Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells

T cells interacting with antigen-presenting cells (APCs) form an "immunological synapse" (IS), a bull's-eye pattern composed of a central supramolecular activation cluster enriched with T cell receptors (TCRs) surrounded by a ring of adhesion molecules (a peripheral supramolecular activation cluster). The mechanism responsible for segregating TCR and adhesion molecules remains poorly understood. Here, we show that immortalized Jurkat T cells interacting with a planar lipid bilayer (mimicking an APC) will form an IS, thereby providing an accessible model system for studying the cell biological processes underlying IS formation. We found that an actin-dependent process caused TCR and adhesion proteins to cluster at the cell periphery, but these molecules appeared to segregate from one another at the earliest stages of microdomain formation. The TCR and adhesion microdomains attached to actin and were carried centripetally by retrograde flow. However, only the TCR microdomains penetrated into the actin-depleted cell center, whereas the adhesion microdomains appeared to be unstable without an underlying actin cytoskeleton. Our results reveal that TCR and adhesion molecules spatially partition from one another well before the formation of a mature IS and that differential actin interactions help to shape and maintain the final bull's-eye pattern of the IS.     

Cluster size regulates protein sorting in the immunological synapse

During antigen recognition by T cells, signaling molecules on the T cell engage ligands on the antigen-presenting cell and organize into spatially distinctive patterns. These are collectively known as the immunological synapse (IS). Causal relationships between large-scale spatial organization and signal transduction have previously been established. Although it is known that receptor transport during IS formation is driven by actin polymerization, the mechanisms by which different proteins become spatially sorted remain unclear. These sorting processes contribute a facet of signal regulation; thus their elucidation is important for ultimately understanding signal transduction through the T cell receptor. Here we investigate protein cluster size as a sorting mechanism using the hybrid live T cell−supported membrane system. The clustering state of the co-stimulatory molecule lymphocyte function-associated antigen-1 (LFA-1) is modulated, either by direct antibody crosslinking or by crosslinking its intercellular adhesion molecule-1 ligand on the supported bilayer. In a mature IS, native LFA-1 generally localizes into a peripheral ring surrounding a central T cell receptor cluster. Higher degrees of LFA-1 clustering, induced by either method, result in progressively more central localization, with the most clustered species fully relocated to the central zone. These results demonstrate that cluster size directly influences protein spatial positioning in the T cell IS. We discuss a sorting mechanism, based on frictional coupling to the actin cytoskeleton, that is consistent with these observations and is, in principle, extendable to all cell surface proteins in the synapse.     

IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo

Biogenesis of the immune synapse at the interface between antigen-presenting cells and T cells assembles and organizes a large number of membrane proteins required for effective signaling through the T-cell receptor. We showed previously that the intraflagellar transport protein 20 (IFT20), a component of the intraflagellar transport system, controls polarized traffic during immune synapse assembly. To investigate the role of IFT20 in primary CD4⁺ T cells in vitro and in vivo, we generated mice bearing a conditional defect of IFT20 expression in T cells. We show that in the absence of IFT20, although cell spreading and the polarization of the centrosome were unaffected, T-cell receptor (TCR)-mediated signaling and recruitment of the signaling adaptor LAT (linker for activation of T cells) at the immune synapse were reduced. As a consequence, CD4⁺ T-cell activation and proliferation were also defective. In vivo, conditional IFT20-deficient mice failed to mount effective antigen-specific T-cell responses, and their T cells failed to induce colitis after adoptive transfer to Rag^−/− mice. IFT20 is therefore required for the delivery of the intracellular pool of LAT to the immune synapse in naive primary T lymphocytes and for effective T-cell responses in vivo.Vesicular traffic has emerged as a central player in the assembly and function of the immune synapse (IS), the specialized interface that forms at the T-cell membrane on contact with an antigen-presenting cell (APC) bearing cognate peptide–MHC complexes. Indeed, polarized recycling of several receptors (1, 2), including the T-cell receptor (TCR) (3), has been shown to control the clustering of these receptors at the synaptic zone and to sustain signaling initiated by their engagement. It is now well established that vesicular traffic to the IS is coopted not only by receptors but also by membrane-associated signaling mediators that are required for signal initiation and amplification, the most prominent being the kinase Lck and the adaptor LAT (linker for activation of T cells) (4, 5). These molecules are carried to the IS by recycling endosomes that move along microtubular tracks toward the centrosome, which polarizes just below the IS on contact with the cognate APC (6).We have recently provided evidence that the intraflagellar transport protein 20 (IFT20) (7) and other components of the intraflagellar transport (IFT) system, which regulates the assembly of the primary cilium (8), act as unconventional players in IS assembly by selectively controlling the polarized traffic of recycling TCRs and other recycling receptors, such as the transferrin receptor (TfR) (9, 10). We investigate here the role of IFT20 in T-cell activation, using a conditional knockout mouse carrying a null IFT20 allele in T cells. We show that IFT20 is required for TCR signaling, IS formation, and recruitment to the TCR activation sites of vesicular LAT downstream of centrosome polarization. These defects in TCR-induced signaling of IFT20-deficient T lymphocytes translate to an inability to mount an antigen-specific T-cell response both ex vivo and in vivo. We also provide evidence of the physiological relevance of these findings by showing that IFT20 deficiency affects disease severity and outcome in a mouse model of CD4⁺ T-cell-driven colitis.     

