sq-23377 and Lymphoproliferative-Disorders

Deficiency of SAP (SLAM (signaling lymphocyte activation molecule)-associated protein) protein is associated with a severe immunodeficiency, the X-linked lymphoproliferative disease (XLP) characterized by an inappropriate immune reaction against Epstein-Barr virus infection often resulting in a fatal clinical course. Several studies demonstrated altered NK and T cell function in XLP patients; however, the mechanisms underlying XLP disease are still largely unknown. Here, we show that non-transformed T cell lines obtained from XLP patients were defective in several activation events such as IL-2 production, CD25 expression, and homotypic cell aggregation when cells were stimulated via T cell antigen receptor (TCR).CD3 but not when early TCR-dependent events were bypassed by stimulation with phorbol 12-myristate 13-acetate/ionomycin. Analysis of proximal T cell signaling revealed imbalanced TCR.CD3-induced signaling in SAP-deficient T cells. Although phospholipase C gamma 1 phosphorylation and calcium response were both enhanced in T cells from XLP patients, phosphorylation of VAV and downstream signal transduction events such as mitogen-activated protein kinase phosphorylation and IL-2 production were diminished. Importantly, reconstitution of SAP expression by retroviral-mediated gene transfer completely restored abnormal signaling events in T cell lines derived from XLP patients. In conclusion, SAP mutation or deletion in XLP patients causes profound defects in T cell activation, resulting in immune deficiency. Moreover, these data provide evidence that SAP functions as an essential integrator in early TCR signal transduction.

Topics: Calcium; Carrier Proteins; CD3 Complex; Cell Adhesion; Cell Division; Cell Line, Transformed; Chromosomes, Human, X; Cytoplasm; Flow Cytometry; Humans; I-kappa B Proteins; Interleukin-2; Intracellular Signaling Peptides and Proteins; Ionomycin; Lymphoproliferative Disorders; MAP Kinase Signaling System; Mutagens; Mutation; NF-KappaB Inhibitor alpha; Phospholipase C gamma; Phosphorylation; Receptors, Antigen, T-Cell; Receptors, Interleukin-2; Retroviridae; Signal Transduction; Signaling Lymphocytic Activation Molecule Associated Protein; T-Lymphocytes; Tetradecanoylphorbol Acetate; Time Factors; Type C Phospholipases

Posttransplant patients undergoing prolonged cyclosporine A (CsA) immunosuppressive therapy have been reported to have increased incidence of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. We undertook experiments to analyze the possible actions of CsA during EBV-infection of human peripheral blood mononuclear cells (PBMC). EBV-infected B cells cultured with CsA demonstrated increased EBV B-cell outgrowth as compared with those cultured without CsA. PBMC, after infection with EBV and CsA treatment, demonstrated increased interleukin-6 (IL-6) activity in the culture supernatant. The induction of IL-6 appears to differ within the various lymphocyte populations. In monocytes, IL-6 expression appears preferentially induced by EBV and is initiated by the binding of the two major virion glycoproteins, gp350 and gp220. Expression of IL-6 in T cells appears to be due mainly to CsA. B cells also express IL-6 after EBV exposure, but not after CsA treatment. EBV-immortalized B-cell lines cultured with CsA exhibited both an increased number of cells expressing viral lytic-cycle antigens and increased amounts of lytic-cycle proteins. IL-6, which is induced by CsA in PBMC, was also capable of inducing the lytic viral cycle in several EBV-immortalized cells. CsA, in promoting both increased numbers of lytic EBV B cells and an EBV paracrine factor, IL-6, within the microenvironment of EBV B cell:T cell and EBV B cell:monocyte interactions, may result in increased EBV B-cell immortalization and ultimately lead to the promotion of B-cell lymphomas in immunosuppressed patients.

Topics: Antigens, Viral; B-Lymphocytes; Cell Transformation, Viral; Cells, Cultured; Cyclosporine; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Humans; Immunocompromised Host; Interleukin-6; Ionomycin; Lymphoma, B-Cell; Lymphoproliferative Disorders; Monocytes; Polymerase Chain Reaction; Receptors, Complement 3d; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Tumor Virus Infections; Viral Matrix Proteins; Virus Activation

