αβ T Cell Receptor Mechanosensing Forces out Serial Engagement
Authors: Yinnian Feng, Ellis L. Reinherz, Matthew J. Lang
Journal: Trends in Immunology (2018)

The αβ TCR is a mechanosensor whose force-dependent structural transition and allostery regulate peptide discrimination and pMHC bond lifetime. Application of mechanical force on the TCR during ligand recognition promotes its molecular translocation and initiates T cell immunological synapse formation. Synergy of external (cell motility based) and internal (cytoskeletal motor based) forces supports a nonequilibrium (energized) model for T cell activation through reconfiguration of the αβ TCR complex at a critical force threshold. A digital mechanosensing mechanism defines physicochemical thresholds with significant implications for CTL-based vaccines and immunotherapies. That knowledge affords new insights relative to earlier αβ TCR activation models based on equilibrium processes. T lymphocytes use αβ T cell receptors (TCRs) to recognize sparse antigenic peptides bound to MHC molecules (pMHCs) arrayed on antigen-presenting cells (APCs). Contrary to conventional receptor–ligand associations exemplified by antigen–antibody interactions, forces play a crucial role in nonequilibrium mechanosensor-based T cell activation. Both T cell motility and local cytoskeleton machinery exert forces (i.e., generate loads) on TCR–pMHC bonds. We review biological features of the load-dependent activation process as revealed by optical tweezers single molecule/single cell and other biophysical measurements. The findings link pMHC-triggered TCRs to single cytoskeletal motors; define the importance of energized anisotropic (i.e., force direction dependent) activation; and characterize immunological synapse formation as digital, revealing no serial requirement. The emerging picture suggests new approaches for the monitoring and design of cytotoxic T lymphocyte (CTL)-based immunotherapy. T lymphocytes use αβ T cell receptors (TCRs) to recognize sparse antigenic peptides bound to MHC molecules (pMHCs) arrayed on antigen-presenting cells (APCs). Contrary to conventional receptor–ligand associations exemplified by antigen–antibody interactions, forces play a crucial role in nonequilibrium mechanosensor-based T cell activation. Both T cell motility and local cytoskeleton machinery exert forces (i.e., generate loads) on TCR–pMHC bonds. We review biological features of the load-dependent activation process as revealed by optical tweezers single molecule/single cell and other biophysical measurements. The findings link pMHC-triggered TCRs to single cytoskeletal motors; define the importance of energized anisotropic (i.e., force direction dependent) activation; and characterize immunological synapse formation as digital, revealing no serial requirement. The emerging picture suggests new approaches for the monitoring and design of cytotoxic T lymphocyte (CTL)-based immunotherapy. αβ T cells specifically recognize foreign peptides displayed on infected or otherwise perturbed cells through a process that discriminates with exquisite specificity. In so doing, T cells can discern a single amino acid difference between two antigens. At the heart of this process is a receptor–ligand interaction between variable domains on the αβ TCR and a peptide cradled in the groove of a major histocompatibility molecule, pMHC ([1Wang J.H. Reinherz E.L. The structural basis of αβ T-lineage immune recognition: TCR docking topologies, mechanotransduction, and co-receptor function.Immunol. Rev. 2012; 250: 102-119Crossref PubMed Scopus (79) Google Scholar, 2Yin L. et al.T cells and their eons-old obsession with MHC.Immunol. Rev. 2012; 250: 49-60Crossref PubMed Scopus (54) Google Scholar] and references therein). APCs displaying peptides at single-molecule (SM) levels can be recognized by T cells [3Purbhoo M.A. et al.T cell killing does not require the formation of a stable mature immunological synapse.Nat. Immunol. 2004; 5: 524-530Crossref PubMed Scopus (428) Google Scholar, 4Huang J. et al.A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4+ T cells.Immunity. 