Disruption of the interaction of T cells with antigen‐presenting cells by the active leflunomide metabolite teriflunomide: Involvement of impaired integrin activation and immunologic synapse formation

Objective

Leflunomide, a potent disease‐modifying antirheumatic drug of the isoxazole class, exhibits antiinflammatory, antiproliferative, and immunosuppressive effects by largely unknown mechanisms, although alterations of pyrimidine synthesis have been proposed. Successful immune responsiveness requires T cell activation by interaction with antigen‐presenting cells (APCs), and integrin activation and formation of an immunologic synapse (IS). In this study, we evaluated the impact of the active leflunomide metabolite teriflunomide on T cell integrin activation, evolution of the IS, and antigen‐specific formation of stable T cell/APC conjugates.

Methods

Effects of pharmacologic concentrations of teriflunomide on CD3/CD28‐ and lymphocyte function–associated antigen 1–induced signal transduction and activation of primary human T cells were investigated. Furthermore, T cells were stimulated with superantigen‐ and antigen‐pulsed APCs to study relocalization of molecules to the IS and T cell/APC conjugate formation.

Results

Teriflunomide inhibited T cell receptor (TCR)/CD3–mediated calcium mobilization, but other critical T cell signaling events, including activation of MAPK and NF‐κB, remained unaltered. In contrast, inhibition of TCR/CD3‐triggered β1,2 integrin avidity and integrin‐mediated costimulation (outside‐in signaling) by teriflunomide revealed a striking interference with integrin function that was independent of altered pyrimidine synthesis. Moreover, teriflunomide abolished molecule relocalization to the IS and induction of T cell/APC conjugates.

Conclusion

These data show that the active metabolite of leflunomide prevents the interaction of T cells with APCs to form an IS. Since IS formation is crucial for eliciting an immune response, this novel mechanism could underlie the beneficial effects of leflunomide in immune‐mediated disorders such as rheumatoid arthritis.

     

T cell receptor (TCR) clustering in the immunological synapse integrates TCR and costimulatory signaling in selected T cells

During T cell activation, T cell receptors (TCR) cluster at the center of the T cell/antigen-presenting cell interface forming a key component of the immunological synapse. The function of this TCR clustering is still unresolved. A comprehensive search for such a function yielded a very limited and specific result. A micrometer-scale receptor clustering integrated the TCR and CD28 signals required for IL-2 secretion in primary 5C.C7 T cells, a low-affinity/avidity TCR system. 5C.C7 TCR signaling itself was not affected. In addition, central TCR accumulation was not required for any T cell effector function tested in three other TCR transgenic models. Central TCR accumulation thus had a specific role in signaling integration in low-affinity T cells.     

Cytoskeletal remodeling mediated by WASp in dendritic cells is necessary for normal immune synapse formation and T-cell priming

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.     

