Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76

T cell receptor (TCR) activation and signaling precede immunological synapse formation and are sustained for hours after initiation. However, the precise physical sites of the initial and sustained TCR signaling are not definitively known. We report here that T cell activation was initiated and sustained in TCR-containing microclusters generated at the initial contact sites and the periphery of the mature immunological synapse. Microclusters containing TCRs, the tyrosine kinase Zap70 and the adaptor molecule SLP-76 were continuously generated at the periphery. TCR microclusters migrated toward the central supramolecular cluster, whereas Zap70 and SLP-76 dissociated from these microclusters before the microclusters coalesced with the TCR-rich central supramolecular cluster. Tyrosine phosphorylation and calcium influx were induced as microclusters formed at the initial contact sites. Inhibition of signaling prevented recruitment of Zap70 into the microclusters. These results indicated that TCR-rich microclusters initiate and sustain TCR signaling.     

Human immunodeficiency virus type-1 infection impairs the formation of the immunological synapse

HIV-1-infected lymphocytes improperly respond to T cell antigen receptor (TCR) stimulation. To document this phenomenon, we studied the capacity of HIV-1-infected lymphocytes to form immunological synapses. We show here that HIV-1-infected T cells poorly conjugated with antigen-presenting cells, and when they formed conjugates, the synapses were abnormal. TCR and Lck accumulated in the recycling endosomal compartment, and their clustering at the synapse was severely reduced. These phenomena were, to a large extent, caused by Nef, a viral protein affecting intracellular trafficking and signaling pathways. Concomitantly, in HIV-infected cells, tyrosine phosphorylation at the synapse and the patterns of tyrosine phosphorylated proteins were disturbed in a Nef-dependent manner. These findings underscore the importance of Lck and TCR endosomal trafficking in synapse formation and early T cell signaling. Alteration of endocytic and signaling networks at the immunological synapse likely impacts the function and fate of HIV-1-infected cells.     

TCR dynamics on the surface of living T cells.

T lymphocyte activation by specific antigen requires prolonged TCR occupancy and sustained signaling. This is accomplished by the formation of a specialized signaling domain, the immunological synapse, at the T cell-antigen-presenting cell contact site. Surface receptors and signaling components are progressively recruited into this domain where they are organized in defined three-dimensional structures. To better understand how TCR are supplied to the signaling domain during the activation process, we measured (using confocal microscopy and photo-bleaching recovery techniques) lateral mobility of GFP-tagged TCR on living Jurkat cell surface. We show that: (i) surface-expressed TCR exhibit an intrinsic, actin cytoskeleton-independent, lateral mobility which allows them to passively diffuse over the entire T cell surface within approximately 60 min and (ii) non-stimulated TCR rapidly enter the signaling domain. Our results indicate that TCR lateral mobility per se is sufficient to ensure TCR supply to the immunological synapse in the course of sustained T cell activation.     

Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation

T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.     

Induction of the NF-kappaB cascade by recruitment of the scaffold molecule NEMO to the T cell receptor

The mechanism by which TCR signaling activates NF-kappaB is poorly understood. We demonstrate here that the IKK kinase complex is recruited to the immunological synapse and can be coprecipitated with the TCR after T cell activation. Using ZAP-70-deficient T cells expressing a hybrid molecule between the SH2 domain of ZAP-70 and NEMO/IKKgamma, we showed that targeting NEMO to the immunological synapse, and more specifically its 120 N-terminal amino acids, was sufficient to selectively restore NF-kappaB activation in response to TCR ligation. Finally, we demonstrated that targeting of NEMO to the membrane of T cells was sufficient to induce constitutive NF-kappaB activation. This study shows that the localization of NEMO to the immunological synapse is important for TCR-induced NF-kappaB activation and offers a powerful system to dissect the NF-kappaB cascade in T cells.     

The immunological synapse: a cause or consequence of T-cell receptor triggering?