2013; 39: 846-857Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar]. Equilibrium between a bound and unbound receptor satisfies the law of mass action and mathematically relates the relative population of species found in the bound and unbound states; the ratio of the forward and reverse state transitions; or similarly the ratio of the state lifetimes. From an equilibrium perspective and our basic understanding of receptor–ligand associations, one expects high affinity. Paradoxically, however, TCR–pMHC affinities as conventionally measured by free-solution methods such as surface plasmon resonance reveal low affinity receptor–ligand interactions; typically in the low to high micromolar 3D affinity range. Notwithstanding, the paradox that a mere handful of foreign peptides is sufficient for CTLs to mount a deadly response or helper T cells to activate despite apparent weak affinity was thought to be explained through a concept known as serial engagement [5Valitutti S. The serial engagement model 17 years after: from TCR triggering to immunotherapy.Front. Immunol. 2012; 3: 272Crossref PubMed Scopus (38) Google Scholar]. Conceptualized 25 years ago, serial engagement (or serial triggering) recycles a single pMHC through multiple sequential TCR binding events to collectively stimulate a T cell over a time period (from seconds to hours) [6Valitutti S. et al.Serial triggering of many T-cell receptors by a few peptide–MHC complexes.Nature. 1995; 375: 148-151Crossref PubMed Scopus (987) Google Scholar]. One ligand on an APC with intermediate affinity (KD = ∼1–5 μM) can thus activate in series a multiplicity of TCRs on a given T cell where the sum of integrated receptor activation collectively suffices to turn the T cell 'on' [7Irving M. et al.Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8(+) T cell responsiveness.J. Biol. Chem. 2012; 287: 23068-23078Crossref PubMed Scopus (89) Google Scholar]. A fundamental limitation of this model is that it is not based on direct visualization and continuous measurement of the in situ dynamic interactions between TCRs and pMHCs at the live cell membrane. Instead it utilizes downregulation of TCR copy number as a parameter of TCR occupancy, assuming reutilization of one pMHC by many TCRs over a period of hours [8Valitutti S. et al.The space and time frames of T cell activation at the immunological synapse.FEBS Lett. 2010; 584: 4851-4857Crossref PubMed Scopus (34) Google Scholar]. T cell activation is an energized, nonequilibrium process. Physiologically, T cells patrolling lymph nodes or inflamed tissues are highly mobile [9Henrickson S.E. et al.In vivo imaging of T cell priming.Sci. Signal. 2008; (Published online March 25, 2008)https://doi.org/10.1126/stke.112pt2Crossref PubMed Scopus (38) Google Scholar]. 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After TCR activation, a structure known as an immunological synapse (IS) on stationary cell interaction or an immunological kinapse on motile cell interaction is formed [19Dustin M.L. T-cell activation through immunological synapses and kinapses.Immunol. Rev. 2008; 221: 77-89Crossref PubMed Scopus (195) Google Scholar]. Internal forces associated with these structures can be directly visualized by traction force microscopy (such as deformation of underlying elastic substrate or PDMS pillars) [20Bashour K.T. et al.CD28 and CD3 have complementary roles in T-cell traction forces.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 2241-2246Crossref PubMed Scopus (151) Google Scholar, 21Basu R. et al.Cytotoxic T cells use mechanical force to potentiate target cell killing.Cell. 2016; 165: 100-110Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 22Hui K.L. et al.Cytoskeletal forces during signaling activation in Jurkat T-cells.Mol. Biol. Cell. 2015; 26: 685-695Crossref PubMed Scopus (107) Google Scholar] or by signal changes in tension gauge tethers (TGTs) (such as DNA/peptide force sensors) that open under nominal threshold forces to emit a fluorescent signal (Figure 1B) [23Liu Y. et al.DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 5610-5615Crossref PubMed Scopus (183) Google Scholar]. Technologies for applying external force and monitoring local stiffness include atomic force microscopy (AFM) [24Hu K.H. Butte M.J. T cell activation requires force generation.J. Cell Biol. 2016; 213: 535-542Crossref PubMed Scopus (103) Google Scholar], biomembrane force probe (BFP) [25Husson J. et al.Force generation upon T cell receptor engagement.PLoS One. 2011; 6e19680Crossref PubMed Scopus (130) Google Scholar, 26Li Y.-C. et al.Cutting edge: mechanical forces acting on T cells immobilized via the TCR complex can trigger TCR signaling.J. Immunol. 