T cell signaling: Protein kinase Cθ the immunological synapse and characterization of SLAT a novel T helper 2-specific adapter protein

Triggering of the antigen-specific T cell receptor (TCR) can lead to various functional outcomes, such as activation and proliferation, anergy or cell death. This differential signaling is mainly determined by the quality and quantity of TCR signals, the nature of accessory signals and the differentiation/maturation status of the T cell. In this regard, T cell development and differentiation of the two major T helper (Th) subsets, namely Th1 and Th2 cells, can also be viewed as examples of differential signaling. In the present report, we review two T cell-selective signaling molecules (protein kinase C (PKC) θ and SLAT), which we have studied extensively and that appear to play important roles in the process of differential signaling. The novel PKC isoform PKCθ is selectively expressed in T lymphocytes and is essential for TCR-triggered activation of mature T cells via activation of the nuclear factor-κB and activator protein-1 pathways. Productive engagement of T cells by antigen-presenting cells (APC) results in recruitment of PKCθ to the T cell-APC contact area, the immunological synapse (IS), where it interacts with several signaling molecules to induce activation signals essential for productive T cell activation and interleukin-2 production. These events are associated with PKCθ translocation to membrane lipid rafts, which also localize to the IS. The Vav/Rac pathway promotes the recruitment of PKCθ to the IS or lipid rafts as well as its activation. SLAT is a novel adapter protein, which we isolated recently. It is selectively expressed in Th2 lineage cells, where it is found associated with the TCR-coupled protein tyrosine kinase ZAP-70. Our initial characterization of SLAT indicates that, by regulating the overall strength of TCR signaling, it may play an important role in differential signaling processes, which promote the differentiation and activation of allergy promoting and anti-inflammatory Th2 cells.     

Disruption of the interaction of T cells with antigen-presenting cells by the active leflunomide metabolite teriflunomide: involvement of impaired integrin activation and immunologic synapse formation

OBJECTIVE: Leflunomide, a potent disease-modifying antirheumatic drug of the isoxazole class, exhibits antiinflammatory, antiproliferative, and immunosuppressive effects by largely unknown mechanisms, although alterations of pyrimidine synthesis have been proposed. Successful immune responsiveness requires T cell activation by interaction with antigen-presenting cells (APCs), and integrin activation and formation of an immunologic synapse (IS). In this study, we evaluated the impact of the active leflunomide metabolite teriflunomide on T cell integrin activation, evolution of the IS, and antigen-specific formation of stable T cell/APC conjugates. METHODS: Effects of pharmacologic concentrations of teriflunomide on CD3/CD28- and lymphocyte function-associated antigen 1-induced signal transduction and activation of primary human T cells were investigated. Furthermore, T cells were stimulated with superantigen- and antigen-pulsed APCs to study relocalization of molecules to the IS and T cell/APC conjugate formation. RESULTS: Teriflunomide inhibited T cell receptor (TCR)/CD3-mediated calcium mobilization, but other critical T cell signaling events, including activation of MAPK and NF-kappaB, remained unaltered. In contrast, inhibition of TCR/CD3-triggered beta1,2 integrin avidity and integrin-mediated costimulation (outside-in signaling) by teriflunomide revealed a striking interference with integrin function that was independent of altered pyrimidine synthesis. Moreover, teriflunomide abolished molecule relocalization to the IS and induction of T cell/APC conjugates. CONCLUSION: These data show that the active metabolite of leflunomide prevents the interaction of T cells with APCs to form an IS. Since IS formation is crucial for eliciting an immune response, this novel mechanism could underlie the beneficial effects of leflunomide in immune-mediated disorders such as rheumatoid arthritis.     

Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy

During adaptive immune responses, T lymphocytes recognize antigenic peptides presented by MHC molecules on antigen-presenting cells (APCs). This recognition results in the formation of a so-called immune synapse (IS) at the T-cell/APC interface, which is crucial for T-cell activation. The molecular composition of the IS has been extensively studied, but little is known about the biophysics and interaction forces between T cells and APCs. Here, we report the measurement of interaction forces between T cells and APCs employing atomic force microscopy (AFM). For these investigations, specific T cells were selected that recognize an antigenic peptide presented by MHC-class II molecules on APCs. Dynamic analysis of T-cell/APC interaction by AFM revealed that in the presence of antigen interaction forces increased from 1 to 2 nN at early time-points to a maximum of ≈14 nN after 30 min and decreased again after 60 min. These data correlate with the kinetics of synapse formation that also reached a maximum after 30 min, as determined by high-throughput multispectral imaging flow cytometry. Because the integrin lymphocyte function antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule-1 (ICAM-1) are prominent members of a mature IS, the effect of a small molecular inhibitor for LFA-1, BIRT377, was investigated. BIRT377 almost completely abolish the interaction forces, emphasizing the importance of LFA-1/ICAM-1-interactions for firm T-cell/APC adhesion. In conclusion, using biophysical measurements, this study provides precise values for the interaction forces between T cells and APCs and demonstrates that these forces develop over time and are highest when synapse formation is maximal.Cell-cell contacts play a crucial role in triggering the body''s immune system. During adaptive immune responses, antigen-presenting cells (APCs) process foreign antigens into peptides, which are loaded into major histocompatibility complex (MHC) molecules. T cells patrolling the body scan APC and establish intercellular contacts when their antigen-specific T-cell receptors (TCR) recognize a foreign peptide/MHC complex on the APC. Elegant two-photon microscopy studies have revealed the dynamics of this process in lymph nodes. There, T cells move through the network of dendritic cells (DCs) and scan DCs for foreign antigen. In the absence of antigen brief transient interactions are observed, whereas upon recognition of a cognate antigen T cells are arrested and interactions prolonged to >1 h (1, 2). Similarly, during antibody responses, long-lasting antigen driven interactions between T helper cells and B cells have been observed in lymph nodes (3). Subsequently, at the contact zone between T cells and APC spatially organized molecular clusters develop, referred to as immune synapse (IS), which is crucial for T-cell activation and effector cells development (4).Formation of an IS includes the coordinated translocation of several protein complexes, among others TCR and its ligand pMHC, and the integrin lymphocyte function-associated antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule 1 (ICAM-1). This orchestrated reorganization of membrane proteins involves many cytoplasmic molecules and is presumably supported by cytoskeletal factors like actin (5). Although many important aspects of IS formation have been identified, little is known about the underlying biophysics and interaction forces between T cells and APCs. Integrins represent a family of major cell adhesion proteins used by cells to tune their adhesion propensity. This tuning is achieved by controlling the number of proteins present at the cell''s interaction face and by the activation state of the adhesion proteins themselves. Switch blade-type heterodimeric integrins are known to exist in different activation states, which are transmitted from the cytoplasmic tail to the extracellular domain (6). It is believed that activation state changes are triggered by inside-out-signaling, for instance when a TCR recognizes a peptide presented by MHC molecules (7). The activation of LFA-1 upon TCR-triggering is mainly mediated by PKC and the small GTPases Ras and Rap1 [(8) and references therein]. The association of actin to LFA-1 accompanies this process. Subsequent motor protein motion yields a cytoskeleton contraction, which exerts low forces on LFA-1 to induce occupied integrin activation and to fully arrest the two cells for adhesion. By actio et reactio, this force has to be counterbalanced on the APC side, resulting in a high interaction force between T cells and APCs.Cell–cell adhesion has been studied by micropipette aspiration techniques (9, 10) and atomic force microscopy (AFM) (11–13). The recent years have seen a significant increase of AFM-related studies in biological systems, and single-cell force spectroscopy (SCFS) by AFM has been established as an important tool for the study of cell adhesion (14). This technique allows for the analysis of adhesion processes and adhesion forces under near-physiological conditions. To the best of our knowledge, the interaction forces between T cells and APC have not yet been investigated by SCFS. In the present study, we have modified and adjusted SCFS techniques for the measurement of long-time interaction forces between T cells and APC. These force spectroscopy measurements were complemented by conjugate and high-throughput fluorescence assays relating the kinetics of IS formation to the development of interaction forces between T cells and APCs.     

