The immunological synapse: kinases in T cell signalling as potential drug targets

This international workshop on key signalling molecules in lymphocyte activation and immune regulation was held in Grossziethen, Germany from November 02-04, 2005 and brought together molecular, cellular, and clinical immunologists whose common goal is to develop ways of manipulating the immune response in order to avert T cell effector functions that are of significant relevance for pathogenesis in different diseases, including dermatological (psoriasis, atopic dermatitis and allergic contact allergy) and other indications (e.g. asthma, rheumatoid arthritis, multiple sclerosis and transplant rejection).     

CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse

How T cells respond with extraordinary sensitivity to minute amounts of agonist peptide and major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells bearing large numbers of endogenous pMHC molecules is not understood. Here we present evidence that CD4 affects the responsiveness of T helper cells by controlling spatial localization of the tyrosine kinase Lck in the synapse. This finding, as well as further in silico and in vitro experiments, led us to develop a molecular model in which endogenous and agonist pMHC molecules act cooperatively to amplify T cell receptor signaling. At the same time, activation due to endogenous pMHC molecules alone is inhibited. A key feature is that the binding of agonist pMHC molecules to the T cell receptor results in CD4-mediated spatial localization of Lck, which in turn enables endogenous pMHC molecules to trigger many T cell receptors. We also discuss broader implications for T cell biology, including thymic selection, diversity of the repertoire of self pMHC molecules and serial triggering.     

免疫突触形成过程中T细胞受体的重定向运动:涡旋驱动模型

目的：建立T细胞受体（TCR）在免疫突触形成过程中作重定向（reorientation）运动的机制模型.方法：基于经典流体力学环境中的双分子反应传能原理,提出了T细胞受体的涡旋驱动模型,利用免疫突触内耦合的受体或配体分子作为涡源驱动T细胞受体分子的募集.结果：模型计算的结果表明,在强度及作用频率同时具备一定范围的涡旋连续驱动下,TCR重定向运动速度可达到实验测定的范围（0.04～0.1 μm/s）.结论：本模型证明突触内受体/配体对耦合时通过将其结合自由能转化为细胞内外流体涡旋运动的机械能可能直接提供了TCR重定向运动的驱动力.     

Activation-induced proteolysis of the cytoplasmic domain of ζ in T cell receptors and Fc receptors

The CD3-T cell receptor (TCR) complex on T cells and the Fcγ receptor type III (FcγRIII)-ζ-γ complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits ζ and γ. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized ζ-p14 heterodimer. ζ-p14 is a component of both CD3-TCR and FcγRIII-ζ-γ. Peptide mapping analysis shows that p14 is structurally related to ζ, suggesting that it is either: (i) derived from ζ proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding ζ express ζ-p14 supports the former possibility. The expression of CD3-TCR complexes including ζ-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of ζ may contribute to receptor modulation or desensitization.     

Synthetic surfaces as artificial antigen presenting cells in the study of T cell receptor triggering and immunological synapse formation

T cell activation occurs when T cell receptors engage peptide-major histocompatibility complex (pMHC) molecules displayed on the surface of antigen presenting cells (APCs). Clustering of TCRs and other receptors in physical patterns at the T-APC interface forms a structure known as an immunological synapse (IS). Studies of the IS are challenging due to the cell-cell contact context of the governing interactions. Model surfaces as synthetic APCs have thus been developed, where the type, quantity, and physical arrangement of ligands displayed to T cells are precisely controlled. These model systems have provided important insights into the structure and function of the IS.     

