Activation of human B lymphocytes. XII. Differential effects of in vitro cyclophosphamide on human lymphocyte subpopulations involved in B-cell activation.

The differential effects of in vitro cyclophosphamide (CY) on subpopulations of normal human peripheral blood lymphocytes involved in the pokeweed mitogen-induced plaque-forming cell (PFC) response against sheep red blood cells were examined. It was found that the plaque-forming B cells in this system are sensitive to CY over a wide concentration range including concentrations which have a minimal effect on overall cell viability. Kinetic experiments revealed that CY exerts its inhibitory effect on the PFC response only if added very early in culture. Thus, it appears that in vitro CY must exert its inhibitory influence on an early phase of polyclonal B-cell activation. When T-cell enriched (TCE) populations were incubated overnight with high concentration CY and then added back in co-culture to fresh autologous B cells, significant enhancement of PFC responses was observed suggesting a selective inhibition or elimination of a regulatory suppressor cell population found in TCE lymphocyte preparations. Helper T cells are relatively resistant to the inhibitory actions of CY. Thus, human B cells appear to be most sensitive to CY, followed in sensitivity by the suppressor cell populations in the T-cell fraction with relative resistance of the helper T cells. These observations have direct relevance in understanding the mechanisms of selective action of CY on normal human lymphocyte subpopulations with possible application to disease states in man.     

Solid-phase monoclonal antibodies. A novel method of directing the function of biologically active molecules by presenting a specific orientation.

Monoclonal antibodies were produced against fibronectin and vitronectin, using the extracellular matrix from cultured bovine corneal endothelial cells as immunogen. Some antibodies inhibited the adhesion of BHK-21 cells to these molecules, in a standard assay with fibronectin or vitronectin coated on a plastic surface. By first coating the plastic surface with the monoclonal antibodies and subsequently binding vitronectin, the inhibitory effect was markedly increased. Divalent fibronectin required the presence of antibody in the medium as well as coated on the surface for maximal inhibition of cell adhesion. The inhibitory effect of surface-coated antibodies was stable in the presence of cells up to at least 24 h in vitro. Using this antibody coating method the surface could either be made compatible for cell adhesion (using non-inhibitory antibodies) or refractory. Assays performed by this method have considerable potential for quantifying particular functions of extracellular matrix molecules.     

Ovine and Human Placental IgG inhibit Human Natural Killer Cell Cytotoxicity in vitro

To determine the effect of ovine and human placental IgG on human Natural Killer (NK) cell cytotoxicity in vitro placental IgG was eluted at acidic pH and purified by ion exchange and subsequently by affinity chromatography on protein G and protein A sepharose columns. These antibodies were analysed for presense of IgG by immuno-electrophoresis and relative purity determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). The effect of these antibodies on human NK cell cytotoxicity was investigated by slChromium Release Assay using human K562 cells as targets and human peripheral blood lymphocytes as effector cells. Both ovine and human placental IgG inhibited human NK cell cytotoxicity in a dose dependent manner. Placental IgG may down-regulate the cytotoxic effects of NK cells in vivo by competitively excluding the binding of NK cells to their respective targets on the trophoblast. Alternatively, these antibodies may themselves be toxic to NK cells. Either way, the presence of these antibodies on the placental trophoblast may prevent the binding of NK cells and subsequent immunological rejection of the fetal allograft. Also, ovine placental IgG may be functionally similar to its human counterpart and may therefore be suitable as a model for the study of maternal fetal interaction during pregnancy in humans.     