Respiratory syncytial virus impairs T cell activation by preventing synapse assembly with dendritic cells

Respiratory syncytial virus (RSV) infection is one of the leading causes of infant hospitalization and a major health and economic burden worldwide. Infection with this virus induces an exacerbated innate proinflammatory immune response characterized by abundant immune cell infiltration into the airways and lung tissue damage. RSV also impairs the induction of an adequate adaptive T cell immune response, which favors virus pathogenesis. Unfortunately, to date there are no efficient vaccines against this virus. Recent in vitro and in vivo studies suggest that RSV infection can prevent T cell activation, a phenomenon attributed in part to cytokines and chemokines secreted by RSV-infected cells. Efficient immunity against viruses is promoted by dendritic cells (DCs), professional antigen-presenting cells, that prime antigen-specific helper and cytotoxic T cells. Therefore, it would be to the advantage of RSV to impair DC function and prevent the induction of T cell immunity. Here, we show that, although RSV infection induces maturation of murine DCs, these cells are rendered unable to activate antigen-specific T cells. Inhibition of T cell activation by RSV was observed independently of the type of TCR ligand on the DC surface and applied to cognate-, allo-, and superantigen stimulation. As a result of exposure to RSV-infected DCs, T cells became unresponsive to subsequent TCR engagement. RSV-mediated impairment in T cell activation required DC-T cell contact and involved inhibition of immunological synapse assembly among these cells. Our data suggest that impairment of immunological synapse could contribute to RSV pathogenesis by evading adaptive immunity and reducing T cell-mediated virus clearance.     

Low T cell receptor expression and thermal fluctuations contribute to formation of dynamic multifocal synapses in thymocytes

Mature T cell activation and selection of immature T cells (thymocytes) are both initiated by binding of T cell receptor (TCR) molecules on the surface of T cells to MHC peptide (MHCp) molecules on the surface of antigen-presenting cells. Recent experiments have shown that the spatial pattern of receptors and ligands in the intercellular junction (synapse) is different during thymocyte selection compared with mature T cell activation. Using a statistical mechanical model, we show that lower TCR expression in thymocytes contributes to effecting these differences. An analogy with the phase behavior of simple fluids helps clarify how, for low TCR expression, thermal fluctuations lead to the dynamic synapse patterns observed for thymocytes. We suggest that a different synapse pattern resulting from lower TCR expression, which could mediate differential signaling, may be the reason why TCR expression level is low in thymocytes.     

Galectin-3 negatively regulates TCR-mediated CD4+ T-cell activation at the immunological synapse

