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O Leo

Publications and source records attributed to O Leo.

At least 55 records · Page 3Linked to original sources
PubMed1996

A model for antigen-induced T cell unresponsiveness based on autophosphorylative protein tyrosine kinase activity.

Helper T cell signaling is initiated by the aggregation of TCR with the induction of tyrosine kinase activity as one of the earliest consequences. Here, a theoretical model for antigen-induced unresponsiveness is presented that relies on a cascade of tyrosine phosphorylation-dephosphorylation cycles. A mechanism is described for both desensitization in the presence of antigen and persistent lowering of cell responsiveness after stimulus removal. An important component of the model, leading to bistability, is the presence of autophosphorylating protein tyrosine kinases in the early steps of TCR signaling. One of its predictions is that, following stimulation, the net phosphorylative activity of these receptor-associated tyrosine kinases will remain above background level after removal of the antigen. It is proposed that this residual tyrosine kinase activity is linked to a deficient signal transduction capacity of the TCR system that leads to a state of prolonged unresponsiveness. In addition, the present analysis defines the notion of a signaling threshold for hyporesponsiveness induction, associated with a durable switch and amplification of the net tyrosine kinase activity. This approach emphasizes the role of tyrosine kinases in the down-regulation of cellular competence.

Antigen Presentation↗
PubMed1995

Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth.

Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals.

Animals↗
PubMed1995

Lack of T cell tolerance in mice exposed to a protein antigen through lactation.

Offspring of mother mice treated immediately after delivery with deaggregated human gamma-globulins (dHGG) are unable to produce HGG-specific antibodies when challenged with immunogenic forms of HGG (HGG/CFA) in adulthood. Despite a defective antibody response, animals rendered tolerant to HGG as neonates retain tolerogen-specific T cells able to proliferate and secrete lymphokines. The pattern of IL-2 and IL-4 secretion by T cells isolated from tolerant animals could not be distinguished from the corresponding cells in control mice, suggesting that neonatal exposure to dHGG did not affect T cell reactivity or Th1/Th2 in vivo balance. Moreover, immunization of tolerant animals with haptenated HGG confirmed the presence of tolerogen-specific helper T cells in vivo. Functional T cell depletion by anti-CD3 mAbs during lactation failed to modify induction of B cell tolerance, suggesting that T cells are neither affected nor required to induce the selective tolerance status observed in this model. Based on the finding that antigen-presenting cell functions in secondary organs (spleen, peritoneal cavity) are a late acquisition during ontogeny and reach adult-like levels at weaning, we propose that most soluble proteins elude T cell recognition during lactation due to defective antigen presentation.

Animals↗
PubMed1995

Assessment of a functional role of auto-anti-idiotypes in idiotype dominance.

We have used a well-defined idiotypic system, the cross-reactive idiotype of A strain (CRIA) (Ab1) idiotype generated in A/J mice injected with arsonate coupled to keyhole limpet hemocyanin (ARS-KLH), to determine the frequency of precursors for auto-anti-idiotypic antibodies (auto-Ab2) in naive and immunized A/J mice by limiting dilution analysis after polyclonal activation by lipopolysaccharide. In naive animals, the precursor frequencies of auto-Ab2 B cells were below the limit of sensitivity of the technique in the majority of A/J mice, and could be detected in only 20% of the animals. Upon immunization with ARS-KLH, a large increase in auto-Ab2 precursor frequency was observed. This shift in frequency was not found when A/J mice were injected with KLH alone, or when BALB/c mice, which do not express the CRIA idiotype, were injected with ARS-KLH. To study the functional role of the auto-Ab2 B cells, we injected neonatal A/J mice with polyclonal rabbit Ab3 antibodies directed against a recurrent idiotype of auto-Ab2. Thereafter, these mice were injected with ARS-KLH. Although the anti-arsonate response level was normal, the CRIA Ab1 expression was reduced tenfold. Thus, the suppression of auto-Ab2 affects Ab1 dominance. We further show that the presence of maternal Ab1 can strongly modify the immune response of the offspring by inducing higher levels of the idiotype after immunization. Furthermore, IgM anti-arsonate antibodies were detected before immunization with antigen. From these data, we conclude that the affinity of antigen alone cannot explain the dominance of CRIA. Network selection is important in the shaping of the available repertoire.

