T Cell Antigen Receptor Triggering Research Articles

Unique roles of co-receptor-bound LCK in helper and cytotoxic T cells

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4–LCK and CD8–LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor–LCK interactions as promising targets for immunomodulation.

Actin polymerization regulates recruitment of Nck to CD3ε upon T-cell receptor triggering.

Following T-cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T-cell activation. The non-catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR-induced actin polymerization. Further, Nck is recruited to the CD3ε subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3ε was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3ε phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3ε.

Small size of membrane-tethered ligands regulates phosphatase segregation in γδ TCR triggering

Abstract Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not recognize pMHC antigens or use coreceptors CD4/8. Instead they recognize invariant membrane-tethered ligands, which are upregulated at sites of inflammation and infection. The substantial structural similarities between αβ and γδ TCRs prompted us to investigate whether they share triggering mechanisms. However, recent studies show that binding-induced γδ TCR conformational changes are not induced by physiological ligands, leaving open the possibility that other mechanisms are involved. We investigated the stress-inducible murine surface protein T22, that is recognized by the G8 γδ TCR - since detailed biophysical and structural information is available for this system. Here we tested whether ‘size-based’ kinase-phosphatase segregation (also known as the kinetic-segregation model) mediates G8 TCR signal initiation. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 release. Electron microscopy showed that elongated T22/G8 leads to increased intermembrane spacing at the immunological synapse. Conversely, truncation of the large CD45 ectodomain attenuated triggering, pointing to a critical dependence on ‘close contact’ for phosphatase exclusion. To establish the physiological relevance of our findings, we confirmed that T22 elongation abrogates triggering in primary G8 γδ T cells, and using a second γδ TCR (KN6). We are currently completing investigations on the role of kinetic-segregation in hapten-specific and human γδ TCR triggering. Taken together, our findings establish that γδ TCR triggering occurs primarily through the kinetic-segregation mechanism.

TCR Triggering Induces the Formation of Lck-RACK1-Actinin-1 Multiprotein Network Affecting Lck Redistribution.

The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1–Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR–CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1–Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1–pY394Lck–α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1–Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.

Small size of membrane-tethered ligands regulates kinase-phosphatase segregation in γδ TCR triggering.

Abstract Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not undergo canonical thymic selection, and consequently are not restricted by pMHC antigens or the co-receptors CD4/8. Some of these unconventional T cells recognize invariant membrane-tethered ligands, and their rapid activation and recruitment to sites of inflammation serves as a ‘first line of defense’ against incipient infection. The substantial structural and stoichiometric similarities between αβ and γδ TCRs prompted us to investigate whether γδ TCR ligand-recognition and triggering shared basic physicochemical constraints with conventional αβ TCRs. We chose as a model system the stress-inducible murine surface protein T22 that is recognized by the G8 γδ TCR - for which detailed biophysical and structural information is available. Since T22 is similar in size to pMHC molecules, we tested whether ‘size-based’ kinase-phosphatase segregation, following G8 engagement by T22, controls initiation of TCR signaling. In our ongoing studies, we have established that the small axial size of T22 is critical for G8 triggering. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 responses. Using optical and electron microscopy we show that elongated T22/G8 complexes increases access of inhibitory phosphatases to sites of TCR engagement at cell-cell contacts. We are testing whether truncation of the ectodomain of the large cell surface phosphatases CD45/CD148, which are normally sterically segregated from engaged TCR, affects triggering. Finally, we are adapting these strategies, to investigate mechanisms of triggering by γδ T cells that recognize small-molecule organic compounds.

Recognition of Microbial Glycolipids by Natural Killer T Cells

T cells can recognize microbial antigens when presented by dedicated antigen-presenting molecules. While peptides are presented by classical members of the major histocompatibility complex (MHC) family (MHC I and II), lipids, glycolipids, and lipopeptides can be presented by the non-classical MHC member, CD1. The best studied subset of lipid-reactive T cells are type I natural killer T (iNKT) cells that recognize a variety of different antigens when presented by the non-classical MHCI homolog CD1d. iNKT cells have been shown to be important for the protection against various microbial pathogens, including B. burgdorferi, the causative agents of Lyme disease, and S. pneumoniae, which causes pneumococcal meningitis and community-acquired pneumonia. Both pathogens carry microbial glycolipids that can trigger the T cell antigen receptor (TCR), leading to iNKT cell activation. iNKT cells have an evolutionary conserved TCR alpha chain, yet retain the ability to recognize structurally diverse glycolipids. They do so using a conserved recognition mode, in which the TCR enforces a conserved binding orientation on CD1d. TCR binding is accompanied by structural changes within the TCR binding site of CD1d, as well as the glycolipid antigen itself. In addition to direct recognition of microbial antigens, iNKT cells can also be activated by a combination of cytokines (IL-12/IL-18) and TCR stimulation. Many microbes carry TLR antigens, and microbial infections can lead to TLR activation. The subsequent cytokine response in turn lower the threshold of TCR-mediated iNKT cell activation, especially when weak microbial or even self-antigens are presented during the cause of the infection. In summary, iNKT cells can be directly activated through TCR triggering of strong antigens, while cytokines produced by the innate immune response may be necessary for TCR triggering and iNKT cell activation in the presence of weak antigens. Here, we will review the molecular basis of iNKT cell recognition of glycolipids, with an emphasis on microbial glycolipids.