Androgens inhibit proliferation of human peripheral blood lymphocytes in vitro

The effect of sex hormones on human lymphocytes was examined in vitro on cell cultures of human peripheral blood mononuclear cells (HPBM). Cells were stimulated using T- and B-cell mitogens, and hormones in physiological (nM) or pharmacological (microM) concentrations were added. Proliferation was determined by measuring the incorporation of tritiated thymidine. It was found that both testosterone and dihydrotestosterone, in physiological concentrations, can attenuate DNA synthesis. The effect was dose dependent in that pharmacological concentrations of both testosterone and dihydrotestosterone caused a strong inhibitory effect on proliferation of in vitro cultured HPBM. However, cell cultures of a few individuals were insensitive to the androgens even at pharmacological concentrations. Also, no difference could be detected in the response between cultured cells of females and males. Although a slight reduction in antibody production was evident in pokeweed mitogen-stimulated cultures, in the presence of both testosterone and dihydrotestosterone, it was, however, not statistically significant.     

Cytomegalovirus-specific B cell activation as a potential marker for the diagnosis of cytomegalovirus infection

In vitro secretion of antibodies to cytomegalovirus was investigated by analysis of peripheral blood mononuclear cell culture supernatants from subjects infected with cytomegalovirus. Patients with primary or recurrent cytomegalovirus infection showed transient in vitro cytomegalovirus-specific antibody secretion. A high proportion of patients infected with human immunodeficiency virus and seropositive for cytomegalivirus showed in vitro cytomegalovirus-specific antibody secretion. All peripheral blood mononuclear cell cultures from patients with symptomatic human immunodeficiency virus infection were found to secrete in vitro antibodies to cytomegalovirus, despite the fact that isolation of cytomegalovirus from some of these patients was not achieved. In human immunodeficiency virus-infected patients, in vitro secretion of anti-cytomegalovirus antibodies appeared to be persistent. In vitro cytomegalovirus-specific antibody secretion by peripheral blood lymphocytes probably reflects an in vitro cytomegalovirus-specific B cell activation. This new assay could be considered an interesting method for detecting both acute or chronic cytomegalovirus infection, with potential use in routine laboratory practice.     

CD23 and CD21 function as adhesion molecules in homotypic aggregation of human B lymphocytes

We have previously found that interleukin-4 and CD40 monoclonal antibodies (mAb) are strong potentiatiors of homotypic B cell aggregation which is dependent on LFA-1. We show here that CD23 mAb were also able to inhibit aggregation to a similar extent as LFA-1 antibodies. This inhibition was restricted to the MHM6 epitope of CD23 and antibodies to other epitopes [Epstein-Barr virus (EBV) CS-1, EBV CS-2, EBV CS-5 and mAb 25] or occupation of the Fc-binding site by IgE had no or a slightly enhancing effect on aggregation. When testing two antibodies to CD21, the recently defined ligand for CD23, one of these (BU32) was found to be inhibitory whereas the other (THB5) had no effect. By combining antibodies to LFA-1 and CD23, aggregation was often completely inhibited. These data suggest that LFA-1/ICAM-1 and CD23/CD21 are the major molecules involved in homotypic aggregation of human B cells.     

In vitro transformation by Epstein-Barr virus induces a switch in growth factor and anti-IgM responsiveness in a human leukemic B cell clone

By in vitro transformation with Epstein-Barr virus (EBV), we have previously established EBV+ lymphoblastoid cell lines (LCL) from a patient with leukemic centrocytic B cell lymphoma. EBV-transformed LCL and EBV genome-negative leukemic B cells showed identical chromosome aberrations and IgH gene rearrangements. In the present study we have analyzed the effect of exogenous cytokines [interleukin (IL) 1, 2, 3, 4, 6, tumor necrosis factor, lymphotoxin, transforming growth factor beta, (TGF-beta)] and anti-IgM antibodies on the in vitro proliferation of EBV- leukemic B cells and EBV-converted LCL. In contrast to conventional chronic lymphocytic leukemia, B cells of the patient DUL spontaneously proliferated for up to two weeks in the absence of exogenous lymphokines. The spontaneous proliferative capacity of clonal DUL B cells was not modulated by IL 1, IL 3, IL 6, TNF or LT. In vitro growth of DUL B cells was increased, however, by exogenous recombinant (r)IL 2, and was abrogated by TGF-beta, rIL 4 and anti-IgM. rIL 4 not only inhibited spontaneous B cell proliferation but also neutralized the enhancing effect of rIL 2. In contrast, growth of the EBV-transformed DUL LCL was not affected by any of these factors. These data demonstrate that in vitro infection and transformation of a clonal B cell population by EBV induces a switch in responsiveness to rIL 4, TGF-beta and anti-IgM. In addition, this report is the first to demonstrate an inhibitory effect of rIL 4 on a spontaneously proliferating human leukemic B cell clone.     