We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3^−/−) CD4⁺ T cells secreted more IFN-γ and IL-4 than gal3^+/+CD4⁺ T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3^+/+ T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3^−/− T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alix''s function at the IS.Galectins are beta-galactoside-binding proteins with evolutionarily conserved carbohydrate-recognition domains (CRD). The family members are expressed by organisms from nematodes to mammals. Currently, 15 members have been identified in mammals (reviewed in ref. 1). Each member contains either one or two CRDs, but galectin-3 is unique in that it contains a single CRD in the C-terminal region connected to an N-terminal domain consisting of tandem repeats of short proline-rich motifs. Galectins play important roles in immune responses and tumor progression and other physiological and pathological processes (reviewed in refs. 2–5).Galectin-3 is widely distributed and is expressed by various immune cells (reviewed in ref. 6). Like other galectins, it does not have a classical signal sequence and is found in the cytosol and nucleus, but is also detected extracellularly. Recombinant galectin-3 has been shown to either induce or suppress cell activation and promote or inhibit cell adhesion in vitro when delivered exogenously, depending on the experimental systems (reviewed in ref. 1). Endogenous galectin-3 has been shown to inhibit apoptosis (reviewed in refs. 7 and 8), promote mediator release and cytokine production by mast cells (9), promote phagocytosis by macrophages (10), and drive alternative macrophage activation (11). While it is clear recombinant galectin-3 exerts its functions by engaging cell surface glycoproteins or glycolipids, the mechanisms by which endogenous galectin-3 functions are largely unknown.With regard to T cells, galectin-3 is expressed by CD4⁺ and CD8⁺ T cells after these cells are activated by anti-CD3 antibody or mitogens (12). Exogenously delivered galectin-3 has been shown to induce IL-2 production by Jurkat cells (13) and cause apoptosis in activated T cells (14, 15). Endogenous galectin-3, however, inhibits apoptosis in Jurkat cell transfectants overexpressing the protein (16). Other than this, the function of endogenous galectin-3 in the T-cell response is largely unknown.Activation of T cells by TCR engagement is associated with the recruitment of many receptors and signaling molecules to the stable contact region between T cells and antigen-presenting cells (APCs) called the immunological synapse (IS), which is important in tolerance and immunity (17). T-cell receptor signaling in the IS involves continual formation of TCR microclusters that recruit signaling molecules (18, 19). These microclusters rapidly coalesce to form supramolecular activation clusters (SMAC) (20, 21). There is a central zone (cSMAC) containing TCR/CD3, which is surrounded by a peripheral zone (pSMAC) marked by lymphocyte function-associated antigen-1 (LFA-1), and a distal zone (dSMAC) (22). Current models suggest that cSMAC is engaged in TCR degradation and costimulation, pSMAC in adhesion and TCR microcluster transport, and dSMAC in TCR and LFA-1 microcluster formation (23, 24).Here, we report that gal3^−/− CD4⁺ T cells secreted higher levels of IFN-γ and IL-4 compared with gal3^+/+ cells. Galectin-3 was recruited to the cytoplasmic side of the IS in CD4⁺ T cells after TCR engagement and was primarily located at the pSMAC. Our findings suggest that galectin-3 destabilizes IS formation. We also obtained evidence that galectin-3 suppresses the activation of the early events in TCR-mediated signal transduction and potentiates down-regulation of TCR in cells activated by engagement of the receptor. Finally, we found that galectin-3 is associated with a component of the endosomal sorting complex required for transport (ESCRT), Alix, known to regulate cell surface expression of certain receptors.     

CD28 plays a critical role in the segregation of PKC theta within the immunologic synapse

The signaling pathways that lead to the localization of cellular protein to the area of interaction between T cell and antigen-presenting cell and the mechanism by which these molecules are further sorted to the peripheral supramolecular activation cluster or central supramolecular activation cluster regions of the immunologic synapse are poorly understood. In this study, we investigated the functional involvement of CD28 costimulation in the T cell receptor (TCR)-mediated immunologic synapse formation with respect to protein kinase C (PKC)theta; localization. We showed that CD3 crosslinking alone was sufficient to induce PKC theta; capping in naive CD4(+) T cells. Studies with pharmacologic inhibitors and knockout mice showed that the TCR-derived signaling that drives PKC theta; membrane translocation requires the Src family kinase, Lck, but not Fyn. In addition, a time course study of the persistence of T cell molecules to the immunologic synapse indicated that PKC theta;, unlike TCR, persisted in the synapse for at least 4 h, a time that is sufficient for commitment of a T cell to cell division. Finally, by using TCR-transgenic T cells from either wild-type or CD28-deficient mice, we showed that CD28 expression was required for the formation of the mature immunologic synapse, because antigen stimulation of CD28(-) T cells led to a diffuse pattern of localization of PKC theta; and lymphocyte function-associated antigen-1 in the immunologic synapse, in contrast to the central supramolecular activation cluster localization of PKC theta; in CD28(+) T cells.     

Involvement of Rap-1 activation and early termination of immune synapse in CTLA-4-mediated negative signal

Abstract

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell co-stimulation receptor that delivers inhibitory signals upon activation. This inhibitory effect by CTLA-4 requires activation of small GTPase Rap-1. However, the precise mechanism underlying these negative signals remains unclear. Here, we show that CTLA-4-induced suppression of IL-2 production correlates with rapid destabilization of immunological synapse (IS) formation in murine normal T cell clones. Overexpression of Spa-1, a Rap-1-specific GTPase activating protein (GAP), abolished both Rap-1 activation and IL-2 suppression induced by CTLA-4. Although we failed to find any specific inhibition of activation of early signals upon CTLA-4 engagement, we found that CTLA-4 specifically up-regulates cell motility and suppresses prolonged accumulation of Talin at the contact area with antigen presenting cells upon antigen stimulation. These results suggest that Rap-1 is activated upon CTLA-4 ligation and mediates inhibitory signals through prevention of IS formation.     

