Biomedical subjects

J A Ledbetter

Publications and source records attributed to J A Ledbetter.

At least 37 records · Page 2Linked to original sources

PubMed1997

HLA-DR-triggered inhibition of hemopoiesis involves Fas/Fas ligand interactions and is prevented by c-kit ligand.

The function of MHC class II (HLA-DR) Ags in hemopoiesis is not well defined. Here we investigated the effect of anti-HLA-DR mAb H81.9 on human marrow cells. mAb H81.9 inhibited colony formation from purified CD34+ marrow cells in long term culture-initiating cell assays. Inhibition was prevented, however, if c-kit ligand (stem cell factor (SCF)) was added to cultures concurrently with H81.9. DNA histograms from cultured untreated marrow mononuclear cells showed 2+/-1.2% apoptotic nuclei, whereas 14.1+/-5.4% were apoptotic after 12-h exposure to mAb H81.9. The apoptotic peak was reduced to 1.2+/-0.8% when SCF was added to cultures concurrently with mAb H81.9. The addition of Fas-Ig, a fusion protein that neutralizes Fas ligand (Fas-L), also prevented mAb H81.9-induced apoptosis. As determined by terminal deoxynucleotidyl transferase assays, agonistic anti-Fas mAb also induced apoptosis (in 13+/-4% of cells), and combined treatment with anti-Fas mAb and H81.9 was additive (27% apoptotic nuclei). The extent of apoptosis induced by anti-Fas mAb was significantly reduced by SCF. After H81.9 exposure, Fas was up-regulated on CD34+ cells, and Fas-L expression was 2.5-fold higher than in controls or CD34- cells, particularly within a small cell window with low orthogonal scatter (lymphocyte gate). These findings show that HLA-DR-mediated signals inhibit hemopoiesis in human marrow by a mechanism involving Fas/Fas-L-dependent signals that are blocked by c-kit ligand. These data suggest a possible role for MHC class II molecules in the regulation of hemopoiesis.

Antibodies, Monoclonal↗

PubMed1997

Human monocytic cells contain high levels of intracellular Fas ligand: rapid release following cellular activation.

Human monocytes express both Fas and Fas ligand (FasL) on the cell surface, and the interaction of these molecules induces spontaneous apoptosis. In this report we present a study of monocytic cells by FACS and confocal microscopy using anti-FasL Abs that reveals high levels of preformed FasL within the intracellular compartment. Further analysis by immunoblotting of cell cytoplasmic proteins confirmed the presence of a 37-kDa protein recognized by anti-FasL Abs. Stimulation of the monocytic cells with immune complexes, PHA, or superantigen gave rise to the rapid release of soluble FasL from within the cells. The presence of high levels of FasL within human monocytes suggests that, upon stimulation, the cells can rapidly translocate intracellular FasL to the cell surface and release it into the extracellular milieu. These findings indicate a novel mechanism for monocytes to respond rapidly to environmental changes, resulting in the release of active, soluble FasL.

Cell Line↗

PubMed1997

4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses.

The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

Animals↗

PubMed1997

In vivo efficacy and toxicity of a single-chain immunotoxin targeted to CD40.

G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37 degrees C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and monkeys, with the maximum tolerated dose being 0.48, 1.0, and 1.67 mg/kg, respectively. Comparative immunohistology showed that the G28-5 specificity was qualitatively similar between human and monkey tissue. In summary, G28-5 sFv-PE40 was effective at inducing complete antitumor responses in lymphoma xenografted mice at doses that were well tolerated in mice, rats, and monkeys.

Animals↗

PubMed1997

Role of oxidative stress in the action of vanadium phosphotyrosine phosphatase inhibitors. Redox independent activation of NF-kappaB.

