Amnon Altman

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Papers by Amnon Altman

Constitutive and mitogen-induced production of T cell growth factor by stable T cell hybridoma lines

The Journal of Immunology

Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructe... more Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructed by fusing murine alloantigen-activated T cells with the 8-azaguanine-resistant lymphoma line, BW5147. Many, but not all, clones of one of these hybridomas, i.e., hybridoma 24, secreted TCGF constitutively, but production was markedly enhanced by stimulation with T cell mitogens. Large numbers of TCGF-secreting hybridoma cells in a stable functional state could be obtained from histocompatible mice inoculated with cloned T cell hybridomas. Moreover, such in vivo-derived hybridoma cells could be stimulated sequentially with mitogen at least twice to secrete their biologically-active product, resulting in larger TCGF yields from the same cells. The secreted product of these T cell hybridoma lines resembled TCGF isolated from other cellular sources in that it: a) supported the growth of a TCGF-dependent T cell line; b) provided help for the induction of alloantigen-reactive cytotoxic T lym...

Macrophage-activating factor produced by a T cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon

The Journal of Immunology

Biochemical and biosynthetic evidence has been obtained which indicates that the macrophage activ... more Biochemical and biosynthetic evidence has been obtained which indicates that the macrophage activating factor (MAF) produced by the murine T cell hybridoma clone 24/G1, which primes macrophages for nonspecific tumoricidal activity, is a form of gamma-interferon (IFN gamma). MAF and antiviral activities were generated by the clone in proportional amounts under a variety of culture conditions. Production of both activities showed identical dependence on cell density even when production precipitously ceased at cell concentrations greater than 1.2 X 10(6) cells/ml. MAF and antiviral activities displayed identical sensitivities to pH and temperature and were indistinguishable on the basis of binding to insolubilized polynucleotides. Dye-ligand chromatography of a stimulated hybridoma supernatant on a column of Matrex Gel Red A resulted in the 1500-fold purification and 100% recovery of the MAF activity. A qualitatively identical elution profile was also obtained for the antiviral activi...

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Protein Kinase Cθ Cooperates with Calcineurin to Induce Fas Ligand Expression During Activation-Induced T Cell Death

The Journal of Immunology

Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) ... more Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKCθ, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKCθ mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKCθ mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKCθ synergized with calcineurin to provide a potent stimulus ...

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SLAT-IP3R interaction: implications and functional relevance in T cell activation and calcium signaling (P1172)

The Journal of Immunology

SWAP-70-like adaptor of T cells (SLAT) is a guanine nucleotide exchange factor for Rho GTPases th... more SWAP-70-like adaptor of T cells (SLAT) is a guanine nucleotide exchange factor for Rho GTPases that regulates the activation and differentiation of CD4+ T cells by controlling the Ca2+-NFAT signaling pathway. In response to T cell receptor (TCR)/CD28 costimulation, SLAT promotes calcium signaling by regulating calcium release from endoplasmic reticulum (ER) stores. Hence, SLAT-deficient T cells display a severe defect in Ca2+ release from the ER, despite normal activation of PLCg1 and intact production of inositol-1,4,5-trisphosphate (IP3). Here we report a direct and TCR-induced association between SLAT and the (IP3) receptor type 1 (IP3R1), an ER-resident calcium channel, which mediates calcium release from intracellular stores. Using biochemical approaches, we found that both a N-terminal domain, containing an EF-hand motif, and the pleckstrin homology (PH) domain of SLAT bind directly a defined ~20 amino acid region in the regulatory domain of IP3R1. Subcellular fractionation an...

Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells

The Journal of Immunology

Ag receptor triggering in B cells stimulates the activity of receptor-associated tyrosine protein... more Ag receptor triggering in B cells stimulates the activity of receptor-associated tyrosine protein kinases (TPK), leading to tyrosine phosphorylation of several cellular substrates, one of which is the Vav proto-oncogene product. We have recently determined that Vav is a TPK-regulated guanine nucleotide exchange factor for Ras in T cells. Here, we show that B cell extracts or Vav immunoprecipitates contain a Ras GDP/GTP exchange activity that is stimulated upon surface Ig (slg) triggering. The receptor-mediated stimulation of Vav exchange activity was blocked by the TPK antagonist, herbimycin A. Furthermore, immunodepletion of Vav from the B cell extracts removed approximately 80% of the Ras GDP/GTP exchange activity. These findings indicate, first, that B cell-derived Vav possesses GDP/GTP exchange activity for Ras; second, that the exchange activity of Vav is accelerated by a slg-triggered, herbimycin A-sensitive TPK and, third, that Vav accounts for most of the receptor-stimulated...