The immunological synapse forms as a result of the tight apposition of a T cell with an antigen-presenting cell (APC) and it is the site where the T-cell receptor (TCR) is triggered by its antigen ligand, the peptide-MHC complex present in the APC membrane. The immunological synapse was initially characterized in the T-cell membrane as three concentric rings of membrane receptors and their underlying cytoskeletal and signalling proteins. The inner circle, or central supramolecular activation cluster (cSMAC), concentrates most of the TCR and CD28, and it is surrounded by the peripheral SMAC that is formed by integrins. Finally, the most external ring or distal SMAC (dSMAC) is where proteins with large ectodomains are located, such as CD43 and CD45, far from the cSMAC. This arrangement was initially thought to be responsible for maintaining sustained TCR signalling, however, this typical concentric bull's-eye pattern is not found in the immunological synapses formed with the APCs of dendritic cells. Interestingly, TCR signalling has been detected in microclusters formed in the dSMAC area and it extinguishes as the TCRs reach the cSMAC. Hence, it appears that TCR signalling and full T-cell activation do not require the formation of the cSMAC and that this structure may rather play a role in TCR down-regulation, as well as participating in the polarized secretion of lytic granules. Here, we shall review the historical evolution of the role of the cSMAC in T-cell activation, finally discussing our most recent data indicating that the cSMAC serves to internalize exhausted TCRs by phagocytosis.     

T-cell activation is accompanied by an ubiquitination process occurring at the immunological synapse

The immunological synapse (IS) is a specialized signaling area formed at the contact site between T-cells and antigen-presenting cells (APC), where sustained engagement and signaling of TCR and accessory molecules occur. A key feature of T-cell antigen recognition is that the process of TCR/peptide-MHC interaction is self-limited by the internalization and degradation of triggered TCR and recruited signaling components. The mechanism of signaling component degradation involves their ubiquitination and targeting for degradation. Yet, the relationship between the ubiquitination process and TCR signaling as well as the cellular localization of TCR-induced ubiquitination are still elusive. In the present work, we visualize for the first time ubiquitination at the TCR signaling area. We show an enrichment of ubiquitin staining in TCR/CD3 caps in T-lymphocytes stimulated by anti-CD3 antibodies. Remarkably, we also show the recruitment of the ubiquitin ligase Cbl-b and a significant ubiquitination at the immunological synapse in antigen-stimulated T-cells. Our results identify the immunological synapse as the cellular area where TCR-induced protein ubiquitination occurs. They imply that the synapse is a specialized site where the activation process is not only triggered, but also controlled via ubiquitination of signaling actors.     

Dynamic regulation of T-cell costimulation through TCR–CD28 microclusters

Summary:  T-cell activation requires contact between T cells and antigen-presenting cells (APCs) to bring T-cell receptors (TCRs) and major histocompatibility complex peptide (MHCp) together to the same complex. These complexes rearrange to form a concentric circular structure, the immunological synapse (IS). After the discovery of the IS, dynamic imaging technologies have revealed the details of the IS and provided important insights for T-cell activation. We have redefined a minimal unit of T-cell activation, the 'TCR microcluster', which recognizes MHCp, triggers an assembly of assorted molecules downstream of the TCR, and induces effective signaling from TCRs. The relationship between TCR signaling and costimulatory signaling was analyzed in terms of the TCR microcluster. CD28, the most valuable costimulatory receptor, forms TCR–CD28 microclusters in cooperation with TCRs, associates with protein kinase C θ, and effectively induces initial T-cell activation. After mature IS formation, CD28 microclusters accumulate at a particular subregion of the IS, where they continuously assemble with the kinases and not TCRs, and generate sustained T-cell signaling. We propose here a 'TCR–CD28 microcluster' model in which TCR and costimulatory microclusters are spatiotemporally formed at the IS and exhibit fine-tuning of T-cell responses by assembling with specific players downstream of the TCR and CD28.     

Antigen decoding by T lymphocytes: from synapses to fate determination

Na?ve T lymphocytes sense foreign antigens by establishing contacts with dendritic cells (DCs). At the immunological synapse between the T cell and a DC, T cell receptors (TCRs) are serially engaged and triggered by specific ligands. The amount and duration of TCR triggering and the efficiency of signal amplification determine T cell commitment to proliferation and differentiation. The nature and availability of DCs bearing antigen and costimulatory molecules shape the T cell response, giving rise to distinct functional outputs such as effector and memory T cell generation or T cell tolerance.     

