The T cell receptor (TCR)–peptide-MHC (pMHC) interaction is the only antigen-specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR–pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, first, 2D TCR–pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6 to 15 pN) previously associated with catch–slip transitions and, second, 2D TCR–pMHC association kinetics, for which the method is well suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6- to 15-pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR–pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation.

• The large extracellular domains of the tyrosine phosphatases CD45 and CD148 prevent them from inhibiting T-cell receptor triggering.• These domains are required for optimal segregation from the engaged T-cell receptor, supporting the kineticsegregation model of triggering.T-cell receptor (TCR) triggering results in a cascade of intracellular tyrosine phosphorylation events that ultimately leads to T-cell activation. It is dependent on changes in the relative activities of membrane-associated tyrosine kinases and phosphatases near the engaged TCR. CD45 and CD148 are transmembrane tyrosine phosphatases with large ectodomains that have activatory and inhibitory effects on TCR triggering. This study investigates whether and how the ectodomains of CD45 and CD148 modulate their inhibitory effect on TCR signaling. Expression in T cells of forms of these phosphatases with truncated ectodomains inhibited TCR triggering. In contrast, when these phosphatases were expressed with large ectodomains, they had no inhibitory effect. Imaging studies revealed that truncation of the ectodomains enhanced colocalization of these phosphatases with ligated TCR at the immunological synapse. Our results suggest that the large ectodomains of CD45 and CD148 modulate their inhibitory effect by enabling their passive, size-based segregation from ligated TCR, supporting the kinetic-segregation model of TCR triggering. (Blood. 2013;121(21):4295-4302) Introduction T cells are stimulated through the T-cell receptor (TCR) when it binds cognate peptide presented by a major histocompatibility complex molecule (pMHC) on another cell. As a consequence of ligation, immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of the TCR/CD3 complex are phosphorylated by lymphocyte-specific protein tyrosine kinase (Lck). These phosphorylated ITAMs recruit z-chain-associated protein tyrosine kinase 70 to the membrane, and Zap-70 phosphorylates substrates such as linker of activated T cells (LAT). 1Despite the extensive research in this field, the mechanism by which the binding of TCR to pMHC leads to phosphorylation of TCR/CD3 ITAMs is still contested, and several models have been proposed. 2One common feature of some of these models is that TCR triggering is initiated by changes in the relative concentrations of membrane tyrosine kinases and phosphatases in the vicinity of ligated TCR. 2The principal membrane tyrosine phosphatases involved in regulating TCR-induced tyrosine phosphorylation are CD45 and CD148. 3 The importance of this dynamic equilibrium between kinase and phosphatase activity in TCR triggering was highlighted in studies that use phosphatase inhibitors such as pervanadate. [4][5][6] Treatment of T cells with these inhibitors alone, in the absence of any TCR ligand, was sufficient to induce full activation of TCR signaling pathways, ranging from early events such as phosphorylation of TCR ITAMs, Zap-70, and LAT to late events such as interleukin 2 (IL-2) production. [4][5][6] Several mechanisms have been propos...

Tethering of the cytoplasmic tyrosine phosphatase SHP-1 to clustered receptors increases its activity 900-fold.

Tethered enzymatic reactions are ubiquitous in signalling networks but are poorly understood. Here, a novel mathematical analysis is established for tethered signalling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides 5 biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increased the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. The work establishes a new framework for studying tethered signalling processes and highlights the tether as a control parameter in clustered signalling.

Dose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently limited by the difficulty in manipulating the surface density of ligands at a cell–cell interface. Here, we describe a generic cell surface ligand system that allows precise manipulation of cell surface ligand densities over several orders of magnitude. These densities are robustly quantifiable, a major advance over previous studies. We validate the system for a range of immunoreceptors, including the T-cell receptor (TCR), and show that this generic ligand stimulates via the TCR at a similar surface density as its native ligand. We also extend our work to the activation of chimeric antigen receptors. This novel system allows the effect of varying the surface density, valency, dimensions, and affinity of the ligand to be investigated. It can be readily broadened to other receptor–cell surface ligand interactions and will facilitate investigation into the activation of, and signal integration between, cell surface receptors.