2010; 184: 5959-5963Crossref PubMed Scopus (152) Google Scholar, 27Liu B. et al.Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.Cell. 2014; 157: 357-368Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar], optomechanical actuator nanoparticles (OMA Nps) [28Liu Z. et al.Nanoscale optomechanical actuators for controlling mechanotransduction in living cells.Nat. Methods. 2016; 13: 143-146Crossref PubMed Scopus (86) Google Scholar], and optical trap/tweezers (OTs) among others (Figure 1C) [29Feng Y. et al.Mechanosensing drives acuity of αβ T-cell recognition.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8204-E8213Crossref PubMed Scopus (101) Google Scholar, 30Kim S.T. et al.The αβ T cell receptor is an anisotropic mechanosensor.J. Biol. Chem. 2009; 284: 31028-31037Crossref PubMed Scopus (276) Google Scholar]. Such methods increasingly refine the link between TCR–pMHC binding and activation in a way to make probing serial engagement feasible. Using traction force microscopy, no evidence for serial engagement was found [31O'Donoghue G.P. et al.Direct single molecule measurement of TCR triggering by agonist pMHC in living primary T cells.eLife. 2013; 2e00778Crossref PubMed Scopus (119) Google Scholar] but later invoked to explain an active feedback mechanism for early T cell activation that globally modulates TCR–pMHC binding [32Pielak R.M. et al.Early T cell receptor signals globally modulate ligand:receptor affinities during antigen discrimination.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 12190-12195Crossref PubMed Scopus (43) Google Scholar]. In recent papers about BFP [27Liu B. et al.Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.Cell. 2014; 157: 357-368Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar] and AFM [24Hu K.H. Butte M.J. T cell activation requires force generation.J. Cell Biol. 2016; 213: 535-542Crossref PubMed Scopus (103) Google Scholar], serial engagement, through repeat pulling on TCRs, is suggested to be required for T cell activation. Collectively, these advances failed to afford an unambiguous conclusion. With increasing spatiotemporal resolution among the spectrum of SM force spectroscopy techniques [33Neuman K.C. Nagy A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.Nat. Methods. 2008; 5: 491-505Crossref PubMed Scopus (1728) Google Scholar] (in particular through OT-based methods [34Lang M.J. et al.Simultaneous, coincident optical trapping and single-molecule fluorescence.Nat. Methods. 2004; 1: 133-139Crossref PubMed Scopus (180) Google Scholar], as shown in Figure 2A), direct testing of molecular association, structural transition, and bond lifetime of receptor–ligand interactions and characterization of binding, unbinding, and rebinding events is possible. An OT is formed by tightly focusing a laser beam typically to a diffraction-limited micron-sized volume [35Neuman K.C. Block S.M. Optical trapping.Rev. Sci. Instrum. 2004; 75: 2787-2809Crossref PubMed Scopus (2078) Google Scholar]. If a tiny object such as a micron-sized plastic bead is near the focus, it is moved by light entering and exiting this region in a way that pulls it towards the central axis of the laser beam. OTs effectively represent a light-based 'pick and place' tool for manipulating small objects. If one moves the laser, one moves the trapped object. Using position-sensing systems akin to super-resolution methods, one can determine the center location of an object to nanometer level precision. The manipulation ability of tweezers paired with the ability to directly visualize in situ dynamic interactions makes directly testing serial engagement mechanisms now feasible. Beads that mimic APCs are coated with pMHCs at known molecular densities. By directly labeling the pMHCs and using SM fluorescence imaging of these beads one can explicitly visualize the molecular density. Beads are then trapped and transported to a surface-bound cell where TCR–pMHC associations are actively facilitated. In Feng et al. [29Feng Y. et al.Mechanosensing drives acuity of αβ T-cell recognition.