Early T cell activation: integrating biochemical, structural, and biophysical cues.

T cells carry out the formidable task of identifying small numbers of foreign antigenic peptides rapidly and specifically against a very noisy environmental background of endogenous self-peptides. Early steps in T cell activation have thus fascinated biologists and are among the best-studied models of cell stimulation. This remarkable process, critical in adaptive immune responses, approaches and even seems to exceed the limitations set by the physical laws ruling molecular behavior. Despite the enormous amount of information concerning the nature of molecules involved in the T cell antigen receptor (TCR) signal transduction network, and the description of the nanoscale organization and real-time analysis of T cell responses, the general principles of information gathering and processing remain incompletely understood. Here we review currently accepted key data on TCR function, discuss the limitations of current research strategies, and suggest a novel model of TCR triggering and a few promising ways of going further into the integration of available data.

Signaling at the immune synapse: vesicular trafficking takes the stage

In this issue of Nature Immunology, Larghi and colleagues1 report the involvement of the SNARE (soluble N-ethylmaleimide-sensitive protein-attachment protein receptor) vesicle-associated membrane protein 7 (VAMP7), which regulates vesicular trafficking to the neurological synapse, in signaling downstream of the T-cell antigen receptor (TCR) and T-cell activation. Namely, they demonstrate that VAMP7 promotes the recruitment to TCR activation sites of the intracellular vesicular pool of the LAT (linker for activation of T cells) adaptor, thereby assisting the assembly of the LAT signalosome. This finding highlights vesicular trafficking to the immunological synapse as a central determinant in the translation of TCR triggering into the assembly of multimolecular signaling complexes that are known to orchestrate not only the activation of T cells, but also their development and function.

VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites

The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.

Organization of the resting TCR in nanoscale oligomers

Despite the low affinity of the T-cell antigen receptor (TCR) for its peptide/major histocompatibility complex (pMHC) ligand, T cells are very sensitive to their antigens. This paradox can be resolved if we consider that the TCR may be organized into pre-existing oligomers or nanoclusters. Such structures could improve antigen recognition by increasing the functional affinity (avidity) of the TCR-pMHC interaction and by allowing cooperativity between individual TCRs. Up to approximately 20 TCRs become tightly apposed in these nanoclusters, often in a linear manner, and such structures could reflect a relatively generalized phenomenon: the non-random concentration of membrane receptors in specific areas of the plasma membrane known as protein islands. The association of TCRs into nanoclusters can explain the enhanced kinetics of the pMHC-TCR interaction in two dimensional versus three dimensional systems, but also their existence calls for a revision of the TCR triggering models based on pMHC-induced TCR clustering. Interestingly, the B-cell receptor and the FcεRI have also been shown to form nanoclusters, suggesting that the formation of pre-existing receptor oligomers could be widely used in the immune system.

Conformational states of the kinase Lck regulate clustering in early T cell signaling

Phosphorylation of the T cell antigen receptor (TCR) by the tyrosine kinase Lck is an essential step in the activation of T cells. Because Lck is constitutively active, spatial organization may regulate TCR signaling. Here we found that Lck distributions on the molecular level were controlled by the conformational states of Lck, with the open, active conformation inducing clustering and the closed, inactive conformation preventing clustering. In contrast, association with lipid domains and protein networks were not sufficient or necessary for Lck clustering. Conformation-driven Lck clustering was highly dynamic, so that TCR triggering resulted in Lck clusters that contained phosphorylated TCRs but excluded the phosphatase CD45. Our data suggest that Lck conformational states represent an intrinsic mechanism for the intermolecular organization of early T cell signaling.

Elementary Steps in T Cell Receptor Triggering

The mechanism by which antigen binding to the T cell antigen receptor (TCR) generates intracellular signaling, a process termed TCR triggering, is incompletely understood. A large body of experimental evidence has implicated multiple biophysical/biochemical effects and multiple molecules in the process of TCR triggering, which likely reflect the uniquely demanding role of the TCR in recognizing diverse antigenic ligands. In this perspective, I propose that breaking down the process of TCR triggering into tractable elementary steps may be a useful approach in building mechanistic TCR triggering models. Once these elementary steps are understood, they can be recombined to build a unified model of TCR triggering.