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Stimulation of PIP2 hydrolysis by aluminum fluoride in resting T cell subsets of normal and autoimmune-prone lpr mice

The Journal of Immunology

T cells of autoimmune-prone mice homozygous for the lpr mutation respond poorly to mitogens in te... more T cells of autoimmune-prone mice homozygous for the lpr mutation respond poorly to mitogens in terms of proliferation and of IL-2 production. In a previous study, we have correlated this deficient activation with the inability of mitogens to stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate in lpr T cells, although these cells bind mitogen and express the TCR/CD3 complex. In order to determine whether activation-deficient lpr T cells contain functional GTP-binding (G) protein(s) and phospholipase C, we examined the effects of the G protein activating agent sodium fluoride plus Al+3 (AlF-4). AlF-4 stimulated phosphatidylinositol turnover, a response characteristic of TCR/CD3 occupancy, in mature L3T4+ and Ly2+ T cells. Second, and more important, AlF-4 stimulated the same biochemical events in L3T4-, Ly2- (double-negative) T cells from the normal thymus or from the enlarged lymph nodes of autoimmune-prone mice homozygous for the lpr mutation. However, these double-negativ...

Suppression of T lymphocyte function by measles virus is due to cell cycle arrest in G1

The Journal of Immunology

Measles virus suppresses T lymphocyte functions in vitro. When measles virus-infected T lymphocyt... more Measles virus suppresses T lymphocyte functions in vitro. When measles virus-infected T lymphocytes are stimulated with PHA or 12-O-tetradecanoylphorbol-13-acetate, plus calcium ionophore, the cells secrete IL-2 and express the IL-2R or Tac Ag to a similar extent as uninfected cells, yet proliferation is reduced by 50 to 90%. Stimulated infected T cells also express the cell surface activation Ag 4F2, transferrin R, and HLA-DR. The secretion of IFN-gamma by infected T cells in response to PHA is not suppressed at 24 to 72 h after stimulation. Total RNA synthesis at 48 and 72 h after stimulation is reduced in infected T lymphocytes. Infectious measles virus progeny are produced during this interval. Thus infected T lymphocytes can become activated in response to mitogenic stimuli and the cells support efficient viral replication before the block in cell proliferation.

Production of interleukin 2 (IL 2) by salivary gland lymphocytes in Sjögren's syndrome. Detection of reactive cells by using antibody directed to synthetic peptides of IL 2

The Journal of Immunology

Because abnormalities in interleukin 2 (IL 2) production have been reported in the blood of patie... more Because abnormalities in interleukin 2 (IL 2) production have been reported in the blood of patients with certain autoimmune diseases, we have examined the lymphocytes from patients with Sjögren's syndrome (SS) in which it is possible to obtain simultaneous samples of inflammatory site (i.e., salivary gland) lymphocytes and blood lymphocytes. We found that IL 2 production by peripheral blood lymphocytes (PBL) after mitogen stimulation was markedly diminished (4 +/- 2 U/ml) in 8/32 SS patients. However, salivary gland lymphocytes (SGL) from six out of six SS patients (including three patients with low IL 2 production by their PBL) had a high level of IL 2 production (97 +/- 32 U/ml), suggesting that IL 2 production by inflammatory site lymphocytes may differ from blood lymphocytes in the same patients. Low IL 2 production by a patient's PBL was not correlated with the patient's age, duration of disease, immunoglobulin level, or presence of antinuclear antibodies. Low IL 2...

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Protein Kinase C Activation Inhibits Tyrosine Phosphorylation of Cbl and Its Recruitment of Src Homology 2 Domain-Containing Proteins

The Journal of Immunology

One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3... more One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKCα and PKCθ can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is...

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Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes

The Journal of Immunology

Antigen binding to its specific receptor on T cells initiates a series of intracellular events th... more Antigen binding to its specific receptor on T cells initiates a series of intracellular events that result in cell differentiation, activation, and clonal expansion. However, the mechanism by which these antigen-occupied receptors induce the transmembrane signal transduction needs clarification. Because this mechanism appears to involve an increase in intracellular free Ca2+ concentration and activation of protein kinase C (PKC), we tested the effect of Ca2+ ionophores and PKC activators on alloantigen-specific primary mixed leukocyte culture cells. Both calcium ionophores, A23187 and ionomycin, in conjunction with 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of antigen or interleukin 2 (IL 2) by inducing strong proliferative and alloantigen-specific cytotoxic responses. In addition, Ca2+ ionophore and TPA induced IL 2 receptor expression and IL 2 secretion. The capacity of other phorbol esters or a non-phorbol ester tumor promoter (teleocidin) to replace TPA in in...