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8204-E8213Crossref PubMed Scopus (101) Google Scholar], we systematically monitored the ability of T cells to activate with different numbers of pMHCs at the bead–cell interface, from high molecular densities (nonphysiological) to SM interactions. In addition to pick and place, binding and unbinding, an OT can be used to exert forces on an object. By measuring the displacement of the bead from the center of the trap (ΔX) and multiplying this by the spring constant of the trap k, the restoring force towards trap center is obtained (Force = k·ΔX). The trap stiffness is measured straightforwardly and is typically expressed in units of pN/nm. It is this force-exerting ability that revealed an alternative mechanism for TCR activation based on mechanosensing, as shown in the relevant experiment in Figure 2B. Pioneering work by Kim et al. demonstrated that T cell activation could be achieved when subjected to a low magnitude oscillatory force in the shear (tangential) but not normal (perpendicular) direction relative to the T cell surface [30Kim S.T. et al.The αβ T cell receptor is an anisotropic mechanosensor.J. Biol. Chem. 2009; 284: 31028-31037Crossref PubMed Scopus (276) Google Scholar]. Force was thought to push and pull on the TCR, permitting activation. Later, a type of binding interaction known as a catch bond was predicted [36Kim S.T. et al.TCR mechanobiology: torques and tunable structures linked to early T cell signaling.Front. Immunol. 2012; 3: 76Crossref PubMed Scopus (62) Google Scholar] and subsequently confirmed with elegant BFP assays and OT-based measurements [27Liu B. et al.Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.Cell. 2014; 157: 357-368Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 37Das D.K. et al.Force-dependent transition in the T-cell receptor β-subunit allosterically regulates peptide discrimination and pMHC bond lifetime.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 1517-1522Crossref PubMed Scopus (152) Google Scholar], as illustrated in Figure 2C,D, left. Conventionally, force accelerates bond release through an exponential dependence given by Bell [38Bell G.I. Models for the specific adhesion of cells to cells.Science. 1978; 200: 618Crossref PubMed Scopus (3410) Google Scholar]. Catch bonds however exhibit an increase in lifetime in the presence of force, followed by a decrease at high forces. Direct measurement of the catch bond was pioneered by the Zhu laboratory using AFM and BFP; shown elegantly in systems such as P-selectin and its ligand [39Marshall B.T. et al.Direct observation of catch bonds involving cell-adhesion molecules.Nature. 2003; 423: 190Crossref PubMed Scopus (772) Google Scholar], as well as many others. Mechanisms for such nonlinear bonding responses have been mathematically explained [40Evans E.A. Calderwood D.A. Forces and bond dynamics in cell adhesion.Science. 2007; 316: 1148Crossref PubMed Scopus (399) Google Scholar] and elaborated as force driven allostery, geometry and increased bonding at the receptor–ligand interface [41Rakshit S. Sivasankar S. Biomechanics of cell adhesion: how force regulates the lifetime of adhesive bonds at the single molecule level.Phys. Chem. Chem. Phys. 2014; 16: 2211-2223Crossref PubMed Scopus (58) Google Scholar]. Catch bonds were observed for isolated SM TCR–pMHC interactions (Figure 2A, top left) and also directly on cells in an single molecule on single cell (SMSC) configuration (Figure 2A, top right) [37Das D.K. et al.Force-dependent transition in the T-cell receptor β-subunit allosterically regulates peptide discrimination and pMHC bond lifetime.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 1517-1522Crossref PubMed Scopus (152) Google Scholar]. While catch bonds show an increase in lifetime, the mathematical effect on equilibrium based models is linear. Thus, it is not clear that the increase in lifetime from catch bonds alone, which may be tenfold, can explain the observed increase in sensitivity and selectivity among ligands, which can be many orders of magnitude [42Sasada T. et al.Thymic selection is influenced by subtle structural variation involving the p4 residue of an MHC class I-bound peptide.Eur. J. Immunol. 