C57BL/6Kh mice were infected with a single i.p. injection of 1 x 10(5) FFU of LP-BM5 MuLV. The development and progress of the virus-induced lymphoproliferative disease was followed for 12 weeks after infection. As anticipated, progressive splenomegaly and lymphadenopathy, as well as almost total abrogation of immune responsiveness ensued. In contrast to previous reports, there was a dramatic increase in the frequency of CD4+ cells in spleens among which over 20% expressed V beta 5 TCR, as compared with fewer than 3% in spleens of normal mice. Spleen cells from infected mice retained their in vitro ability to proliferate upon stimulation with IL-2 and anti-CD3, but were unable to respond when stimulated with phorbol ester and either a low dose of IL-2 or calcium ionophore (ionomycin). A similar pattern of in vitro proliferative responses was obtained when normal spleen cells were treated with K252a compound, a known inhibitor of protein kinase C activity. Together with the observations that viral infection impaired down-regulation of the phorbol-induced kinase activity and that the kinase inhibitor only marginally enhanced suppression of virus-infected cells proliferation, this finding suggests that disturbances of protein kinase C activity may underly the pathological effects seen after viral infection. However, since no apparent quantitative and qualitative changes in protein kinase C itself and its translocation were observed, it is more likely that the virus may interfere with either the substrate or product of kinase activity.

Topics: Animals; Antibody-Producing Cells; Calcium; Cell Count; Cells, Cultured; Female; Flow Cytometry; Ionomycin; Lymph Nodes; Lymphoproliferative Disorders; Male; Mice; Mice, Inbred C57BL; Murine Acquired Immunodeficiency Syndrome; Protein Kinase C; Spleen; Tetradecanoylphorbol Acetate

Peripheral lymphoid tissues of lpr mice contain a large proportion of TCR alpha beta/CD3+CD4-CD8- T cells that lack surface CD2 and express the B cell isoform of CD45, B220. This subset of T cells does not proliferate or produce IL-2 in response to mitogenic signals or TCR-CD3 ligation. At the same time, these abnormal T cells display several characteristics of an activated phenotype. Collectively, these properties of lpr CD4-CD8- T cells have functional parallels with anergic T cells. A critical co-stimulatory molecule implicated in the prevention of or recovery from anergy is CD28, which binds the ligand BB1/B7 on certain accessory cells. lpr CD4-CD8- T cells express normal levels of CD28 which is capable of transducing a strong proliferative signal to these cells in co-stimulation with mitogens. However, proliferation of lpr CD4-CD8- T cells in response to CD28 co-stimulation does not reach the levels observed in normal T cells stimulated under similar conditions. Stimulation with anti-CD28 mAb in conjunction with phorbol myristate acetate and ionomycin promotes cell cycling in the CD2- subset of CD4-CD8- T cells, and results in a slight induction of CD2 levels during the course of the culture period. However, the majority of cells obtained at the end of the culture period remain TCR alpha beta+ CD4-CD8-, CD2low/- and B220high, similar to freshly isolated CD4-CD8- lpr T cells. In contrast, if IL-2 is included in the cultures, a strong shift toward a CD2+ phenotype is observed by a majority of the lpr T cells. Upon repeat stimulation, these lpr CD4-CD8- T cells can now proliferate in an IL-2-dependent manner when stimulated with only anti-CD3 mAb or mitogens, in the absence of exogenous IL-2 or anti-CD28 mAb. These data show that the hyporesponsiveness of lpr CD4-CD8- T cells does not result from a lack of CD28 expression, that it is not a fixed state, and that it can be reversed by the induction of cell cycling in the presence of IL-2. These observations extend the parallels between lpr CD4-CD8- T cells and anergic T cells.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; CD2 Antigens; CD28 Antigens; Cells, Cultured; Clonal Anergy; Flow Cytometry; Interleukin-2; Ionomycin; Lymphocyte Activation; Lymphoproliferative Disorders; Mice; Mice, Inbred Strains; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Immunologic; T-Lymphocytes; Tetradecanoylphorbol Acetate

Mice homozygous for the lymphoproliferation (lpr) gene spontaneously develop autoimmune syndrome. These mice were characterized by the massive accumulation of double negative (DN) T cells. Although peripheral T cells in normal mice do not express J11d antigen, those abnormal DN T cells in autoimmune-prone mice express J11d antigen. In this study, the mechanisms that control the expression of J11d antigen are analyzed. High concentration of calcium ionophore alone induces the expression of J11d antigen, but not of CD4, CD8, and activation antigens such as interleukin 2 receptor as well as transferrin receptor by J11d- DN T cells from lpr mice. The expression of J11d antigen is primarily regulated at the transcription level rather than the post transcription level. Experiments using metabolic inhibitors reveal that the induction of J11d antigen requires the activation of not only a Ca2+/calmodulin- but also protein kinase C-dependent signaling pathway. Furthermore, J11d- DN thymocytes from control mice share the similar functional property with DN lpr T cells in J11d antigen inducibility.

Topics: Animals; Antigens, CD; Antigens, Differentiation; Autoimmune Diseases; Base Sequence; Calcimycin; CD24 Antigen; CD4 Antigens; CD8 Antigens; Female; Ionomycin; Lymphoproliferative Disorders; Membrane Glycoproteins; Mice; Molecular Sequence Data; T-Lymphocytes