Adjuvants, Immunologic↗
PubMed1995

B7.2 provides co-stimulatory functions in vivo in response to staphylococcal enterotoxin B.

Excessive T cell activation induced by bacterial superantigens plays an important role in the pathology associated with Gram-positive bacteremia. To gain insight into the early phases of T cell activation by bacterial enterotoxins in vivo, we investigated the ability of antibodies to well-defined co-stimulatory molecules to inhibit T cell activation and the subsequent toxic shock syndrome induced in BALB/c mice following the injection of staphylococcal enterotoxin B (SEB). We demonstrate here that a single dose of anti-B7.2 antibodies, but not anti-B7.1 antibodies, significantly inhibits T cell activation, as judged by lower systemic IL-2 release, blastogenesis and IL-2 receptor expression, and reduces the lethal effect of SEB in D-galactosamine-sensitized mice. These results demonstrate that co-stimulation through the B7.2 molecule plays an important role in the activation of T cells in response to SEB in vivo and suggest alternative therapies for septic shock caused by bacterial enterotoxins based on blocking antibodies to co-stimulatory molecules.

Animals↗
PubMed1995

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo.

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively down-regulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.

Animals↗
PubMed1995

Activation of murine T cells by bacterial superantigens requires B7-mediated costimulation.

Staphylococcus enterotoxins bind class II MHC molecules on antigen-presenting cells (APC) and stimulate T cells expressing appropriate V beta gene products. Although the role of non-TcR-associated costimulatory receptors during antigen-specific T cell stimulation has been clearly established, the involvement of costimulatory activity in T cell activation by superantigens (SAgs) has been the matter of controversy. The aim of this study was to evaluate the role of the costimulatory-receptor ligand molecules CD28/B7 on bacterial SAg-mediated activation of naive murine T cells. We demonstrate in this report that a combination of monoclonal antibodies to murine B7.1 and B7.2 molecules inhibits the in vitro response of naive T cells to SAgs SEA, SEB, and TSST-1. The inhibition of T cell responses required simultaneous blocking of B7.1 and B7.2, suggesting that either B7.1 or B7.2 is sufficient to provide costimulatory signals to naive T cells in response to bacterial exotoxins. Inhibition of T cell activation by antibodies to B7-related molecules can be overcome by antibodies to CD28, a finding in agreement with the hypothesis that CD28-mediated signals participate in T cell activation by bacterial SAgs. These observations suggest that, as demonstrated for conventional antigen, T cell activation by SAgs requires the coordinated participation of TcR- and CD28-derived signals.

Animals↗
PubMed1995

Production and characterization of bispecific single-chain antibody fragments.

We report the construction, expression and purification of a bispecific single-chain Fv antibody fragment produced in Escherichia coli. The protein possesses a dual specificity: the single-chain FvB1 portion is directed to the Idiotype of BCL1 lymphoma cells, the single-chain Fv2C11 moiety binds to the CD3 marker on T cells. The two domains are joined by a flexible peptide linker. Using Immobilized Metal Affinity Chromatography, the recombinant protein was purified from bacterial insoluble membrane fractions. After refolding of the bispecific protein, it was affinity-purified. As demonstrated by flow cytometry, both binding sites are retained in the refolded protein. Retargeted cytotoxicity and T cell proliferation assays further prove the biological activity and specificity of the bispecific single-chain Fv. Thus, these bispecific molecules show a potential anti-tumor activity.

Animals↗
PubMed1995

The induction of human T cell unresponsiveness by soluble anti-CD3 mAb requires T cell activation.