The tumor promoter teleocidin induces IL 2 receptor expression and IL 2-independent proliferation of human peripheral blood T cells

The Journal of Immunology

In testing the recently discovered tumor promoter teleocidin (TCD), we found that like phorbol es... more In testing the recently discovered tumor promoter teleocidin (TCD), we found that like phorbol esters, TCD was mitogenic to human peripheral blood lymphocytes (PBL) and preferentially stimulated sheep erythrocyte-rosetted (ER) T cell-enriched populations. Stimulation of PBL with TCD induced synthesis and expression of receptors for interleukin 2 (IL 2), as shown by dot-blot analysis with the use of a synthetic oligonucleotide probe, cell surface staining with anti-Tac antibody followed by fluorescence-activated cell sorter analysis, and a functional proliferation assay in which TCD-stimulated cells were washed free of TCD and were recultured with human recombinant IL 2 (rIL 2). Increased expression of cell surface markers after TCD stimulation of PBL is not general, because TCD did not affect the expression of Leu-2a antigen, and it also reduced the density of Leu-3a and Leu-4 antigens. Stimulation of cultured, IL 2 receptor-positive PBL with rIL 2, but not TCD, was blocked by anti-...

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Human T lymphocyte activation by tumor promoters: role of protein kinase C

The Journal of Immunology

Protein kinase C (PKC) has a major role in a ligand-receptor-mediated signal transduction system ... more Protein kinase C (PKC) has a major role in a ligand-receptor-mediated signal transduction system in a variety of cell types including T lymphocytes. One of the early phenotypic changes associated with T cell activation is the expression of cell surface receptors for interleukin 2 (IL 2). To test the role of PKC in regulation of IL 2 receptor (IL 2-R) expression and T cell activation in general, we used tumor promoters (TP) as modulators of PKC and compared their effects on intact human T cells and on the enzymatic activity of T cell-derived PKC in a cellfree system. In T cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) induced IL 2-R expression and proliferation associated with cytosol-to-membrane PKC translocation. A dose of TPA (1 to 4 ng/ml) that induced about 50% of the maximal activation of PKC in the enzymatic assay also induced half-maximal effects on cell proliferation, IL 2-R expression, and PKC redistribution in intact T cells. Structure-function studie...

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Inhibition of phorbol ester-induced T cell proliferation by bryostatin is associated with rapid degradation of protein kinase C

The Journal of Immunology

The bryostatins (Bryo) represent a group of immunomodulators that counteract the tumor-promoting ... more The bryostatins (Bryo) represent a group of immunomodulators that counteract the tumor-promoting effects of PMA. In contrast to the mitogenic effect of PMA on human peripheral blood T lymphocytes, Bryo was nonmitogenic and, furthermore, it inhibited PMA-induced T cell proliferation in a dose-dependent manner when added up to 2 days after PMA. Because both Bryo and PMA bind to, and activate, protein kinase C (PKC), we compared their effects on PKC expression and activity in human PBL or leukemic T cells (Jurkat). After treatment for 5 to 60 min, both Bryo and PMA were found to: a) activate PKC in vitro with similar dose-response curves; b) induce a nearly complete cytosol-to-membrane translocation of enzymatically active, Ca(2+)-dependent PKC and of distinct immunoreactive PKC isoforms in intact PBL; and c) stimulate similar patterns of protein phosphorylation. After a longer, 20-h treatment with PMA (20 nM), a considerable portion of PKC was still membrane-associated, and the total ...

The biologic effects of allogeneic effect factor on T lymphocytes. I. The mitogenic activity and the autonomous induction of cytotoxic T lymphocytes by AEF

The Journal of Immunology

Studies presented herein illustrate the capacity of the soluble mediator, allogeneic effect facto... more Studies presented herein illustrate the capacity of the soluble mediator, allogeneic effect factor (AEF), which is derived from histoincompatible cell interactions, to induce the in vitro differentiation of normal murine splenic lymphocytes into mature cytotoxic cells capable of exerting activity on H-2-identical target cells. This process requires the presence of T lymphocytes during the sensitization phase, and the lytic activity on tumor cells is mediated by cytotoxic T lymphocytes (CTL). The capacity of AEF to induce differentiation of such CTL does not require the presence of stimulating target cells in the sensitization phase. The induction of CTL requires the presence of AEF at the initiation of culture, although exposure to AEF as brief as 1 hr is sufficient to induce fresh spleen cells to differentiate into CTL during the subsequent 5 days in culture. In addition to its ability to induce CTL, AEF is highly mitogenic for T lymphocytes. However, the mitogenic and the CTL-indu...

The biologic effects of allogeneic factor on T lymphocytes. II. The specificity of AEF-induced cytotoxic T lymphocytes

The Journal of Immunology

The accompanying communication documented the ability of allogeneic effect factor (AEF) to induce... more The accompanying communication documented the ability of allogeneic effect factor (AEF) to induce a murine primary cytotoxic T lymphocyte (CTL) response in vitro in the absence of an exogenous antigen source. In this study we investigated the specificity of CTL induced by AEF. The activity of such CTL was not directed against fetal calf serum (FCS) determinants since they were fully efficient in lysing target cells that were not exposed to FCS. Furthermore, FCS was not required for the induction of a primary CTL response in vitro since a substantial level of CTL activity was induced by AEF in medium containing fresh mouse serum. However, this activity was lower than that induced in FCS-containing medium, and the mouse serum appeared to contain factor(s) capable of inhibiting the AEF-induced CTL response. In addition to tumor target cells, lymphoblasts induced by B or T cell mitogens were also susceptible to lysis by AEF-induced CTL. Addition of H-2-alloantisera during the effector p...