Molecular Mechanism of T Cell Signaling

T cell activation begins with the formation of signaling complexes at the cell surface involving among others, the T cell antigen receptor (TCR), the Src family kinase Lck and the adaptor protein, linker for activation of T cells (LAT). We have established photo-activatable localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), dual channel PALM/direct STORM (dSTORM), live cell PALM and quantitative statistical cluster analysis to determine how TCR engagement reorganizes the T cell membrane on the molecular scale.In quiescent T cells, LAT is already segregated into clusters on the cell surface raising the question how TCR triggering initiates signaling through these pre-existing domains. We demonstrated that pre-existing LAT domains are neither phosphorylated nor laterally transported to the TCR activation site suggesting that these clusters do not participate in TCR signaling. Instead, TCR activation results in the recruitment, and phosphorylation of new LAT clusters from sub-synaptic vesicles. Our data suggests that TCR ligation preconditions the membrane for vesicle recruitment and bulk activation of the LAT signaling network.We next demonstrated that TCR triggering re-organizes Lck into distinct signaling clusters. Lck clustering, driven by self-association was independent of the kinase activation state but TCR activation segregated Lck clusters from clusters of the phosphatase CD45. In addition, de-phosphorylation of Lck, either at Tyr 394 or Tyr 505, resulted in larger but fewer clusters. Phosphorylated TCR and Lck co-clustered at the centre of the activation area suggesting that molecular re-organization may result in unique TCR signaling domains in the plasma membrane.In conclusion, single-molecule imaging provides insights into T cell signaling that could not have been obtained by any other means.

PKC-theta-mediated signal delivery from the TCR/CD28 surface receptors

Protein kinase C-theta (PKCθ) is a key enzyme in T lymphocytes, where it plays an important role in signal transduction downstream of the activated T cell antigen receptor (TCR) and the CD28 costimulatory receptor. Interest in PKCθ as a potential drug target has increased following recent findings that PKCθ is essential for harmful inflammatory responses mediated by Th2 (allergies) and Th17 (autoimmunity) cells as well as for graft-versus-host disease (GvHD) and allograft rejection, but is dispensable for beneficial responses such as antiviral immunity and graft-versus-leukemia (GvL) response. TCR/CD28 engagement triggers the translocation of the cytosolic PKCθ to the plasma membrane (PM), where it localizes at the center of the immunological synapse (IS), which forms at the contact site between an antigen-specific T cell and antigen-presenting cells (APC). However, the molecular basis for this unique localization, and whether it is required for its proper function have remained unresolved issues until recently. Our recent study resolved these questions by demonstrating that the unique V3 (hinge) domain of PKCθ and, more specifically, a proline-rich motif within this domain, is essential and sufficient for its localization at the IS, where it is anchored to the cytoplasmic tail of CD28 via an indirect mechanism involving Lck protein tyrosine kinase (PTK) as an intermediate. Importantly, the association of PKCθ with CD28 is essential not only for IS localization, but also for PKCθ-mediated activation of downstream signaling pathways, including the transcription factors NF-κB and NF-AT, which are essential for productive T cell activation. Hence, interference with formation of the PKCθ-Lck-CD28 complex provides a promising basis for the design of novel, clinically useful allosteric PKCθ inhibitors. An additional recent study demonstrated that TCR triggering activates the germinal center kinase (GSK)-like kinase (GLK) and induces its association with the SLP-76 adaptor at the IS, where GLK phosphorylates the activation loop of PKCθ, converting it into an active enzyme. This recent progress, coupled with the need to study the biology of PKCθ in human T cells, is likely to facilitate the development of PKCθ-based therapeutic modalities for T cell-mediated diseases.

The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Control of T cell activation by vitamin D

