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There is growing interest in understanding antibody (Ab) function beyond neutralization. The non‐structural protein 1 (NS1) of Zika virus (ZIKV) is an attractive candidate for an effective vaccine as Abs against NS1, unlike the envelope or premembrane, do not carry the risk of mediating antibody‐dependent enhancement. Our aim was to evaluate whether ZIKV NS1 Abs elicited following natural infection in humans can mediate antibody‐dependent cellular cytotoxicity (ADCC). We evaluated the isotype specificity of ZIKV‐specific Abs in immune sera and supernatants from stimulated immune PBMC and found that Abs against ZIKV NS1 and virus‐like particles were predominantly of the IgG1 isotype. Using a recently developed FluoroSpot assay, we found robust frequencies of NS1‐specific Ab‐secreting cells in PBMC of individuals who were naturally infected with ZIKV. We developed assays to measure both natural killer cell activation by flow cytometry and target cell lysis of ZIKV NS1‐expressing cells using an image cytometry assay in the presence of ZIKV NS1 Abs. Our data indicate efficient opsonization of ZIKV NS1‐expressing CEM‐NKR cell lines using ZIKV‐immune but not ZIKV‐naïve sera, a prerequisite of ADCC. Furthermore, sera from immune donors were able to induce both NK cell degranulation and lysis of ZIKV NS1 CEM‐NKR cells in vitro. Our data suggest that ADCC is a possible mechanism for ZIKV NS1 Abs to eliminate virally infected target cells. [ABSTRACT FROM AUTHOR]

Summary: B lymphocytes, known as antibody producers, mediate tumor cell destruction in the manner of antibody‐dependent cell‐mediated cytotoxicity; however, their anti‐tumor function seems to be weakened during tumorigenesis, while the underlying mechanisms remain unclear. In this study, we found that IgG mediated anti‐tumor effects, but IgG‐producing B cells decreased in various tumors. Considering the underlying mechanism, glycometabolism was noteworthy. We found that tumor‐infiltrating B cells were glucose‐starved and accompanied by a deceleration of glycometabolism. Both inhibition of glycometabolism and deprivation of glucose through tumor cells, or glucose‐free treatment, reduced the differentiation of B cells into IgG‐producing cells. In this process, special AT‐rich sequence‐binding protein‐1 (SATB1) was significantly silenced in B cells. Down‐regulating SATB1 by inhibiting glycometabolism or RNA interference reduced the binding of signal transducer and activator of transcription 6 (STAT6) to the promoter of germline Cγ gene, subsequently resulting in fewer B cells producing IgG. Our findings provide the first evidence that glycometabolic inhibition by tumorigenesis suppresses differentiation of B cells into IgG‐producing cells, and altering glycometabolism may be promising in improving the anti‐tumor effect of B cells. IgG‐producing B cells exerted an anti‐tumor effect, but dramatically decreased in various tumors. Glucose deprivation caused by tumorigenesis down‐regulated the glucose metabolism of B cells and then impaired their generation into IgG‐producing B cells. SATB1, known as a cell‐type‐specific 'genomic organizer', was down‐regulated in the glucose‐starved B cells and responsible for the deficiency generation of IgG‐producing B cells in tumors. [ABSTRACT FROM AUTHOR]

Fc receptors for immunoglobulin G (IgG) (FcγR) mediate several defence mechanisms in the course of inflammatory and infectious diseases. In Gram-negative infections, cellular wall lipopolysaccharides (LPS) modulate different immune responses. We have recently demonstrated that murine LPS in vivo treatment significantly increases FcγR-dependent clearance of immune complexes (IC). In addition, we and others have reported the induction of adhesion molecules on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-α (TNF-α) in vitro. The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancing effects on Fcγ-dependent functionality of tissue macrophages. Our results have demonstrated that LPS can enhance antibody-dependent cellular cytotoxicity (ADCC) and IC-triggered cytotoxicity (IC-Ctx), two reactions which involve the Fcγ-receptor but different lytic mechanisms. In vitro incubation of splenocytes from LPS-treated mice with anti-CD11b/CD18 abrogated ADCC and IC-Ctx enhancement, without affecting FcγR expression. Similar results were obtained with physiological concentrations of fibrinogen. In this way cytotoxic values of LPS-splenocytes decreased to the basal levels of control mice. Time and temperature requirements for such inhibition strongly suggested that anti-CD11b/CD18 could modulate intracellular signals leading to downregulation of FcγR functionality. Data presented herein support the hypothesis that functional and/or physical associations between integrins and FcγR could be critical for the modulation of effector functions during an inflammatory response. [ABSTRACT FROM AUTHOR]