Manipulation of Cytotoxic T Lymphocyte Responses to Syngeneic Tumors

The Journal of Immunology

This study was designed to extend a recent report about the ability of hapten-primed T lymphocyte... more This study was designed to extend a recent report about the ability of hapten-primed T lymphocytes to serve as helper cells, capable of augmenting the generation of tumor-specific CTL in vivo when the priming hapten was coupled to the immunizing tumor cells. Spleen cells of mice primed with 2,4,6-trinitrophenyl-mouse γ-globulin (TNP-MGG) conjugate under conditions known to elicit hapten-reactive T lymphocytes, and subsequently immunized with TNP-derivatized, mitomycin C-treated syngeneic tumor cells, generated, upon in vitro stimulation with the relevant tumor cells, substantially higher CTL responses than did spleen cells from mice that were not primed with TNP-MGG. Furthermore, we demonstrated directly that such help resulted from T-T cell cooperation by showing that the addition of TNP-MGG-primed spleen cells in culture augmented the in vitro induction of a secondary tumorspecific CTL response upon sensitization of tumor-primed spleen cells against TNP-derivatized tumor cells. Th...

Natural Cell-Mediated Cytotoxicity in Guinea Pigs: Properties and Specificity of Natural Killer Cells

The Journal of Immunology

Lymphoid cells of guinea pigs were found to be naturally cytotoxic against a variety of target ce... more Lymphoid cells of guinea pigs were found to be naturally cytotoxic against a variety of target cells. Natural killer (NK) cell activity was high in peripheral blood and spleen, low in lymph nodes and peritoneal exudates, and absent in the thymus. Significant activity was found between the ages of 3 and 40 weeks, and strain 2 guinea pigs had a higher NK activity than strain 13 guinea pigs. Peripheral blood NK cells were sensitive to treatment with ammonium chloride solution, whereas splenic NK cells were resistant to the same treatment. The NK cells were found to be nonadherent to nylon wool and plastic and were enriched among cells bearing receptors for the Fc portion of IgG. The NK activity of guinea pig lymphocytes was selective in that only some, but not all, of the target cells used were susceptible to the cytotoxic effector cells. Susceptible target cells included syngeneic, allogeneic, and xenogeneic tumor cell lines, especially the human myeloid cell line K562, and some passa...

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Existence of T cells manifesting self-reactivity indistinguishable from alloreactivity

The Journal of Immunology

The studies reported here were designed to analyze the phenotypic characteristics of self-reactiv... more The studies reported here were designed to analyze the phenotypic characteristics of self-reactive T lymphocytes induced in culture by allogeneic effect factor (AEF), as well as the control of their functional activities by the major histocompatibility complex (MHC). Unprimed T cells cultured with AEF in the absence of exogenous stimulating target cells become activated against self-antigens, as evidenced by their ability to manifest two distinct activities. First, such cells could lyse syngeneic target cells. This cytolytic activity was directed against H-2K antigens and was mediated by Lyt-2+ T cells. Second, the AEF-activated T cells could be stimulated in a secondary culture to high levels of proliferative activity by irradiated syngeneic spleen cells. The stimulator cells in this syngeneic mixed lymphocyte reaction (MLR) were found to be Thy-1-negative, Ia-positive splenic adherent cells. Stimulation in the secondary syngeneic MLR was provided by I-region specificities, and the...

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Production of antigen-nonspecific immunoregulatory lymphokines by T cell hybridomas

Current topics in microbiology and immunology, 1982

A large number of non-immunoglobulin soluble mediators or lymphokines (LK) with immunoregulatory ... more A large number of non-immunoglobulin soluble mediators or lymphokines (LK) with immunoregulatory and other biological activities have been studied over the last 12 years. The majority of these LK are products of T cells activated by antigens or polyclonal mitogens. Studies on T cell-derived LK have been severely hampered by the limited amounts of LK available from conventional cultures and the heterogeneity of the cell populations producing these LK which, in turn, resulted in heterogeneous mixtures of many distinct LK. As a result of these limitations, it has been difficult to delineate the exact chemical nature of LK, the structure-function relationship among various biological activities and their biological significance.

Antigen-induced translocation of SLAT to immunological synapse is required for NFAT activation and Th1/Th2 immune responses

The FASEB Journal, 2008

Amnon Altman

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