In order to obtain an in vitro model of human T cell unresponsiveness induced by soluble anti-CD3 mAb in the presence of accessory cells, T cells purified from peripheral blood of healthy volunteers were cultured for 4 days with irradiated accessory cells and OKT3. After a 48 h resting period allowing TCR-CD3 complex re-expression, T cells were rechallenged with plastic-immobilized OKT3, and their proliferative response as well as their secretion of IL-2, IFN-gamma and IL-10 measured. Primary culture with OKT3 induced a state of unresponsiveness characterized by defective responses to OKT3 rechallenge but normal or enhanced responses to PMA and A23187 calcium ionophore, indicating a defect in the early steps of TCR-CD3-mediated signal transduction. Indeed, we found that unresponsive T cells displayed an impaired mobilization of intracellular calcium stores upon TCR-CD3 ligation. In order to determine whether the development of unresponsiveness depends on the initial T cell activation triggered by OKT3, we compared several versions of OKT3 differing in their ability to bind Fc receptors. We found that only the activating antibodies that bind Fc receptors on accessory cells induced T cell unresponsiveness. We conclude that human resting T cells can be rendered unresponsive by anti-CD3 mAb in soluble form provided that they trigger T cell activation.

Antigen-Presenting Cells↗
PubMed1995

FcR cross-linking on monocytes results in impaired T cell stimulatory capacity.

Presentation of antigen to T lymphocytes without the appropriate co-stimulatory signals results in a state of antigen-specific unresponsiveness. Despite the presumed importance of the B7-CD28 interaction for the initiation and maintenance of T cell-mediated immune responses, relatively few studies have addressed the regulation of B7 expression. We have studied the expression of the CD80 (B7-1) and B7-2 molecules on peripheral blood monocytes following different activation signals, and it was demonstrated that not only IFN-gamma, but also granulocyte macrophage colony stimulating factor can induce CD80 expression on monocytes. In addition, we found that cross-linking of FcR on monocytes strongly inhibits the up-regulation of CD80 and B7-2, with as a functional consequence that the capacity to function as antigen presenting cells (APC) and to stimulate T cell activation is severely impaired. When cultures were prepared in 96-well plates coated with human IgG, stimulation of T cells with allogeneic monocytes resulted only in modest T cell proliferation and no detectable IL-2 secretion as compared with untreated culture plates or plates coated with Fab fragments of human IgG. Under these conditions cross-linking of CD28 on the T cells with specific mAb completely reverted the inhibitory effect observed after culture on IgG-coated plates. Furthermore, FcR cross-linking on monocytes strongly inhibited the capacity of monocytes to induce a specific memory T cell response to viral, bacterial and fungal antigens, whereas the treatment did not impair the capacity of the T cells to respond to pokeweed mitogen, phytohemagglutinin and concanavalin A. We conclude that after FcR cross-linking, the impaired APC function is most likely due to the inability of monocytes to provide the essential co-stimulatory signals to the T cells via the B7-CD28/CTLA-4 interaction.

Antibodies, Monoclonal↗
PubMed1995

Induction of Th2 responses to soluble proteins is independent of B cell tolerance status.

Injection of high doses of monomeric human gamma globulins (dHGG) in naive, adult mice causes antigen-specific tolerance of B cell and Th1 lymphocytes, while inducing the selective expansion of antigen-specific Th2 cells. Several parameters of tolerance induction were analyzed in this work, in order to establish whether B cell tolerance and Th1 unresponsiveness were functionally related in this in vivo model. By varying the antigen form and site of injection, we demonstrate in this work that Th1 unresponsiveness to HGG is not a consequence of peripheral B cell tolerance. In particular, mice pretreated with heat-aggregated antigen (HAHGG) or F(ab')2 HGG were found to develop a strong humoral response while displaying a defective Th1 response. In fact, these animals developed a strong Th2 response in vivo, demonstrating that selective expansion of antigen-specific Th2 cells in this model is not a consequence of B cell tolerance or antigen capture by Fc receptor-expressing cells. We conclude that while B cell tolerance in this model is only observed in response to deaggregated antigen, injection of all forms of adjuvant-free, protein antigens induces T helper precursor cells to differentiate into Th2-type helper cells in vivo irrespectively of the B cell tolerance status.