2000; 30: 1281-1289Crossref PubMed Scopus (21) Google Scholar]. In OT studies, another clue arose. A structural transition was observed for the αβ TCR both in SM and SMSC assays (Figure 2D, right), manifest as an extension of the molecule under force, thus reducing the displacement observed (ΔX). This transition not only requires work done on the TCR–pMHC bond, but it correlates with the presence of a catch bond state [43Das D.K. et al.Pre-T cell receptors (Pre-TCRs) leverage Vβ complementarity determining regions (CDRs) and hydrophobic patch in mechanosensing thymic self-ligands.J. Biol. Chem. 2016; 291: 25292-25305Crossref PubMed Scopus (33) Google Scholar]. The structural transition likely alters the loading pathway, changing the distribution of force sustained through each bond. It may also allosterically alter the bonding interface between the TCR and pMHC (Figure 2C). Furthermore, as shown schematically in Figure 2E, over time the loaded TCR reversibly transitions between compact and extended states (CSs and ESs, respectively). Structural transitions may also impact larger assemblies including that of the TCR and its co-receptor working in tandem during a pMHC interaction [44Reinherz E.L. Wang J.H. Codification of bidentate pMHC interaction with TCR and its co-receptor.Trends Immunol. 2015; 36: 300-306Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. Of note, H57 Fab, an antibody fragment binding to the FG loop of the β TCR constant region, generally blocks the primary transition (and signaling), constraining the TCR in a compact state, compared to the multiple reversible transitions observed for wild-type αβ TCR. This force-based (energized) mechanism is by definition distinct from purely equilibrium binding mechanisms, as compared head to head in Feng et al. [29Feng Y. et al.Mechanosensing drives acuity of αβ T-cell recognition.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8204-E8213Crossref PubMed Scopus (101) Google Scholar]. Additional studies have tied external force to activation. These include: Li et al. in which a micropipette and a shear force were associated with activation [26Li Y.-C. et al.Cutting edge: mechanical forces acting on T cells immobilized via the TCR complex can trigger TCR signaling.J. Immunol. 2010; 184: 5959-5963Crossref PubMed Scopus (152) Google Scholar]; Husson et al. where pushing and pulling with a BFP activated cells [25Husson J. et al.Force generation upon T cell receptor engagement.PLoS One. 2011; 6e19680Crossref PubMed Scopus (130) Google Scholar]; Hu and Butte where T cells were triggered by a pMHC-functionalized AFM tip [24Hu K.H. Butte M.J. T cell activation requires force generation.J. Cell Biol. 2016; 213: 535-542Crossref PubMed Scopus (103) Google Scholar]; Liu et al. where catch bonds were observed and repeat pulling on the apical tip of T cells activated them [27Liu B. et al.Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.Cell. 2014; 157: 357-368Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar]; and Liu et al. where OMA Nps were used to stretch cell surfaces, leading to activation [28Liu Z. et al.Nanoscale optomechanical actuators for controlling mechanotransduction in living cells.Nat. Methods. 2016; 13: 143-146Crossref PubMed Scopus (86) Google Scholar]. Here we outline results involving mechanical measurements. These findings reveal that force enhances the sensitivity of T cell activation and that the direction of force differentially distributes load on individual TCRs, impacting the signaling requirements of pMHC copy number on APCs (Box 1).Box 1Internally Generated Force, Externally Applied Force and Direction-Based T Cell ActivationInternally Generated Force ActivationActivation occurring passively through forces native to the cell generally refers to the methods shown in Figure 1B in main text, where forces arising from internal cell cytoskeleton motions activate T cells. Such forces are monitored and quantified by force sensors on the coverslip surface [45Liu Y. et al.Molecular tension probes for imaging forces at the cell surface.Acc. Chem. Res. 2017; 50: 2915-2924Crossref PubMed Scopus (86) Google Scholar] or deflection of pillars [20Bashour K.T. et al.CD28 and CD3 have complementary roles in T-cell traction forces.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 2241-2246Crossref PubMed Scopus (151) Google Scholar, 21Basu R. et al.Cytotoxic T cells use mechanical force to potentiate target cell killing.Cell. 2016; 165: 100-110Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar]. However, higher local pMHC density and nN force magnitudes typical of these assays have masked direct elucidation of the molecular associations underpinning activation. Estimates based on the total force and local pMHC density suggest each TCR sustains tens of pN. This force threshold (10–20 pN/TCR) for T cell activation has been observed in the recent publications by the molecular tension fluorescence microscopy [23Liu Y. et