Lack of immunosuppression by ketoconazole and itraconazole.

The antifungal drugs ketoconazole and itraconazole were evaluated for their effects in the following test systems: in vitro, phytohaemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells and IL-2-driven proliferation of CTLL-2 cells; in vivo, antibody response to sheep red blood cells (SRBC) and delayed-type hypersensitivity (DTH) reaction to oxazolone. At a concentration of 10 microM, ketoconazole moderately and itraconazole strongly inhibited thymidine (Thd) incorporation in human peripheral blood mononuclear cells cultured in medium supplemented with 5% human serum. Increasing the serum concentration from 5 to 20% almost completely reversed these inhibitory effects. Also, cell viability, found to be less than 15% in cultures containing 10 microM itraconazole was restored by increasing the serum concentrations in the culture medium. Similar observations were made in experiments using IL-2-stimulated CTLL-2 cells: the growth inhibition in the presence of 10 microM ketoconazole or 1 microM itraconazole could be counteracted by increased serum supplementation. In vivo, subchronic intraperitoneal dosing with 40 mg/kg ketoconazole or itraconazole to mice had no effect on the antibody response to SRBC as measured by the number of splenic IgM and IgG plaque-forming cells and did not significantly affect the DTH response to oxazolone. These data indicate that neither ketoconazole nor itraconazole exert immunosuppressive properties in vivo. Their in vitro inhibitory effects on PHA-induced lymphocyte proliferation and IL-2-dependent CTLL-2 growth are reversed by the serum supplementation to the culture medium and these activities should therefore be considered as in vitro artefacts.     

In vitro immunomodulatory activity of ruthenium complexes

OBJECTIVE AND DESIGN: We have explored the in vitro immunomodulatory effects of pure ruthenium red and a series of pyridine and imidazole substituted ruthenium complexes (RCs). MATERIAL: Human peripheral blood lymphocytes and purified T cells were used in these studies along with various cell lines. METHODS: Cells were treated with dilutions of RCs and assessed in various assays of immune function, cytotoxicity and cell cycle progression. RESULTS: RCs efficiently blocked T cell receptor (TCR)-mediated stimulation (IC(50)'s in the low nM range) of human peripheral blood lymphocytes (hPBL) by various agents, including tetanus toxoid, alloantigens, superantigens, and receptor-specific antibodies. RCs are not cytotoxic to T cells. Antiproliferative activity was also observed for B cells. Some non-lymphoid cell lines or primary cultures showed sensitivity to the RCs, but only at higher concentrations. The inhibitory effect on human T cells was assessed and demonstrated at the level of proliferation (DNA synthesis), IL-2 secretion, and IL-2 receptor (CD25) upregulation. RCs also inhibited IL-2-mediated proliferation of antigen-induced T-cell blasts and the IL-2-dependent T cell line Kit-225. Cell cycle analysis indicates that RCs inhibit the progression of activated T cells from G(0)/G(1) to S phase. CONCLUSIONS: Since the mechanism of T cell inhibition by RCs appears to be different than that of rapamycin (RAP) or cyclosporin A (CsA), they may provide a new tool to investigate intracellular signaling in T cells, and may present novel opportunities for immunosuppression     

IL-4 suppresses IL-1 beta, TNF-alpha and PGE2 production by human peritoneal macrophages.