The balance between T cell receptor signaling and degradation at the center of the immunological synapse is determined by antigen quality

The role of the center of the immunological synapse (the central supramolecular activation cluster or cSMAC) is controversial. One model suggests that the role of the cSMAC depends on antigen quality and can both enhance signaling and receptor downregulation, whereas a second model proposes that the sole function of the cSMAC is to downregulate signaling. An important distinction between the models is whether signaling occurs in the cSMAC. Here, we demonstrate that at early time points, signaling occurs outside the cSMAC, but occurs in the cSMAC at later time points. Additionally, we show that cSMAC formation enhances the stimulatory potency of weak agonists for the TCR. Combined with previous studies showing that cSMAC formation decreases the signaling by strong agonists, our data support a model proposing that signaling and receptor degradation both occur in the cSMAC and that the balance between signaling and degradation in the synapse is determined by antigen quality.     

Endothelial nitric oxide synthase regulates T cell receptor signaling at the immunological synapse

The role of nitric oxide (NO) in T cells remains controversial, and the origin and localization of endogenous NO and whether it regulates lymphocyte activation are unclear. We show here that, within minutes of binding to antigen, T cells produce NO via endothelial nitric oxide synthase (eNOS). This process required increased intracellular Ca2+ and phosphoinositide3-kinase activity. By using an eNOS-green fluorescent fusion protein and fluorescent probes to detect NO, we show that eNOS translocates with the Golgi apparatus to the immune synapse of T helper cells engaged with antigen-presenting cells (APC), where it was fully activated. Overexpression of eNOS prevented the central coalescence of CD3 at the T cell-APC contact site, which was accompanied by increased phosphorylation of CD3zeta chain, ZAP-70, and extracellular signal-regulated kinases and increased IFN-gamma synthesis, but reduced production of IL-2. Therefore, eNOS-derived NO selectively potentiates T cell receptor signaling to antigen at the immunological synapse.     

T cell killing does not require the formation of a stable mature immunological synapse

A notable feature of T lymphocyte recognition on other cell surfaces is the formation of a stable mature immunological synapse. Here we use a single-molecule labeling method to directly measure the number of ligands a cytotoxic T cell engages and track the consequences of that interaction by three-dimensional video microscopy. Like helper T cells, cytotoxic T cells were able to detect even a single foreign antigen but required about ten complexes of peptide-major histocompatibility complex (pMHC) to achieve full calcium increase and to form a mature synapse. Thus, cytotoxic T cells and helper T cells are more uniform in their antigen sensitivities than previously thought. Furthermore, only three pMHC complexes were required for killing, showing that stable synapse formation and complete signaling are not required for cytotoxicity.     

Dynamics of p56lck translocation to the T cell immunological synapse following agonist and antagonist stimulation

To study the spatio/temporal recruitment of lck during immunological synapse formation, we utilize high-speed time-lapse microscopy to visualize green fluorescent protein (GFP) fusions of lck and CD3zeta following agonist or altered peptide ligand (APL) stimulation. The dynamics of lck and CD3zeta recruitment are comparable; however, lck becomes excluded to the periphery of mature synapses, while most CD3zeta is centrally localized, suggesting a limited time frame within which lck can efficiently phosphorylate CD3 molecules during synapse maturation. Exposure of T cells to specific APLs affects the efficiency of conjugate formation and lck accumulation. Most surprisingly, we find an intracellular pool of lck associated with recycling endosomes that translocates to mature synapses within 10 min of calcium flux. This bolus of lck may contribute to intermediate-late signal transduction.     

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.     

The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling

The actin cytoskeleton seems to play two critical roles in the activation of T cells. One of these roles is T cell shape development and movement, including formation of the immunological synapse. The other is the formation of a scaffold for signaling components. This review focuses on the recent convergence of cell biology and immunology studies to explain the role of the actin cytoskeleton in creating the molecular basis for immunological synapse formation and T cell signaling.     