al.DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 5610-5615Crossref PubMed Scopus (183) Google Scholar]. In OT experiments without external applied force, activation requires higher local pMHC density and exhibits large bead motion due to the transport via cytoskeletal machinery where internal forces are likely contributing to activation.Externally Applied Force-Based ActivationBy contrast, force can be applied actively. There are two classes of large forces applied over many molecules and precision forces applied through a few or individual molecules. External forces can be applied to probe the triggering mechanism of individual cells or single receptor–ligand complexes, serially, one bond at a time. With force applied through the OT at 10–20 pN/TCR, cells are able to be activated with as few as two TCR–pMHC interactions at the interface [29Feng Y. et al.Mechanosensing drives acuity of αβ T-cell recognition.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E8204-E8213Crossref PubMed Scopus (101) Google Scholar]. This is strong evidence that the TCR harnesses a force-based mechanism for triggering.Direction-Based ActivationA T cell scanning an APC features highly organized cytoskeletal structures such as leading edge, uropod, and microvilli. Measurements of activation have largely focused on force applied normal or in shear directions relative to the generally spherical cell surface. How these forces interface with the underlying microstructure and distribute their loads across bonded TCRs impacts activation. The direction of force application significantly impacts cell functions [46Nicolas A. et al.Cell mechanosensitivity controls the anisotropy of focal adhesions.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12520Crossref PubMed Scopus (185) Google Scholar]. Microvilli present on the T cell surface may apply shear force when scanning the APC [11Jung Y. et al.Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E5916-E5924Crossref PubMed Scopus (112) Google Scholar, 47Cai E. et al.Visualizing dynamic microvillar search and stabilization during ligand detection by T cells.Science. 2017; 356 (eaal3118)Crossref Scopus (142) Google Scholar], facilitating TCR triggering. In OT experiments on single cells a preference for force applied in the shear direction versus normal direction was observed. In these assays the pMHC-coated bead is placed at the waist of the T cell, far away from the cell coverslip adhesion site where abundant microvilli exist [11Jung Y. et al.Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E5916-E5924Crossref PubMed Scopus (112) Google Scholar, 48Fritzsche M. et al.Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation.Sci. Adv. 2017; 3e1603032Crossref PubMed Scopus (100) Google Scholar]. Normal forces activate more readily with fewer pMHCs at the interface, although shear direction favors more sustained Ca2+ flux. The specific pulling vector on the TCR–pMHC bond must take into account the geometry relative to the cell surface microstructure and likely includes some contribution from both normal and shear components, even in OT experiments where the bead can rotate and weak forces are utilized. In other methods such as AFM and BFP where force magnitudes capable of distorting the T cell are applied, stretch in axes orthogonal to the pulling or pushing directions occurs, and additional care is needed when interpreting direction with respect to both the cell probe frame of reference and microstructure presentation of the TCR–pMHC bond. A T cell held in a micropipette displays abundant microvilli [49Depoil D. Dustin M.L. Force and affinity in ligand discrimination by the TCR.Trends Immunol. 2014; 35: 597-603Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. BFP assays pulling normal with respect to the T cell will inherently apply shear motions along the microvilli. In AFM experiments pushing may apply more shear along the tip needle in these cases so that the correlation between the Ca2+ flux and the force magnitudes is obscured [24Hu K.H. Butte M.J. T cell activation requires force generation.J. Cell Biol. 2016; 213: 535-542Crossref PubMed Scopus (103) Google Scholar]. Internally Generated Force Activation Activation occurring passively through forces native to the cell generally refers to the methods shown in Figure 1B in main text, where forces arising from internal cell cytoskeleton motions activate T cells. Such forces are moni