Human interleukin-4 (IL-4) down-regulates IL-1 and tumour necrosis factor-alpha (TNF-alpha) production by monocytes stimulated in vitro. In contrast, in studies of activation of murine macrophages, both stimulatory and inhibitory functions of murine IL-4 have been documented. To investigate whether opposing activities of IL-4 reflect a difference in the target cell studied, due either to cell maturation or the site from which the cells were isolated, we examined the effect of IL-4 on human peritoneal macrophage production of IL-1 beta, TNF-alpha and prostaglandin E2 (PGE2). Human peritoneal macrophages stimulated with lipopolysaccharide (LPS) produced levels of these mediators that were at least as great as those previously reported for monocytes. Similarly, IL-4 was inhibitory for peritoneal macrophage mediator production after in vitro stimulation. Thus, IL-4 has effects on human peritoneal macrophages similar to those on blood monocytes. In addition, as it down-regulates mediator production by cells that have left the circulation, it may be important in controlling the immune response in tissues.     

GMCSF in the absence of other cytokines sustains human dendritic cell precursors with T cell regulatory activity and capacity to differentiate into functional dendritic cells

Dendritic cell precursors, from human peripheral blood, express epitopes reactive with monoclonal antibodies specific for the empty conformation of HLA-DR1. Expression is substantially up-regulated during GMCSF-induced differentiation to immature dendritic cells, but is strongly down-regulated by IL-4. In the conventional protocol for in vitro generation of human dendritic cells from monocyte precursors, both GMCSF and IL-4 are used together, with IL-4 thought to have an effect on preventing macrophage outgrowth but not substantially altering the dendritic cell maturation pathway, whereas conventional protocols for generation of murine dendritic cells use GMCSF alone. We characterized human monocytes cultured in the presence of GMCSF and in the absence of IL-4, and found that the resultant cultures are stable for long periods in vitro, and have T cell stimulatory and immunodulatory activity characteristic of immature dendritic cells. Their response to maturation stimuli is weak in the absence of IL-4 but these cells retain the ability to differentiate into fully functional mature dendritic cells upon IL-4 treatment. We suggest that these cells may provide a useful model to investigate tolerogenic function as a developmental feature of DC and to understand molecular events involved in IL-4 priming for maturation.     

Role of Ia antigens in the human autologous mixed lymphocyte reaction

The role of Ia antigens in the human autologous mixed lymphocyte reaction (MLR) was analyzed. To do this, different mononuclear cell populations were fractionated according to their Ia antigen expression and used as stimulating cells in autologous MLR. Also examined was the effect of two hybridoma monoclonal antibodies specific for human Ia molecules on the autologous MLR. The results show that the stimulating capacity of peripheral blood mononuclear cells is restricted to Ia-bearing cells. While peripheral blood T cells are unable to function as stimulating cells, highly purified Ia+ mixed lymphocyte culture-activated T cells are very effective. The possibility that Ia molecules are directly involved in the autologous MLR is further substantiated by the strong inhibitory effect of monoclonal anti-Ia antibodies.     

EBV Transformation and Microfusion as the Potential Source of Human Monoclonal Antisperm Antibodies

ABSTRACT: Peripheral blood lymphocytes were obtained from vasectomized men with high serum titers of anti-sperm antibodies. An Epstein-Barr virus (EBV) transformation was performed either with B cells or mononuclear leukocytes. The effect of feeder cells (irradiated umbilical cord blood lymphocytes), cyclosporin A, and in vitro stimulation of lymphocytes with sperm extract on EBV transformation was evaluated. Antibody-producing cells were screened for specificity against human sperm by an enzyme-linked immunosorption assay (ELISA) one to six weeks after transformation. Using B cells or leukocyte mononuclear cells, we found that the percentage of wells containing antibody reactive against human sperm was greatest two weeks after transformation (range 3% to 7.5% positive wells). To increase and maintain antibody synthesis by these transformed cells, microfusions were performed in those wells positive for antisperm antibody using the UC 729-6 lymphoblastoid cell partner. Then resultant hybridomas were expanded, subcloned, and preliminarily characterized.     