The c-Abl tyrosine kinase regulates actin remodeling at the immune synapse

Actin dynamics during T-cell activation are controlled by the coordinate action of multiple actin regulatory proteins, functioning downstream of a complex network of kinases and other signaling molecules. The c-Abl nonreceptor tyrosine kinase regulates actin responses in nonhematopoietic cells, but its function in T cells is poorly understood. Using kinase inhibitors, RNAi, and conditional knockout mice, we investigated the role of c-Abl in controlling the T-cell actin response. We find that c-Abl is required for normal actin polymerization and lamellipodial spreading at the immune synapse, and for downstream events leading to efficient interleukin-2 production. c-Abl also plays a key role in signaling chemokine-induced T-cell migration. c-Abl is required for the appropriate function of 2 proteins known to be important for controlling actin responses to T-cell receptor (TCR) engagement, the actin-stabilizing adapter protein HS1, and the Rac1-dependent actin polymerizing protein WAVE2. c-Abl binds to phospho-HS1 via its SH2 domains and is required for full tyrosine phosphorylation of HS1 during T-cell activation. In addition, c-Abl is required for normal localization of WAVE2 to the immune synapse (IS). These studies identify c-Abl as a key player in the signaling cascade, leading to actin reorganization during T-cell activation.     

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca²⁺ concentration near the interface, indicating Ca²⁺ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca²⁺ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca²⁺ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca²⁺ signaling during subsequent antigen encounter.     

PKC-theta selectively controls the adhesion-stimulating molecule Rap1

The antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunologic synapse (IS) between the membranes of the 2 cells. beta(2) integrins on the T cell, namely, leukocyte function-associated antigen 1 (LFA-1) and its counter ligand, namely, immunoglobulin-like cell adhesion molecule 1 (ICAM-1) on the APC, critically stabilize this intercellular interaction. The small GTPase Rap1 controls T-cell adhesion through modulating the affinity and/or spatial organization of LFA-1; however, the upstream regulatory components triggered by the T-cell receptor (TCR) have not been resolved. In the present study, we identified a previously unknown function of a protein kinase C- theta (PKC-theta)/RapGEF2 complex in LFA-1 avidity regulation in T lymphocytes. After T-cell activation, the direct phosphorylation of RapGEF2 at Ser960 by PKC- theta regulates Rap1 activation as well as LFA-1 adhesiveness to ICAM-1. In OT-II TCR-transgenic CD4(+) T cells, clustering of LFA-1 after antigen activation was impaired in the absence of PKC- theta. These data define that, among other pathways acting on LFA-1 regulation, PKC- theta and its effector RapGEF2 are critical factors in TCR signaling to Rap1. Taken together, PKC- theta sets the threshold for T-cell activation by positively regulating both the cytokine responses and the adhesive capacities of T lymphocytes.     

T cell activation requires mitochondrial translocation to the immunological synapse

T helper (Th) cell activation is required for the adaptive immune response. Formation of the immunological synapse (IS) between Th cells and antigen-presenting cells is essential for Th cell activation. IS formation induces the polarization and redistribution of many signaling molecules; however, very little is known about organelle redistribution during IS formation in Th cells. We show that formation of the IS induced cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. Using total internal reflection microscopy, we found that upon stimulation, the distance between the IS and mitochondria was decreased to values<200 nm. Consequently, mitochondria close to the IS took up more Ca2+ than the ones farther away from the IS. The redistribution of mitochondria to the IS was necessary to maintain Ca2+ influx across the plasma membrane and Ca2+-dependent Th cell activation. Our results suggest that mitochondria are part of the signaling complex at the IS and that their localization close to the IS is required for Th cell activation.     

The T-cell receptor is not hardwired to engage MHC ligands

The bias of αβ T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.