The role of intracellular oxidative stress in the mechanism of action of phosphotyrosine phosphatase (PTP) inhibitors was studied using three vanadium-based compounds. Sodium orthovanadate (Na3VO4), sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen), and bis(maltolato)-oxovanadium(IV) (BMOV) differentially induced oxidative stress in lymphocytes. Treatment with pV(phen), which caused intracellular oxidation, induced strong protein tyrosine phosphorylation compared with Na3VO4 and BMOV. Syk family kinases and the mitogen-activated protein kinase erk2 were rapidly activated by pV(phen) but not by BMOV or Na3VO4. In contrast, both BMOV and pV(phen) strongly activated NF-kappaB. The antioxidant pyrrolidine dithiocarbamate (PDTC) greatly diminished the intracellular oxidation and protein phosphotyrosine accumulation induced by pV(phen). Pretreatment of cells with PDTC reduced and delayed the activation of Syk kinases and erk2. However, NF-kappaB activation by pV(phen) was markedly enhanced in lymphocytes pretreated with PDTC, and another antioxidant, N-acetylcysteine, did not prevent the activation of NF-kappaB by BMOV. These results indicate a role for oxidative stress in the biological effects of some PTP inhibitors, whereas NF-kappaB activation by PTP inhibitors is mediated by mechanisms independent of intracellular redox status.

Antioxidants↗

PubMed1997

Identification of amino acid residues important for ligand binding to Fas.

The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas-FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

Binding Sites↗

PubMed1997

Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages.

Human monocytes undergo spontaneous apoptosis upon culture in vitro; removal of serum from the media dramatically increases the rate of this process. Monocyte apoptosis can be significantly abrogated by the addition of growth factors or proinflammatory mediators. We have evaluated the role of the endogenous Fas-Fas ligand (FasL) interaction in the induction of this spontaneous apoptosis and found that a Fas-immunoglobulin (Ig) fusion protein, an antagonistic anti-Fas monoclonal antibody and a rabbit anti-FasL antibody all greatly reduced the onset of apoptosis. The results indicate that spontaneous death of monocytes is mediated via an autocrine or paracrine pathway. Treatment of the cells with growth factors or cytokines that prevented spontaneous apoptosis had no major effects on the expression of Fas or FasL. Additionally, monocyte-derived macrophages were found to express both Fas and FasL but did not undergo spontaneous apoptosis and were not sensitive to stimulation by an agonistic anti-Fas IgM. These results indicate that protective mechanisms in these cells exist at a site downstream of the receptor-ligand interaction.

Apoptosis↗

PubMed1997

Co-ligation of the antigen and Fc receptors gives rise to the selective modulation of intracellular signaling in B cells. Regulation of the association of phosphatidylinositol 3-kinase and inositol 5'-phosphatase with the antigen receptor complex.

Cross-linking of the Fc receptor (FcR) to surface immunoglobulin (sIg) on B cells inhibits the influx of extracellular calcium and abrogates the proliferative signal. The mechanism by which this occurs is not well understood. In this report we show that co-cross-linking the FcR to the antigen receptor gives rise to very selective modulation of signal transduction in B cells. Co-cross-linking sIg and the FcR enhanced the phosphorylation of the FcR, the adapter protein, Shc, and the inositol 5'-phosphatase Ship. Furthermore, phosphorylation of the FcR induced its association with Ship. Cross-linking of the FcR and sIg decreased the tyrosine phosphorylation of CD19, which led to a reduction in the association of phosphatidylinositol 3-kinase. In addition, the phosphorylation of several other proteins of 73, 39, and 34 kDa was reduced. Activation of the cells with either F(ab')2 or intact anti-IgG induced very similar changes in levels of tyrosine phosphorylation of most other proteins, and no differences in the activation of several protein kinases were observed. These results indicate that the inhibitory signal that is transmitted through the FcR is not mediated by a global shutdown of tyrosine phosphorylation but is, rather, a selective mechanism involving localized changes in the interactions of adapter proteins and the enzymes Ship and phosphatidylinositol 3-kinase with the antigen receptor complex.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Antibody engineering.

