Amnon Altman

Antigen analog-major histocompatibility complexes act as antagonists of the T cell receptor

A novel mechanism for inhibition of T cell responses is described. Using the recognition of the influenza hemagglutinin (HA) 307-319 peptide in the context of DR1 class II major histocompatibility complex molecules, we have found that nonstimulatory analogs of the HA peptide preferentially inhibit HA-specific T cells in inhibition of antigen presentation assays. This antigen-specific effect could be generalized to another DR1-restricted peptide, Tetanus toxoid 830-843. Direct binding and cellular experiments indicated that the mechanism responsible was distinct from competition for binding to DR1 molecules. Likewise, negative signaling and induction of T cell tolerance could also be excluded as effector mechanisms. Thus, the most likely mechanism for this effect is engagement of antigen-specific T cell receptors by DR1-peptide analog complexes, which results in antigen-specific competitive blocking of T cell responses by virtue of their capacity to compete with DR1-antigen complexes for binding to the T cell receptor.

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway

Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

Antigen-induced translocation of PKC-θ to membrane rafts is required for T cell activation

Protein kinase C-θ (PKC-θ) is essential for mature T cell activation; however, the mechanism by which it is recruited to the TCR signaling machinery is unknown. Here we show that T cell stimulation by antibodies or peptide–major histocompatibility complex (MHC) induces translocation of PKC-θ to membrane lipid rafts, which localize to the immunological synapse. Raft translocation was mediated by the PKC-θ regulatory domain and required Lck but not ZAP-70. In addition, PKC-θ was associated with Lck in the rafts. An isolated PKC-θ catalytic fragment did not partition into rafts or activate the transcription factor NF-κB, although addition of a Lck-derived raft-localization sequence restored these functions. Thus, physiological T cell activation translocates PKC-θ to rafts, which localize to the T cell synapse; this PKC-θ translocation is important for its function.

Functional and Physical Interactions of Syk Family Kinases with the Vav Proto-Oncogene Product

Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.

Molecular events mediating T cell activation.

Publisher Summary This chapter reviews the state of the art in the area of T lymphocyte signal transduction. The chapter presents a critical overview and synthesis of the important findings in the field of T cell activation. The ability to maintain functional antigen-specific T cell clones in long-term culture, and the determination of the primary structure of the T cell antigen–receptor complex and other surface activation molecules have been some of the major advances in T cell immunobiology in recent years. This progress has paved the way to analyze the cascade of biochemical events that constitute the transmembrane signaling machinery. Although this aspect of T cell activation is generally accepted by most researchers in this area, nearly all other biochemical events are poorly understood. These include the mechanisms that couple receptor occupancy to PIP2 hydrolysis, the potential role of other signaling pathways, and the critical cytoplasmic and nuclear events that follow second messenger production. Activation of resting T cells can be initiated by a diverse group of cell surface receptors identified in recent years, originally, by appropriate monoclonal antibodies (mAbs) and, subsequently, by gene cloning and sequencing. The conclusion that such receptors play a role in T cell activation is based on the agonist or antagonist properties of the relevant mAb. The physiological ligands for some, but not all, of these receptors are also identified.

Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1 − /−) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.

A Novel Functional Interaction between Vav and PKCθ Is Required for TCR-Induced T Cell Activation

Vav and PKCtheta play an early and important role in the TCR/CD28-induced stimulation of MAP kinases and activation of the IL-2 gene. Vav is also essential for actin cytoskeleton reorganization and TCR capping. Here, we report that PKCtheta function was selectively required in a Vav signaling pathway that mediates the TCR/CD28-induced activation of JNK and the IL-2 gene and the upregulation of CD69 expression. Vav also promoted PKCtheta translocation from the cytosol to the membrane and cytoskeleton and induced its enzymatic activation in a CD3/CD28-initiated pathway that was dependent on Rac and on actin cytoskeleton reorganization. These findings reveal that the Vav/Rac pathway promotes the recruitment of PKCtheta to the T cell synapse and its activation, essential processes for T cell activation and IL-2 production.

Inhibition of the c-Jun N-terminal Kinase/AP-1 and NF-κB Pathways by PICOT, a Novel Protein Kinase C-interacting Protein with a Thioredoxin Homology Domain

Protein kinase C-θ (PKCθ) is a Ca2+-independent PKC isoform that is selectively expressed in T lymphocytes (and muscle), and is thought to play an important role in T cell receptor-induced activation. To gain a better understanding of the function and regulation of PKCθ, we have employed the yeast two-hybrid system to identify PKCθ-interacting proteins. We report the isolation and characterization of a cDNA encoding a novel 335-amino acid (37.5-kDa) PKCθ-interacting protein termed PICOT (for PKC-interactingcousin of thioredoxin). PICOT is expressed in various tissues, including in T cells, where it colocalizes with PKCθ. PICOT displays an N-terminal thioredoxin homology domain, which is required for the interaction with PKC. Comparison of the unique C-terminal region of PICOT with expressed sequence tag data bases revealed two tandem repeats of a novel domain that is highly conserved from plants to mammals. Transient overexpression of full-length PICOT (but not its N- or C-terminal fragments) in T cells inhibited the activation of c-Jun N-terminal kinase (but not extracellular signal-regulated kinase), and the transcription factors AP-1 or NF-κB. These findings suggest that PICOT and its evolutionary conserved homologues may interact with PKC-related kinases in multiple organisms and, second, that it plays a role in regulating the function of the thioredoxin system.

PSGL-1 engagement by E-selectin signals through Src kinase Fgr and ITAM adapters DAP12 and FcRγ to induce slow leukocyte rolling

E-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) can activate the β2 integrin lymphocyte function-associated antigen-1 by signaling through spleen tyrosine kinase (Syk). This signaling is independent of Gαi-protein–coupled receptors, results in slow rolling, and promotes neutrophil recruitment to sites of inflammation. However, the signaling pathways linking E-selectin engagement of PSGL-1 to Syk activation are unknown. To test the role of Src family kinases and immunoreceptor tyrosine-based activating motif (ITAM)–containing adaptor proteins, we used different gene-deficient mice in flow chamber, intravital microscopy, and peritonitis studies. E-selectin–mediated phosphorylation of Syk and slow rolling was abolished in neutrophils from fgr−/− or hck−/− lyn−/− fgr−/− mice. Neutrophils from Tyrobp−/− Fcrg−/− mice lacking both DAP12 and FcRγ were incapable of sustaining slow neutrophil rolling on E-selectin and intercellular adhesion molecule-1 and were unable to phosphorylate Syk and p38 MAPK. This defect was confirmed in vivo by using mixed chimeric mice. Gαi-independent neutrophil recruitment into the inflamed peritoneal cavity was sharply suppressed in Tyrobp−/− Fcrg−/− mice. Our data demonstrate that an ITAM-dependent pathway involving the Src-family kinase Fgr and the ITAM-containing adaptor proteins DAP12 and FcRγ is involved in the initial signaling events downstream of PSGL-1 that are required to initiate neutrophil slow rolling.

Protein kinase Cθ: a new essential superstar on the T-cell stage

Abstract Recent studies have identified protein kinase Cθ (PKCθ), a member of the Ca 2+ -independent PKC family, as an essential component of the T-cell synapse that cooperates with calcineurin to activate the interleukin-2 (IL-2) gene. Several selective functions of PKCθ involved in the activation and survival of T cells are reviewed herein. Among these, the nuclear factor-κB (NF-κB) signaling cascade appears to be the most critical target of PKCθ in the T-cell receptor/CD28 costimulatory pathway that leads to T-cell activation.