theory for the movement of a sphere near a wall in a shear flow. The HA polymer layer exerted long-range repulsive steric force on the beads and the hydrodynamics at the boundary remained more or less unchanged. By incubating HA at various concentrations, the thickness of the layer, as estimated by beads most probable height, was tuned in the range 20-200 nm. Frequency of bond formation was decreased by more than one order of magnitude by increasing the thickness of the repulsive layer, while the lifetime of individual bonds was not affected. This study opens the way for further quantitative studies of the effect of molecular environment and separation distance on ligand-receptor association and dissociation.

The adhesion of giant unilamellar phospholipid vesicles to planar substrates coated with extracellular matrix mimetic cushions of hyaluronan is studied using quantitative reflection interference contrast microscopy. The absolute height of the vesicle membrane at the vicinity of the substrate is measured by considering, for the first time, the refractive indices of the reflecting media. The thickness of the cushion is varied in the range of approximately 50-100 nm, by designing various coupling strategies. On bare protein-coated substrates, the vesicles spread fast (0.5 s) and form a uniform adhesion disk, with the average membrane height approximately 4 nm. On thick hyaluronan cushions (>80 nm), the membrane height is approximately the same as the thickness of the cushion, implying that the vesicle lies on top of the cushion. On a thin and inhomogeneous hyaluronan cushion, the adhesion is modified but not prevented. The spreading is slow ( approximately 20 s) compared to the no-cushion case. The average membrane height is approximately 10 nm and the adhesion disk is studded with blisterlike structures. Observations with fluorescent hyaluronan indicate that the polymer is compressed under, rather than expelled from, the adhesion disk. The adhesion energy density is approximately threefold higher in the no-cushion case (1.2 microJ/m(2)) as compared to the thin-cushion case (0.54 microJ/m(2)). In the thin-cushion case, the presence of short ( approximately 4 nm) glyco-polymers on the vesicles results in a hitherto unreported stable partial adhesion state--the membrane height ranges from zero to approximately 250 nm. The minimal model system presented here mimics in vitro the hyaluronan-modulated early stages of cell adhesion, and demonstrates that the presence of a polymer cushion influences both the final equilibrium adhesion-state and the spreading kinetics.