The immunomodulatory effect of anti-Micrococcus luteus antibodies. I. Effect on in vitro rabbit T cell functions

A range of purified rabbit anti-Micrococcus luteus antibodies (anti-MCAb) were tested for their ability to interfere with a variety of in vitro immune responses. Such antibodies strongly inhibited the secondary IgG antibody response to sheep red blood cells without affecting the IgM response or the proliferative responses to mitogens and antigens. By exposing lymphocyte populations to anti-MCAb, it was found that such reagents exerted a strong mitogenic effect on rabbit T lymphocytes, provided these cells were derived from antigen-activated lymph nodes. This mitogenic effect was also obtained with F(ab')2 fragments of anti-MCAb and with hybridoma-derived anti-MCAb. Collectively, these data indicate that anti-MCAb inhibit the initiation of IgG synthesis possibly through the expansion of immunoregulatory T cell subsets.     

A bispecific antibody against human IgE and human FcgammaRII that inhibits antigen-induced histamine release by human mast cells and basophils

BACKGROUND: FcgammaRIIB are low-affinity immunoglobulin (Ig)G receptors that we previously demonstrated to negatively regulate IgE-induced mast cell activation when coaggregated with FcepsilonRI. Here, we engineered and characterized a bispecific reagent capable of coaggregating FcgammaRIIB with FcepsilonRI on human mast cells and basophils. METHODS: A bispecific antibody was constructed by chemically crosslinking one Fab' fragment against human IgE and one Fab' fragment against human FcgammaRII. This molecule was used to coaggregate FcepsilonRI with FcgammaRII on human mast cells and basophils sensitized with human IgE antibodies, and the effect of coaggregation was examined on mediator release upon challenge with specific antigen. RESULTS: When used under these conditions, this bispecific antibody not only failed to trigger the release of histamine by IgE-sensitized cells, but it also prevented specific antigen from triggering histamine release. Comparable inhibitions were observed with mast cells and basophils derived in vitro from cord blood cells and with peripheral blood basophils. CONCLUSIONS: The bispecific antibody described here is the prototype of similar molecules that could be used in new therapeutic approaches of allergic diseases based on the coaggregation of activating receptors, such as FcepsilonRI, with inhibitory receptors, such as FcgammaRIIB, that are constitutively expressed by mast cells and basophils.     

Cytotoxicity of human mononuclear cells against chicken and human red blood cells,induced by treatment of the effector cells with phospholipase C

Human mononuclear cells from peripheral blood which were treated with phospholipase C (PLC), became cytotoxic against human or chicken red blood cells. PLC- induced cellular cytotoxicity against human red blood cells was further analyzed and compared to anti-D-mediated, antibody-dependent cellular cytotoxicity (ADCC), using the same target cells. ADCC, but not cytotoxicity of PLC-treated effector cells, was inhibited by free IgG. In addition, iodoacetate strongly enhanced PLC-induced cytotoxicity, but blocked ADCC completely. Addition of fetal calf serum or human AB serum impaired PLC-induced cytotoxicity. A similar inhibition was found by adding lecithin liposomes suggesting that the inhibitory effect of sera was also due to their phospholipid content. The data show that cytotoxicity of PLC-treated effector cells can be clearly distinguished from cellular cytotoxicity, occurring spontaneously or induced by target cell antibodies. We favor the notion that cytotoxicity of PLC- treated effector cells against human erythrocytes is due to the action of PLC, adsorbed to the effector cells.     

Inhibition of LFA-1-dependent human B-cell aggregation induced by CD40 antibodies and interleukin-4 leads to decreased IgE synthesis.