The development of recombinant techniques for the rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, and inclusion of self-assembly motifs, novel molecules such as bispecific Abs can now be generated which possess the subset of functional properties designed to fit the intended application. Rapid technological developments in phage display of peptides and proteins have led to a plethora of applications directed towards immunology and antibody engineering. Many of the problems associated with the therapeutic use of Abs are being addressed by the application of these new techniques.

Genetic Engineering↗

PubMed1997

Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors.

The 4-1BB glycoprotein is a member of the tumor necrosis factor receptor superfamily and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. Expression of 4-1BB is restricted to primed CD4+ and CD8+ T cells, and 4-1BB signaling either by binding to 4-1BBL or by antibody ligation delivers a dual mitogenic signal for T-cell activation and growth. These observations suggest an important role for 4-1BB in the amplification of T cell-mediated immune responses. We now show that administration of anti-4-1BB monoclonal antibodies can eradicate established large tumors in mice, including the poorly immunogenic Ag104A sarcoma and the highly tumorigenic P815 masto cytoma. The immune response induced by anti-4- 1BB monoclonal antibodies is mediated by both CD8+ and CD4+ T cells and is accompanied by a marked augmentation of tumor-selective cytolytic T-cell activity. Our data suggest that a similar approach may be efficacious for immunotherapy of human cancer.

Animals↗

PubMed1997

Agonistic activity of a CD40-specific single-chain Fv constructed from the variable regions of mAb G28-5.

A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces.

Animals↗

PubMed1996

Intracellular CD22 rapidly moves to the cell surface in a tyrosine kinase-dependent manner following antigen receptor stimulation.

CD22 is a key accessory molecule for Ag receptor signaling in B cells that becomes tyrosine phosphorylated in the signaling process. CD22 associates with sIg and strongly amplifies sIg-induced signals. During B cell development, CD22 is initially expressed intracellularly, with surface expression appearing with IgD expression. We used confocal laser-scanning microscopy and flow cytometry to analyze CD22 translocation responses to signaling events. Cross-linking surface IgM (sIgM) induced rapid movement of CD22 to the cell surface in both Ramos and Daudi B cells, with a 50 to 100% increase in surface expression observed within 5 min of stimulation. The increase in CD22 surface expression was specific in that CD19 expression was not affected. Both cell surface and intracellular CD22 were directed toward the site of sIgM stimulation. Treatment with the phosphotyrosine phosphatase inhibitor bis(maltolato)oxovanadium(IV) also increased CD22 surface expression. The tyrosine kinase inhibitor tyrphostin A10 inhibited CD22 movement at concentrations that inhibited tyrosine phosphorylation of CD22 and other cellular proteins. In contrast to the B cell lines, mature peripheral blood B cells contained very little intracellular CD22 and showed no significant increase in surface expression following sIgM stimulation. The rapid directed movement of intracellular CD22 provides a new mechanism to dynamically regulate Ag receptor signaling, as well as CD22-mediated adhesion.

Antigens, CD↗

PubMed1996

Apoptosis in hematopoietic cells is associated with an extensive decrease in cellular phosphotyrosine content that can be inhibited by the tyrosine phosphatase antagonist pervanadate.

In the present study, we investigated whether apoptosis in hematopoietic cells is associated with changes in cellular phosphotyrosine content. Murine thymocytes and B cells, human leukemia cells, and normal peripheral blood leukocytes were induced to undergo apoptosis by treatment with specific stimuli or by incubation in growth factor-deprived medium. Multiparameter flow cytometry was used to measure changes in phosphotyrosine content that correlated with the appearance of features of programmed cell death, such as cell shrinkage, DNA fragmentation, and loss of membrane integrity. The results show that conditions that induced apoptosis also induced a dramatic decrease in cellular phosphotyrosine levels. Tyrosine dephosphorylation preceded the loss of plasma membrane integrity and, in most cases, was temporally correlated with the onset of DNA fragmentation. The protein tyrosine phosphatase antagonist pervanadate had a dose-dependent inhibitory effect on both dephosphorylation and apoptosis in murine thymocytes, which were treated with dexamethasone or with the topoisomerase II inhibitor etoposide. The results suggest that extensive tyrosine dephosphorylation is an intrinsic part of the apoptotic process of hematopoietic cells and may be involved mechanistically in the apoptosis induced by certain stimuli.