Specific recruitment of SPA-1 to the immunological synapse: involvement of actin-bundling protein actinin

SPA-1 is involved in the regulation of T cell activation in response to antigens through the control of Rap1 GTPase signaling. In this study, the subcellular localization of SPA-1 in the T cells was examined by using anti-SPA-1 antibody and GFP-SPA-1. While SPA-1 was detected diffusely at the surface cortical region in the floating unpolarized T cells, it was concentrated at the matrix-adhesion region with dense actin-cytoskeleton. Upon interaction with specific antigen-presenting cells, SPA-1 was highly concentrated at the immunological synapse closely co-localizing with actin. By yeast two-hybrid system, SPA-1 was shown to interact with an actin-bundling protein alpha-actinin, and it was indicated that SPA-1 co-localized with alpha-actinin at the immunological synapse. The results have suggested that SPA-1 in the T cells is selectively recruited to the immunological synapse with dense actin-cytoskeletal reorganization and keeps restraining the levels of Rap1GTP at the local TCR-signaling complex for the T cell activation.     

Relationship of B cell Fc receptors to T cell recognition of Mls antigen

The Mls locus on chromosome 1 controls the expression of cellular determinants that are responsible for stimulating mixed lymphocyte reactions between H-2-identical strains. However, the biochemical nature of Mls antigenic determinants remains undefined. It has been proposed that Ly-17 lymphoid cell surface antigens (also known as Ly.m.20.2 and LyM-1) and Mls antigens could be identical because they are both encoded by loci on chromosome 1 and display similar tissue distribution. The Ly-17 locus encodes polymorphic alleles of the IgG Fc receptor (Fc gamma R). In the present study, two approaches were used to address the question of whether Fc gamma R are involved in T cell recognition of Mls antigen. In the first approach we tested the effect of Fc gamma R blockade by heat-aggregated mouse IgG or anti-Fc gamma R monoclonal antibodies (2.4.G2) on the ability of an Ia+, Fc gamma R+ Mlsa-expressing B cell hybrid (LBB.3.4.16) to stimulate interleukin 2 secretion by an anti-Mlsa-specific T cell hybrid. We show that blockade of Fc gamma R does not inhibit the Mlsa-specific stimulation of T cells during a 24-h culture period in which Fc gamma R remain blocked. In the second approach, we derived irradiation-induced variants of LBB.3.4.16 to dissociate Fc gamma R expression and Mls antigen expression. We describe 2 LBB variants which no longer stimulate Mlsa-reactive T cells but do express Fc gamma R. Compared to parental LBB cells, the capacity of variant LBB cells to present soluble antigen to Ia-restricted T cells is unaffected. Collectively, these results indicate that Fc gamma R expression and Mlsa antigen stimulation can be dissociated. We conclude that Fc gamma R expression may be necessary, but not sufficient for T cell recognition of Mls antigen.     

The stimulatory potency of T cell antigens is influenced by the formation of the immunological synapse

T cell activation is predicated on the interaction between the T cell receptor and peptide-major histocompatibility (pMHC) ligands. The factors that determine the stimulatory potency of a pMHC molecule remain unclear. We describe results showing that a peptide exhibiting many hallmarks of a weak agonist stimulates T cells to proliferate more than the wild-type agonist ligand. An in silico approach suggested that the inability to form the central supramolecular activation cluster (cSMAC) could underlie the increased proliferation. This conclusion was supported by experiments that showed that enhancing cSMAC formation reduced stimulatory capacity of the weak peptide. Our studies highlight the fact that a complex interplay of factors determines the quality of a T cell antigen.     

Helper T cell activation for the human B cell response to trinitrophenylated polyacrylamide beads: involvement of the T4 antigen

The monoclonal antibody (mAb) OKT4A (but not OKT4) inhibits the in vitro antibody response of human peripheral blood mononuclear cells. The target of OKT4A mAb is the helper T cell, as the helper cells for the antibody response to trinitrophenylated polyacrylamide beads (TNP-PAA) are exclusively in the T4 subset. The OKT4A mAb is still suppressive when the anti-TNP response of cultures of monocyte-depleted cells is supported by purified interleukin 1. Both the OKT4A mAb and anti-DR mAb suppress the non-specific T cell proliferation in the cultures leading to the in vitro mAb response. A parallel inhibition is observed for the autologous mixed lymphocyte reaction, the nonspecific B cell response, but the T cell response to mitogens is not affected. These results suggest that the recognition of self major histocompatibility complex class II determinants by the T4 molecule plays a major role in the activation of T helper cells for antibody production to this particulate antigen.     

