Mechanism of T-Cell Lymphomagenesis: Transformation of Growth-Factor-Dependent T-Lymphoblastoma Cells to Growth-Factor-Independent T-Lymphoma Cells

In a previous paper we described the induction by x-irradiation or radiation-induced leukemia virus-in-oculation of two classes of lymphoid T-cell neoplasms: The first class, designated T-cell lymphoblastoma (TCLB), consists of growth-factor-dependent eudiploid cells that home to the spleen and give rise to splenic tumors on injection into syngeneic mice; the second class, designated T-cell lymphoma (TCL), consists of growth-factor-independent aneuploid or pseudodiploid cells that give rise to local tumors at the site of subcutaneous injection. This paper describes the generation of a family of growth-factor-independent aneuploid or pseudodiploid TCL cells after the injection into the thymus of growth-factor-dependent diploid TCLB cells. In contrast to the donor TCLB cells, the resulting TCL cells could be cloned in semisolid medium, produced local tumors at the site of subcutaneous injection, and proliferated in a growth-factor-independent fashion in vitro. The induced growth-factor-independent TCL cells were chromosomally and phenotypically unstable and continued to evolve both in vivo and in vitro. After propagation in the thymus, the cells often showed stable translocations in addition to the evolving aneuploidy. We propose that the chromosome abnormalities induced during the proliferation of growth-factor-dependent TCLB cells in the thymus constitute a general mechanism by which neoplastic cells progress from growth-factor dependency to independency

A direct interaction between the adaptor protein Cbl-b and the kinase Zap-70 induces a positive signal in T cells

AbstractEngagement of the T-cell receptor (TCR)–CD3 complex induces a rapid increase in the activities of Src-family and Syk/Zap-70-family kinases [1,2]. These activated kinases then induce the tyrosine phosphorylation of multiple intracellular proteins, eventually leading to T-cell activation. One of the prominent substrates for these kinases is the adaptor protein Cbl [3] and recent studies suggest that Cbl negatively regulates upstream kinases such as Syk and Zap-70 [4,5]. Cbl-b, a homologue of Cbl, is widely expressed in many tissues and cells including hematopoietic cells [6,7]. Cbl-b undergoes rapid tyrosine phosphorylation upon stimulation of the TCR and cytokine receptors [8,9]. The role of Cbl-b is unclear, however. Here, we show that overexpression of Cbl-b in T cells induced the constitutive activation of the transcription factor nuclear factor of activated T cells (NFAT). A loss-of-function mutation in Cbl-b disrupted the interaction between Cbl-b and Zap-70 and nearly completely abrogated the Cbl-b-mediated activation of NFAT. Unlike the proposed role of Cbl as a negative regulator, our results suggest that the Cbl homologue Cbl-b has a positive role in T-cell signaling, most likely via a direct interaction with the upstream kinase Zap-70

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

AbstractVav and PKCθ 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 PKCθ 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 PKCθ 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 PKCθ to the T cell synapse and its activation, essential processes for T cell activation and IL-2 production

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

Translocation of PKCθ in T cells is mediated by a nonconventional, PI3-K– and Vav-dependent pathway, but does not absolutely require phospholipase C

PKCθ plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-κB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCθ to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCθ, we addressed here the question of whether the translocation and activation of PKCθ in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCγ1-deficient T cell line, or a dominant negative PLCγ1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCθ are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCα. Membrane or lipid raft recruitment of PKCθ (but not PKCα) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCθ, but did not associate with it. These results provide evidence that a nonconventional PI3-K– and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCθ in T cells

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

Identification of a Novel Alternatively Spliced Form of Inflammatory Regulator SWAP-70-Like Adapter of T Cells

福岡歯科大学2016年

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

Regulation of immune system cell functions by protein kinase C

Members of the protein kinase C (PKC) family of Ser/Thr kinases are encoded by nine distinct but closely related genes, which give rise to more than 12 different protein isoforms via a mechanism of alternative RNA splicing. Most PKC proteins are ubiquitously expressed and participate in a plethora of functions in most cell types. A majority of PKC isoforms is also expressed in cells of the immune system in which they are involved in signal transduction downstream of a range of surface receptors, including the antigen receptors on T and B lymphocytes. PKC proteins are central to signal initiation and propagation, and to the regulation of processes leading to immune cell proliferation, differentiation, homing and survival. As a result, PKC proteins directly impact on the quality and quantity of immune responses and indirectly on the host resistance to pathogens and tendency to develop immune deficiencies and autoimmune diseases. A significant progress was made in recent years in understanding the regulation of PKC enzymes, their mechanism of action and their role in determining immunocyte behavior This volume reviews the most significant contributions made in the field of immune cell regulation by PKC enzymes. Several manuscripts are devoted to the role of distinct PKC isoforms in the regulation of selected immunocyte responses. Additional manuscripts review more general mechanisms of regulation of PKC enzymes, either by post-translational modifications, such as phosphorylation or controlled proteolysis, or by interaction with different binding proteins that may alter the conformation, activity and subcellular location of PKC. Both types of mechanisms can introduce conformational changes in the molecule, which may affect its ability to interact with cofactors, ATP, or substrates. This topic will be followed by a discussion on the positive and negative impact of individual PKC isoforms on cell cycle regulation. A second section of this volume concentrates on selected topics relevant to role of the novel PKC isoform, PKC-theta, in T lymphocyte function. PKC-theta plays important and some non-redundant roles in T cell activation and is a key isoform that recruits to the immunological synapse - the surface membrane area in T cells that comes in direct contact with antigen presenting cells. The immunological synapse is formed in T cells within seconds following the engagement of the TCR by a peptide-bound MHC molecule on the surface of antigen-presenting cells. It serves as a platform for receptors, adaptor proteins, and effector molecules, which assemble into multimolecular activation complexes required for signal transduction. The unique ability of PKC-theta to activate the NF-kB, AP-1 and NF-AT transcription factors is well established, and recent studies contributed essential information on the mechanisms involved in the recruitment of PKC-theta to the center of the immunological synapse and the nature of its substrates and the role of their phosphorylated forms in signal transduction. Additional review manuscripts will describe the unique behavior of PKC-theta in regulatory T cells and its role in the regulation of other cell populations, including those of the innate immune response. This volume brings together leading experts from different disciplines that review the most recent discoveries and offer new perspectives on the contributions of PKC isoforms to biochemical processes and signaling events in different immune cell populations and their impact on the overall host immune response