Animals↗

PubMed1996

Cytokine activation sensitizes human monocytic and endothelial cells to the cytotoxic effects of an anti-CD40 immunotoxin.

G28-5 sFv-PE40 is a single-chain immunotoxin that is cytotoxic toward malignant B cells expressing CD40. Human monocytes, which also express cell surface CD40, were found to be insensitive to the immunotoxin. Activation of the monocytic cell line THP-1, or freshly isolated peripheral blood monocytes with IFN-gamma, but not IL-3, GM-CSF, IL-6, or TNF-alpha, greatly sensitized the cells toward G28-5 sFv-PE40, lowering the EC50 value from > 10 micrograms/ml to 80 ng/ml. This sensitization could not be explained simply by the two- to threefold increase in cell surface CD40 expression induced by IFN-gamma since TNF-alpha or the combination of TNF-alpha and IL-6 gave similar increases in CD40 expression but did not sensitize the cells to the immunotoxin. Internalization of G28-5 sFv-PE40 after IFN-gamma activation was also increased threefold, reflective of the increase in CD40 expression. IFN-gamma-treated but not -untreated THP-1 cells produced IL-6 and TNF-alpha following incubation with G28-5 sFv-PE40, indicating an association between CD40 signaling, which induces cytokine production, and sensitivity to the immunotoxin. HUVECs also express CD40 but were found to be insensitive to the anti-CD40 immunotoxin. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, sensitized the endothelial cells to G28-5 sFv-PE40. These data show that activation with cytokines can sensitize monocytes and endothelial cells to an immunotoxin targeted to CD40, most likely by altering the trafficking and/or processing of the immunotoxin after receptor-mediated internalization.

ADP Ribose Transferases↗

PubMed1996

Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils.

Human neutrophils, monocytes, and eosinophils are known to undergo apoptotic cell death. The Fas/Fas ligand pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the importance of the Fas/FasL system in normal human phagocytes. Although Fas expression was detected on neutrophils, monocytes, and eosinophils, constitutive expression of FasL was restricted to neutrophils. The three types of phagocytes demonstrated differential sensitivity to Fas-induced apoptosis. Only neutrophils were highly susceptible to rapid apoptosis in vitro after stimulation with activating anti-Fas IgM (mAb CH-11). Fas-mediated neutrophil apoptosis was suppressed by incubation with G-CSF, GM-CSF, IFN-gamma, TNF-alpha, or dexamethasone, as well as the selective tyrosine kinase inhibitors, herbimycin A and genistein. Spontaneous neutrophil death in vitro was partially suppressed by Fas-Ig fusion protein or antagonistic anti-Fas IgG1 (mAb ZB4). In coculture experiments, neutrophils released a soluble factor inducing death in Fas-susceptible Jurkat cells via a mechanism sensitive to the presence of Fas-Ig or anti-Fas IgG1. Immunoblot analysis using specific anti-human FasL IgG1 (mAb No. 33) identified a 37-kD protein in lysates of freshly isolated neutrophils and a 30-kD protein in the culture supernatant of neutrophils maintained in vitro. Our results suggest that mature neutrophils may be irrevocably committed to autocrine death by virtue of their constitutive coexpression of cell-surface Fas and FasL via a mechanism that is sensitive to proinflammatory cytokines, glucocorticoids, and inhibitors of tyrosine kinase activity. Furthermore, neutrophils can serve as a source of soluble FasL, which may function in a paracrine pathway to mediate cell death.

Apoptosis↗

PubMed1996

The role of the B7-1a molecule, an alternatively spliced form of murine B7-1 (CD80), on T cell activation.