Antibodies to CD40 have been shown to induce homotypic aggregation of human resting B cells and B-cell lines via an LFA-1-dependent mechanism. We show here that interleukin-4 (IL-4) is a strong potentiator of this process and stimulation of tonsillar B cells for 4 days with IL-4 and CD40 antibodies resulted in the formation of large, dense aggregates. Also in this case, aggregation appeared to be chiefly dependent on the activation of LFA-1, although the small clusters of cells remaining after blocking with LFA-1 antibodies suggest the involvement of another adhesion system(s). When testing the relationship between aggregation and IgE synthesis, a known consequences of IL-4/CD40 stimulation, IgE levels were found to be significantly decreased in the presence of LFA-1 antibodies. In contrast to these observations, proliferation occurring in response to the IL-4/CD40 stimulation was not inhibitable by LFA-1 antibodies. Rather, in most cases, this was slightly enhanced, suggesting that aggregation may have a limiting effect on cell growth. Isolated aggregates, each of which could comprise more than 10(5) cells, were also examined by electron microscopy. This revealed a tissue-like structure of the aggregates with large contact areas and with minimal intercellular space between the adjacent cells. As the apparent inhibitory effect of aggregation on proliferation may reflect a negative autocrine signalling, which is enhanced by the close cell contact, we also tested the effect of neutralizing antibodies to IL-6, one of the factors known to be produced in the system. Such treatment did not affect aggregation but in most experiments enhanced proliferation. The results suggest that a possible effect of aggregation may be to enhance differentiation of cells and that this may also be associated with the difficulties in growing B cells in vitro.     

Spontaneous thyroglobulin autoantibody production by peripheral blood lymphocytes is regulated by T cells and is augmented by soluble T cell factors

In this study we report the spontaneous production of autoimmune anti-thyroglobulin antibodies (Tg Ab) by peripheral blood lymphocytes from patients with autoimmune thyroid disease and the use of these cells to study the regulation of this production in vitro. Using a mitogen free microculture system we have found a significant correlation between an individuals' serum titre and the amount of Tg Ab produced in vitro. At low B:T cell ratios Tg Ab production decreased. Specific depletion of suppressor T cells and helper T cells was accomplished using the monoclonal antibodies OKT 8 and OKT 4 respectively. Suppressor cell depletions resulted in increases in Tg Ab production while depletion of helper cells had no consistent effect. The addition of pokeweed mitogen had no stimulatory effect on spontaneous thyroglobulin antibody production. In contrast, when T cell conditioned media was added to the cells, Tg Ab production increased substantially in all patient cultures tested but not in normal control cultures. The results of this study are consistent with the existence of functional thyroid-antigen specific suppressor cells in the peripheral blood which are actively involved in the regulation of autoantibody producing B cells. The results obtained with the cultures maintained in conditioned media show that autoimmune lymphocytes are extremely sensitive to one or more stimulatory factors present in the media and suggests an important role for the production of soluble lymphocyte factors and the expression of receptors for these factors in the aetiology of autoimmune thyroid disease.     

Generation and Characterization of Monoclonal Antibodies Specific to Surface Antigens of Human Trophoblast Cells

PROBLEM : To generate and utilize specific monoclonal antibodies for routine fetal cell isolation from the maternal circulation. METHODS : Monoclonal antibodies specific to human trophoblast cell surface antigens were generated and characterized. After cell fusion, antibodies secreted by hybridomas were screened by enzyme-linked immunosorbent assay and immunohistochemical assays. RESULTS : By using cultured BeWo choriocarcinoma cells or the membrane fraction of human placenta as the immunogen, seven (BW-108, 110, 123, 124, HP-15, 16 and 17) antibodies specific to the surface antigens of trophoblast were produced. They were shown to have little cross-reactivity to other human tissues. Among the antibodies raised against human sperm, HSA-10 was also found to cross-react with human trophoblast, but not detected in other tissues. When immobilized to magnetic beads, these antibodies were shown to react only with BeWo cells in suspension, but not blood cells and ovarian carcinoma cell line, OC-3-VGH. CONCLUSION : Therefore, these antibodies may have potential application in fetal trophoblast cell isolation from the maternal circulation for prenatal genetic diagnosis.     

Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus

B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.