To the Editor: We read with great interest the paper by von Essen et al. published in the April 2010 issue of Nature Immunology1. The authors show a critical difference in the expression of the T cell antigen receptor (TCR) pathway signaling molecule PLC-γ1 between naive CD4+ T cells and in vitro–primed CD4+ T cells. However, the suggested role of vitamin D in the induction of PLC-γ1 expression is disputable. First, von Essen et al. demonstrate that expression of the vitamin D receptor (VDR) precedes PLC-γ1 expression after activation of freshly isolated CD4+ T cells and propose a causal relationship between these events. Such a relationship is unlikely, however, as neither the ligand of the VDR, 1,25-dihydroxyvitamin D (1,25(OH)2D3), nor its precursor, 25-hydroxyvitamin D (25(OH)D3), is reported to be present in the culture system. Second, two normocalcemic analogs (ZK191784 and ZK203278) are erroneously used as VDR antagonists. Both analogs have been developed as therapeutic alternatives to 1,25(OH)2D3 because of their lack of effects on calcium homeostasis and their ability to retain the immunomodulatory potential of active vitamin D. Indeed, these molecules are comparable to 1,25(OH)2D3 in their ability to inhibit proliferation and cytokine production of peripheral blood mononuclear cells2,3. The claim that these compounds are VDR antagonists is based on a study with a structurally distinct analog (ZK159222), which indeed has antagonistic immunomodulatory effects4. Third, the conclusions of the experiments using ketocanozole to block the hydroxylase CYP27B1 (which is required for intracellular conversion of the inactive vitamin D precursor 25(OH)D3 into 1,25 (OH)2D3) are premature. Ketoconazole is not a specific inhibitor of CYP27B1 and, moreover, inhibits the proliferation of T cells5. Additionally, 1,25(OH)2D3 and 25(OH)D3 are used in equal concentrations here, despite the over 100-fold greater potency of 1,25(OH)2D3 to inhibit the proliferation of CD4+ T cells6. Finally, the lower T cell proliferation in vitamin D–deficient patients on chronic dialysis relative to that of healthy control subjects might be due to factors other than poor vitamin D status itself. In addition, the promotion of the proliferation of CD4+ T cells after exposure to 1,25(OH)2D3 is in contrast to numerous other published observations2,6,7. In summary, von Essen et al. show that PLC-γ1 is upregulated after TCR triggering in a culture system in the presumed absence of 1,25(OH)2D3 and that the addition of VDR agonists inhibits T cell proliferation and PLC-γ1 expression. The conclusion that vitamin D stimulates the activation of human T cells by controlling the expression of VDR and PLC-γ1 is therefore questionable. Alternatively, as PLC-γ1 is expressed after T cell activation, the observed downregulation of PLC-γ1 in T cells exposed to VDR agonists can be explained by the inhibitory effects of 1,25(OH)2D3 on T cell activation.

Vitamin D controls T cell antigen receptor signaling and activation of human T cells

Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation.

PMHC ligand anchored on a plastic surface via a 61 nm flexible poly(ethylene glycol) linker triggers the TCR: implications for the mechanism of TCR signal initiation (35.18)

Abstract A critical step in the immune response is signal initiation from the T cell antigen receptor (TCR) by its ligand, peptide antigen presented by the major histocompatibility complex (pMHC). How TCR is triggered by ligand binding is still unclear. An interesting aspect of the puzzle is the sharp contrast between the inability of soluble pMHC to trigger TCR and the high triggering efficiency of surface-anchored pMHC. Here, we investigate the boundary between soluble and surface-anchored pMHC by anchoring pMHCs on plastic surfaces via poly(ethylene glycol) (PEG) linkers of varying length. We found that pMHCs anchored on the surface via a 61 nm linker trigger TCR as efficiently as a much shorter 5.6 nm linker. The lack of negative impact on TCR triggering by the long linker does not support the kinetic segregation model of TCR triggering, in which the critical factor is the shortness of the pMHC-TCR bridge between the membranes of T cell and antigen presenting cell (APC). Our result supports the idea that TCR triggering does not require receptor cross-linking, since the length of the linker far exceeds the distance required for two pMHC molecules to cross-link the TCR. Furthermore, this result is consistent with the receptor deformation model - a long linker may be tolerated as long as the T cell and APC membranes detach to a distance greater than the length of the pMHC-TCR bridge, such that mechanical force can still be applied to the TCR. Work supported by NIH R21 AI078387, Joseph L. Hollander Chair, and Joseph Stokes Jr. Res. Inst.

T Cell Receptor Engagement Triggers Its CD3ε and CD3ζ Subunits to Adopt a Compact, Locked Conformation

How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3ε subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3ε and CD3ζ subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3ε and CD3ζ, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.

The receptor deformation model of TCR triggering

Signal initiation from the T cell antigen receptor (TCR) by its ligand, peptide antigen presented by major histocompatibility complex (pMHC), is a critical step in immune responses. How TCR is “triggered” by ligand binding is still unclear. Using lipid bilayer and plastic based artificial antigen presenting systems, we demonstrate that TCR can be triggered by a very low number (<10) of monomeric agonist pMHCs anchored on a surface. TCR triggering was not dependent on the presence of endogenous pMHCs. It was, however, critically dependent on active T cell adhesion to the surface and intact actin cytoskeleton function. Based on these observations, and the dynamic T cell/APC interaction observed in vivo by other groups, we propose the receptor deformation model of TCR triggering. In this model, TCR signaling is initiated by conformational changes of the TCR/CD3 complex, induced by a pulling force originating from the cytoskeleton, and transmitted through pMHC‐TCR binding interactions with enough strength to resist rupture. The increased dissociation rate of pMHC‐TCR interaction under force should facilitate fast and efficient TCR serial triggering by a single agonist pMHC, thus explaining the sensitivity of TCR triggering. This model also offers “pMHC‐TCR binding strength under force” as the parameter that defines the specificity of TCR triggering, i.e., the ability and potency of certain pMHCs to trigger TCR.