B7 molecules (CD80 (B7-1) and CD86 (B7-2/B70)) on APCs provide costimulatory signals for T cell proliferation. We previously described the presence of an alternatively spliced form of murine CD80 (previously termed MB7-2 and renamed as B7-1a) that completely lacks the second Ig-like domain coded by exon 3 in activated murine B cells. in this study, we first examined whether B7-1a mRNA can be detected in vivo by RNase protection assay. The expression of B7-1a mRNA was only detected in lymphoid organs although the level of expression was lesser than that of CD80 mRNA. However, we demonstrated that the expression of B7-1a mRNA like CD80 mRNA was considerably augmented in spleen cells treated with either LPS in vitro or OVA/CFA conjugate in vivo. We next determined the functional activity of B7-1a using Chinese hamster ovary (CHO) cells transfected by B7 genes. When resting T cells were cocultured with CHO cells expressing B7-1a molecules in the presence of PMA/ionomycin, T cell proliferation was not detected, while CHO cells either expressing CD80 or CD86 could promote the proliferation of resting T cells. in contrast to resting T cells, CHO cells expressing B7-1a could support the proliferation of activated T cells. Thus, costimulatory activity of B7-1a molecules was dependent upon the activation stage of T cells. Therefore the IgV-like region of CD80 contains a critical region for functional interaction with its ligands and can transduce a costimulatory signal for T cell proliferation.

Alternative Splicing↗

PubMed1996

CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis.

We investigated the role of CD40-CD40 ligand (CD40L) interactions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Activated helper T cells expressing CD40L (gp39) surface protein were found in MS patient brain sections, but not in brain tissue sections of normal controls or patients with other neurological disease. CD40L-positive cells were co-localized with CD40-bearing cells in active lesions (perivascular infiltrates). Most of these CD40-bearing cells proved to be of the monocytic lineage (macrophages or microglial cells), and relatively few were B cells. To functionally evaluate CD40-CD40L interactions, EAE was elicited in mice by means of proteolipid-peptide immunization. Treatment with anti-CD40L monoclonal antibody completely prevented the development of disease. Furthermore, administration of anti-CD40L monoclonal antibody, even after disease onset, shortly before maximum disability score was reached led to dramatic disease reduction. The presence of helper T cells expressing CD40L in brain tissue of MS patients and EAE animals, together with the functional evidence provided by successful experimental prevention and therapy in an animal model, indicates that blockade of CD40-CD40L-mediated cellular interactions may be a method for interference in active MS.

Animals↗

PubMed1996

The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of self-reactive T cells.

The zona pellucida (ZP), an ovarian extracellular structure, contains three major glycoproteins: ZP1, ZP2, and ZP3. A ZP3 peptide contains both an autoimmune oophoritis-inducing T cell epitope and a B cell epitope that induces autoantibody to ZP. This study investigates two major T cell costimulation pathways in this disease model. Herein we show that blockage of glycoprotein (gp)39 and CD40 interaction with gp39 monoclonal antibody (mAb) results in the failure to induce both autoimmune oophoritis and autoantibody production. Inhibition of ligand binding to the CD28 receptor with the fusion protein, murine CTLA4-immunoglobulin (Ig), also results in failure to generate antibody to ZP and significantly reduces disease severity and prevalence. Surprisingly, the frequencies of antigen-specific T cells in anti-gp39 mAb-treated mice, CTLA4-Ig treated mice, and in mice given control hamster IgG or control fusion protein L6, were equivalent as determined by limiting dilution analysis (approximately equals 1:5,000). These T cells, which produced comparable amounts of interleukin 4 and interferon gamma in vitro, were able to transfer oophoritis to normal recipients. When anti-gp39 mAb and CTLA4-Ig were given together, the effect was additive, leading to inhibition of T cell activation as determined by in vitro proliferation and limiting dilution analysis (approximately equals 1:190,000); disease and antibody responses were absent in these mice. By studying these two costimulatory pathways in parallel, we have shown that autoimmune disease and autoantibody production are inhibitable by blocking either the gp39 or the CD28 pathway, whereas inhibition of clonal expansion of the effector T cell population occurs only when both pathways are blocked.

Abatacept↗