Molecular interactions at the T cell-antigen-presenting cell interface

The development of new imaging techniques has made it possible to investigate the dynamic movements of molecules involved in T-cell signalling. Fluorescence resonance energy transfer (FRET) imaging allows the investigation of protein-protein interactions in live cells, and has demonstrated that T-cell receptors (TCRs) and CD4 are brought together in the immunological synapse during antigen recognition. This interaction is inhibited by antagonist ligands. Antagonism works through competition between agonist and antagonist ligands for TCR binding, as well as through feedback via the SHP-1 tyrosine phosphatase and extracellular signal-related kinase. Early signalling events result in the clustering of co-receptors and TCRs at the synapse, and the activation of various signalling molecules. Recent data show that some T-cell signalling precedes the formation of the mature form of the immunological synapse, but that full T-cell activation depends on sustained signalling, which in turn requires the synapse.     

The membrane-microfilament linker ezrin is involved in the formation of the immunological synapse and in T cell activation.

Dynamic interactions between membrane and cytoskeleton components are crucial for T cell antigen recognition and subsequent cellular activation. We report here that the membrane-microfilament linker ezrin plays an important role in these processes. First, ezrin relocalizes to the contact area between T cells and stimulatory antigen-presenting cells (APCs), accumulating in F-actin-rich membrane protrusions at the periphery of the immunological synapse. Second, T cell receptor (TCR)-mediated intracellular signals are sufficient to induce ezrin relocalization, indicating that this protein is an effector of TCR signaling. Third, overexpression of the membrane binding domain of ezrin perturbs T cell receptor clustering in the T cell-APC contact area and inhibits the activation of nuclear factor for activated T cells (NF-AT).     

Body-barrier surveillance by epidermal γδ TCRs

The surveillance of body barriers relies on resident T cells whose repertoires are biased toward particular γδ T cell antigen receptors (TCRs) according to location. These γδ TCRs can recognize ligands that emerge after stress. Through the use of intravital dynamics-immunosignal correlative microscopy, we found that γ-chain variable region 5 (V(γ)5) TCRs expressed by epidermal T cells were constitutively clustered and functionally activated in vivo at steady state, forming true immunological synapses that polarized and anchored T cell projections at squamous keratinocyte tight junctions. This synaptogenesis depended on TCR variable domains, the kinase Lck and the integrin α(E)β(7) but not the γδ lineage or the receptor NKG2D. In response to tissue stress, TCR-proximal signals did not increase substantially but underwent stress mode-dependent relocalization toward the basal epidermis and Langerhans cells. Thus, the γδ TCR orchestrates barrier surveillance proactively, presumably by recognizing tissue ligands expressed in the steady state.     

Continuous T cell receptor signaling required for synapse maintenance and full effector potential

Although signals through the T cell receptor (TCR) are essential for the initiation of T helper cell activation, it is unclear what function such signals have during the prolonged T cell-antigen-presenting cell contact. Here we simultaneously tracked TCR-CD3 complex and phosphoinositide 3-kinase activity in single T cells using three-dimensional video microscopy. Despite rapid internalization of most of the TCR-CD3, TCR-dependent signaling was still evident up to 10 h after conjugate formation. Blocking this interaction caused dissolution of the synapse and proportional reductions in interleukin 2 production and cellular proliferation. Thus TCR signaling persists for hours, has a cumulative effect and is necessary for the maintenance of the immunological synapse.     

Unusual features of self-peptide/MHC binding by autoimmune T cell receptors

Structural studies on T cell receptors (TCRs) specific for foreign antigens demonstrated a remarkably similar topology characterized by a central, diagonal TCR binding mode that maximizes interactions with the MHC bound peptide. However, three recent structures involving autoimmune TCRs demonstrated unusual interactions with self-peptide/MHC complexes. Two TCRs from multiple sclerosis patients bind with unconventional topologies, and both TCRs are shifted toward the peptide N terminus and the MHC class II beta chain helix. A TCR from the experimental autoimmune encephalomyelitis (EAE) model binds in a conventional orientation, but the structure is unusual because the self-peptide only partially fills the binding site. For all three TCRs, interaction with the MHC bound self-peptide is suboptimal, and only two or three TCR loops contact the peptide. Optimal TCR binding modes confer a competitive advantage for antimicrobial T cells during an infection, whereas altered binding properties may permit survival of a subset of autoreactive T cells during thymic selection.     

