Baoyu Liu

The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness

The T-cell receptor (TCR) interacts with peptide-major histocompatibility complexes (pMHC) to discriminate pathogens from self-antigens and trigger adaptive immune responses. Direct physical contact is required between the T cell and the antigen-presenting cell for cross-junctional binding where the TCR and pMHC are anchored on two-dimensional (2D) membranes of the apposing cells. Despite their 2D nature, TCR–pMHC binding kinetics have only been analysed three-dimensionally (3D) with a varying degree of correlation with the T-cell responsiveness. Here we use two mechanical assays to show high 2D affinities between a TCR and its antigenic pMHC driven by rapid on-rates. Compared to their 3D counterparts, 2D affinities and on-rates of the TCR for a panel of pMHC ligands possess far broader dynamic ranges that match that of their corresponding T-cell responses. The best 3D predictor of response is the off-rate, with agonist pMHC dissociating the slowest. In contrast, 2D off-rates are up to 8,300-fold faster, with the agonist pMHC dissociating the fastest. Our 2D data suggest rapid antigen sampling by T cells and serial engagement of a few agonist pMHCs by TCRs in a large self pMHC background. Thus, the cellular environment amplifies the intrinsic TCR–pMHC binding to generate broad affinities and rapid kinetics that determine T-cell responsiveness.

Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.

TCR-pMHC interactions initiate adaptive immune responses, but the mechanism of how such interactions under force induce T cell signaling is unclear. We show that force prolongs lifetimes of single TCR-pMHC bonds for agonists (catch bonds) but shortens those for antagonists (slip bonds). Both magnitude and duration of force are important, as the highest Ca(2+) responses were induced by 10 pN via both pMHC catch bonds whose lifetime peaks at this force and anti-TCR slip bonds whose maximum lifetime occurs at 0 pN. High Ca(2+) levels require early and rapid accumulation of bond lifetimes, whereas short-lived bonds that slow early accumulation of lifetimes correspond to low Ca(2+) responses. Our data support a model in which force on the TCR induces signaling events depending on its magnitude, duration, frequency, and timing, such that agonists form catch bonds that trigger the T cell digitally, whereas antagonists form slip bonds that fail to activate.

T Cell Receptor Signaling Is Limited by Docking Geometry to Peptide-Major Histocompatibility Complex

T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

Dynamic control of β1 integrin adhesion by the plexinD1-sema3E axis

Significance Cell-expressed integrins mediate adhesion with other cells and with extracellular matrix and are essential for embryonic development and for controlling leukocyte migration in later life. Integrin adhesion depends on conformational change leading to activation, although it remains unknown exactly how integrins alter their conformational state and adhesion in response to guidance cues. We show that the guidance molecule plexinD1 controls clustering of integrins in patches on the cell membrane and that the activation state of individual integrins in these patches can be switched off by binding of sema3E to plexinD1. Disruption of this pathway causes abnormal thymocyte adhesion regulation and migration during development, leading to autoimmune phenomena. Plexins and semaphorins comprise a large family of receptor-ligand pairs controlling cell guidance in nervous, immune, and vascular systems. How plexin regulation of neurite outgrowth, lymphoid trafficking, and vascular endothelial cell branching is linked to integrin function, central to most directed movement, remains unclear. Here we show that on developing thymocytes, plexinD1 controls surface topology of nanometer-scaled β1 integrin adhesion domains in cis, whereas its ligation by sema3E in trans regulates individual β1 integrin catch bonds. Loss of plexinD1 expression reduces β1 integrin clustering, thereby diminishing avidity, whereas sema3E ligation shortens individual integrin bond lifetimes under force to reduce stability. Consequently, both decreased expression of plexinD1 during developmental progression and a thymic medulla-emanating sema3E gradient enhance thymocyte movement toward the medulla, thus enforcing the orchestrated lymphoid trafficking required for effective immune repertoire selection. Our results demonstrate plexin-tunable molecular features of integrin adhesion with broad implications for many cellular processes.

Molecular Force Spectroscopy on Cells

Molecular force spectroscopy has become a powerful tool to study how mechanics regulates biology, especially the mechanical regulation of molecular interactions and its impact on cellular functions. This force-driven methodology has uncovered a wealth of new information of the physical chemistry of molecular bonds for various biological systems. The new concepts, qualitative and quantitative measures describing bond behavior under force, and structural bases underlying these phenomena have substantially advanced our fundamental understanding of the inner workings of biological systems from the nanoscale (molecule) to the microscale (cell), elucidated basic molecular mechanisms of a wide range of important biological processes, and provided opportunities for engineering applications. Here, we review major force spectroscopic assays, conceptual developments of mechanically regulated kinetics of molecular interactions, and their biological relevance. We also present current challenges and highlight future directions.

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10(-12) N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

The cellular environment regulates in situ kinetics of T-cell receptor interaction with peptide major histocompatibility complex

T cells recognize antigens at the two‐dimensional (2D) interface with antigen‐presenting cells (APCs), which trigger T‐cell effector functions. T‐cell functional outcomes correlate with 2D kinetics of membrane‐embedded T‐cell receptors (TCRs) binding to surface‐tethered peptide‐major histocompatibility complex molecules (pMHCs). However, most studies have measured TCR–pMHC kinetics for recombinant TCRs in 3D by surface plasmon resonance, which differs drastically from 2D measurements. Here, we compared pMHC dissociation from native TCR on the T‐cell surface to recombinant TCR immobilized on glass surface or in solution. Force on TCR–pMHC bonds regulated their lifetimes differently for native than recombinant TCRs. Perturbing the cellular environment suppressed 2D on‐rates but had no effect on 2D off‐rate regardless of whether force was applied. In contrast, for the TCR interacting with its monoclonal antibody, the 2D on‐rate was insensitive to cellular perturbations and the force‐dependent off‐rates were indistinguishable for native and recombinant TCRs. These data present novel features of TCR–pMHC kinetics that are regulated by the cellular environment, underscoring the limitations of 3D kinetics in predicting T‐cell functions and calling for further elucidation of the underlying molecular and cellular mechanisms that regulate 2D kinetics in physiological settings.

Dual Biomembrane Force Probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species

Conventional approaches for studying receptor-mediated cell signaling, such as the western blot and flow cytometry, are limited in three aspects: 1) The perturbing preparation procedures often alter the molecules from their native state on the cell; 2) Long processing time before the final readout makes it difficult to capture transient signaling events (<1 min); 3) The experimental environments are force-free, therefore unable to visualize mechanical signals in real time. In contrast to these methods in biochemistry and cell biology that are usually population-averaged and non-real-time, here we introduce a novel single-cell based nanotool termed dual biomembrane force probe (dBFP). The dBFP provides precise controls and quantitative readouts in both mechanical and chemical terms, which is particularly suited for juxtacrine signaling and mechanosensing studies. Specifically, the dBFP allows us to analyze dual receptor crosstalk by quantifying the spatiotemporal requirements and functional consequences of the up- and down-stream signaling events. In this work, the utility and power of the dBFP has been demonstrated in four important dual receptor systems that play key roles in immunological synapse formation, shear-dependent thrombus formation, and agonist-driven blood clotting.

2D Kinetic Analysis of TCR and CD8 Coreceptor for LCMV GP33 Epitopes

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.