Animals↗
PubMed1995

Co-stimulation lowers the threshold for activation of naive T cells by bacterial superantigens.

Staphylococcus enterotoxins bind class II MHC molecules on antigen presenting cells (APC) and stimulate T cells expressing appropriate V beta gene products. Although the role of non-TCR associated co-stimulatory receptors during antigen-specific T cell stimulation has been clearly established, the involvement of co-stimulatory activity in T cell activation by superantigens has been the matter of controversy. In this report, we examine the role of co-stimulation provided by selected APC populations in the response to bacterial exotoxins (staphylococcal enterotoxin A, staphylococcal enterotoxin B and toxic shock syndrome type 1). We demonstrate that the APC population able to activate naive T cells to IL-2 production is heterogeneous, comprising both adherent (presumably dendritic) and non-adherent (mostly B lymphocytes) cells. By stimulating naive T cells in the presence of graded doses of superantigens, we have observed that half-maximal IL-2 production was achieved at lower doses of superantigens in the presence of dendritic cells. Similarly, addition of antibodies to CD28 or B7.1-transfected cell lines increased the sensitivity of naive T cells to lower doses of superantigens. These observations indicate therefore that superantigens can be presented to naive T cells by APC displaying distinct levels of co-stimulatory activity, although with different efficacy. Thus, naive T cells are sensitive to CD28-mediated co-stimulation during superantigen-mediated responses but IL-2 production can be induced by high doses of superantigens in the presence of APC expressing weak co-stimulatory activity. These observations are compatible with the hypothesis that CD28-mediated signals participate in T cell activation by lowering T cell sensitivity to TCR ligands.

Animals↗
PubMed1994

Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor.

A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.

Aniline Compounds↗
PubMed1994

Role of T-cell subsets in the bispecific antibody (anti-idiotype x anti-CD3) treatment of the BCL1 lymphoma.

We reported previously on the successful use of bispecific antibodies in two well characterized B-cell lymphoma models. These bispecific antibodies were hybrid-hybridoma antibodies with dual specificity for the TcR/CD3 complex and for the tumor-specific idiotype of the surface IgM expressed by the lymphoma cells. Class-matched control antibodies, either monovalent for CD3, monovalent for idiotype, or bivalent for these surface markers, were always used in parallel with the bispecific antibodies. We extended our studies to determine the relative contribution of antibody-dependent cellular cytotoxicity and a T-cell-mediated therapeutic effect in the BCL1 lymphoma model. In tumor-bearing mice depleted of CD4+, CD8+ or both T-cell subsets and treated with bispecific antibodies, we could show that both T-cell populations contribute to the therapeutic outcome and have an additive role. In vitro studies demonstrate that bridging BCL1 tumor cells to T-cells by bispecific antibodies induces T-cell activation and secretion of tumor growth inhibiting lymphokines by both CD4+ and CD8+ T-cell populations. Particularly gamma-interferon seems to be the major tumor-inhibiting substance for BCL1 tumor cells. However, in vivo experiments using anti-cytokine antibodies showed that both gamma-interferon and tumor necrosis factor alpha have an effect on the tumor growth. The former acts directly by inhibiting tumor growth, the latter via an indirect mechanism, possibly by activating macrophages. In conclusion, our results show that induction of targeted cytolytic activity by the direct CD3/TcR cross-linking and development of targeted cytotoxic activity, mediated by gamma-interferon, by both T-cell subsets, contribute to the therapeutic success of bispecific antibody therapy.

Animals↗