Clustering of MHC–peptide complexes prior to their engagement in the immunological synapse: lipid raft and tetraspan microdomains

Summary: Protein reorganization at the interface of a T cell and an antigen‐presenting cell (APC) plays an important role in T cell activation. Imaging techniques reveal that reorganization of particular receptor–ligand pairs gives rise to an intercellular junction, termed the immunological synapse. In this synapse antigenic peptides associated with major histocompatibility complex (MHC) molecules form multimolecular arrays on the APC side, engaging an equivalent number of clustered T cell receptors (TCRs) on the T cell. The accumulation of MHC molecules carrying cognate peptide in the APC–T cell interface was thought to depend on the specificity and presence of TCRs. Recent evidence, however, suggests that the APC is equipped to preorganize MHC–peptide complexes in the absence of T cells. To this end, MHC molecules become incorporated into two types of membrane microdomains: (i) cholesterol‐ and glycosphingolipid‐enriched domains, denoted lipid rafts, that preconcentrate MHC class II molecules; and (ii) microdomains made up of tetraspan proteins, such as CD9, CD63, CD81 or CD82, that mediate enrichment of MHC class II molecules loaded with a select set of peptides. It follows that the integrity, composition and dynamics of these microdomains are candidate determinants favoring activation or silencing of T cells.     

T cell receptor internalization from the immunological synapse is mediated by TC21 and RhoG GTPase-dependent phagocytosis

The immunological synapse (IS) serves a dual role for sustained T?cell receptor (TCR) signaling and for TCR downregulation. TC21 (Rras2) is a RRas subfamily GTPase that constitutively associates with the TCR and is implicated in tonic TCR signaling by activating phosphatidylinositol 3-kinase. In this study, we demonstrate that TC21 both cotranslocates with the TCR to the IS and is necessary for TCR internalization from the IS through a mechanism dependent on RhoG, a small GTPase previously associated with phagocytosis. Indeed, we found that the TCR triggers T?cells to phagocytose 1-6?μm beads through a TC21- and RhoG-dependent pathway. We further show that TC21 and RhoG are necessary for the TCR-promoted uptake of major histocompatibility complex (MHC) from antigen-presenting cells. Therefore, TC21 and RhoG dependence underlie the existence of a common phagocytic mechanism that drives TCR internalization from the IS together with its peptide-MHC ligand.     

Multifocal structure of the T cell - dendritic cell synapse

The structure of immunological synapses formed between murine naive T cells and mature dendritic cells has been subjected to a quantitative analysis. Immunofluorescence images of synapses formed in the absence of antigen show a diffuse synaptic accumulation of CD3 and LFA-1. In electron microscopy, these antigen-free synapses present a number of tight appositions (cleft size approximately 15 nm), all along the synapse. These tight appositions cover a significantly larger surface fraction of antigen-dependent synapses. In immunofluorescence, antigen-dependent synapses show multiple patches of CD3 and LFA-1 with a variable overlap. A similar distribution is observed for PKCtheta and talin. A concentric organization characteristic of prototypical synapses is rarely observed, even when dendritic cells are paralyzed by cytoskeletal poisons. In T-DC synapses, the interaction surface is composed of several tens of submicronic contact spots, with no large-scale segregation of CD3 and LFA-1. As a comparison, in T-B synapses, a central cluster of CD3 is frequently observed by immunofluorescence, and electron microscopy reveals a central tight apposition. Our data show that it is inappropriate to consider the concentric structure as a "mature synapse" and multifocal structures as immature.