Imaging synapse formation during thymocyte selection: inability of CD3zeta to form a stable central accumulation during negative selection

TCR signaling can result in cell fates ranging from activation to tolerance to apoptosis. Organization of molecules in an "immunological synapse" between mature T cells and APCs correlates with the strength of TCR signaling. To investigate synapse formation during thymic selection, we have established a reaggregate system in which molecular recruitment of GFP fusion proteins to thymocyte:stromal cell interfaces can be visualized in real time. We demonstrate that negative selection is associated with efficient conjugate formation and rapid recruitment of p56(lck) and CD3zeta to an immunological synapse. Interestingly, CD3zeta-GFP does not accumulate at the center of the synapse, as in mature T cells, but at the periphery across a wide range of ligand densities. This implicates differences in synapse geometry in initiation of alternate signals downstream of the TCR.     

TCR-engineered T cells to treat tumors: Seeing but not touching?

Adoptive transfer of T cells gene-engineered with T cell receptors (TCRs) has proven its feasibility and therapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCR gene therapy, it is necessary to accurately select TCRs that demonstrate antigen-selective responses that are restricted to tumor cells and, at the same time, include strategies that restore or enhance the entry, migration and local accumulation of T cells in tumor tissues. Here, we present the current standing of TCR-engineered T cell therapy, discuss and propose procedures to select TCRs as well as strategies to sensitize the tumor to T cell trafficking, and provide a rationale for combination therapies with TCR-engineered T cells.     

Unique ζ‐chain motifs mediate a direct TCR‐actin linkage critical for immunological synapse formation and T‐cell activation

TCR‐mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ‐chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ‐chain mutated in these motifs lack cytoskeleton (actin) associated (cska)‐TCRs, they express normal levels of non‐cska and surface TCRs as cells expressing wild‐type ζ‐chain. However, such mutant cells are unable to display activation‐dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR‐actin linkage, providing the missing gap linking between TCR‐mediated Ag recognition, specific cytoskeleton orientation toward the T‐cell–APC interacting pole and long‐lived IS maintenance.     

Structural basis for self-recognition by autoimmune T-cell receptors

T-cell receptors (TCRs) recognize peptides presented by major histocompatibility complex molecules (pMHC) to discriminate between foreign and self-antigens. Whereas T-cell recognition of foreign peptides is essential for protection against microbial pathogens, recognition of self-peptides by T cells that have escaped negative selection in the thymus can lead to autoimmune disease. Structural studies of autoimmune TCR–pMHC complexes have provided insights into the mechanisms underlying self-recognition and escape from thymic deletion. Two broad categories of self-reactive TCRs can be clearly distinguished: (i) TCRs with altered binding topologies to self-pMHC and (ii) TCRs that bind self-pMHC in the canonical diagonal orientation, but where there are structural defects or suboptimal anchors in the self-ligand. For both categories, however, the overall stability of the autoimmune TCR–pMHC complex is markedly reduced compared to anti-microbial complexes, allowing the autoreactive T cells to evade negative selection, yet retain the ability to be activated by self-antigens in target organs. Additionally, the structures provide insights into TCR cross-reactivity, which can contribute to autoimmunity by increasing the likelihood of self-pMHC recognition. Efforts are now underway to understand the impact of structural alterations in autoimmune TCR–pMHC complexes on higher order assemblies involved in TCR signaling, as well as on immunological synapse formation.     

Analyses of TCR clustering at the T cell-antigen-presenting cell interface and its impact on the activation of naive CD4+ T cells

The role of micrometer-scale clustering of TCRs at the T cell-antigen-presenting cell (APC) interface in T cell activation is an area of active investigation. Here we have investigated the impact of variations in the extent of TCR clustering on the activation of naive CD4+ T cells. These T cells are derived from transgenic (tg) mice expressing TCRs (172.10 and 1934.4) specific for the N-terminal nonapeptide of MBP bound to I-A(u), and are associated with murine experimental autoimmune encephalomyelitis (EAE). The 172.10 TCR has a approximately 4-fold higher affinity for antigen relative to the 1934.4 TCR, allowing us to compare the properties of two tg T cells of different avidities. We observe that variations in large-scale TCR clustering at the T cell-APC interface do not correlate well with the extent of activation (CD25 or CD69 up-regulation and IL-2 or IFN-gamma production). Efficient activation can also be achieved in the absence of micrometer-scale TCR clustering, indicating that this is not a prerequisite for the effective stimulation of naive T cells.