The role of perforin in cytotoxicity is controversial. This paper characterizes a novel monoclonal antibody (anti-Phu) against human perforin, using murine cell lines transfected with human perforin cDNA. The antibody specifically stains human pefforin in transfected mouse CTLL-2. Anti-Phu blocked granule-mediated haemolysis in an in vitro assay using intact granules isolated from the natural killer (NK)-like human cell line YT, indicating that perforin is a major granule component causing lysis of red blood cells (RBC) in this assay. Inhibition of haemolysis by anti-Phu demonstrated that the antibody binds to undenatured protein as well as fixed perforin molecules. However, the antibody did not inhibit lysis by an allospecific T-cell clone or by YT cells. This could be due to an extremely tight contact between effector and target cell, preventing the antibody from interfering with perforin function by stearic hindrance. Physiologically this may reduce lysis of bystander cells. The anti-Phu antibody is a useful tool for further studies of perforin-induced cytotoxicity in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Two methods are described for linking chlorambucil to antibodies. At low pH an association is formed which is not stable under physiological conditions and the cytotoxicity is the result of the independent action of chlorambucil and antibody. At pH 8 a more stable linkage is formed and this complex shows specific toxicity for cells carrying the appropriate antigen. Uncombined chlorambucil and antibody are more cytotoxic than chlorambucil alone, especially if the cells are exposed first to the chlorambucil. [ABSTRACT FROM AUTHOR]

We used various culture conditions to generate type 1 (Tc1) or type 2 (Tc2) cytotoxic T cells in vitro. T-cell receptor (TCR) transgenic T cells were cultured with antigen and spleen cells, or antigen and dendritic cells (DC), or anti-CD3 and anti-CD28. Tc1 cultures contained interleukin (IL)-2 and IL-6, and Tc2 cultures contained IL-2, IL-6 and IL-4. Tc2 cells generated in each culture condition acquired a CD62Llow CD44high phenotype, had high cytotoxic activity, and secreted IL-4, IL-5 and moderate amounts of interferon-γ (IFN-γ). In contrast, the phenotype and function of Tc1 cells varied depending on culture conditions. Tc1 cells from anti-CD3 and anti-CD28 cultures had high cytotoxic activity and were CD62Llow CD44high, while Tc1 cells from antigen and spleen cell cultures had low cytotoxic activity and were CD62Lhigh CD44low. Tc1 cells from antigen and DC cultures had an intermediate phenotype. All Tc1 cells secreted high amounts of IFN-γ, but only Tc1 from anti-CD3 and anti-CD28 cultures had antitumour activity in vivo. Differences were not caused by suboptimal culture conditions, as Tc1 cells divided at a similar rate whether cultured with antigen and spleen cells or with anti-CD3 and anti-CD28. We conclude that IL-4 not only induces‘type 2’ cytokine secretion in CD8+ T cells, but also affects their expression of surface markers and cytotoxic activity. [ABSTRACT FROM AUTHOR]

P815, a transformed mouse mastocytoma cell line, produced and released cytotoxic factors after stimulation with phorbol 12-myristate 13-acetate (PMA), but not with lipopolysaecharide (LPS), calcium ionophore A23187 and IgE receptor triggering. The cytotoxic activity was reduced 60% by antibodies to mouse turnout necrosis factor (TNF). In addition, we demonstrated that TNF mRNA was already expressed under normal culture conditions and that it increased after stimulation with PMA. Although it was unknown whether factors other than TNF were lymphotoxin or some other unknown factors, it has been suggested that mast cells have cytotoxic activity, and that they contribute to inflammatory response through the release of TNF, which has a wide range of biological activity. [ABSTRACT FROM AUTHOR]

Antibody-dependent cell-mediated cytotoxicity (ADCC) of infectious bovine rhinotracheitis (IBR)-infected bovine kidney cells (MDBK) by neutrophils was demonstrated. Neutrophils from bovine and sheep mammary exudate and peripheral blood, and also from human peripheral blood, were all active in the presence of anti-IBR antibody. The component of the ruminant neutrophil granules which was responsible for cytotoxicity appeared to be cationic protein since purified cationic protein lysed the virus-infected cells and heparin inhibited cytotoxicity. Human neutrophil cytotoxicity to herpes simplex virus (HSV)-infected human Chang liver cells was also inhibited by heparin. Human neutrophil cytotoxicity to IBR-infected bovine kidney cells did not appear to be mediated by cationic protein since it was inhibited by the chelators of oxidative intermediates DMSO, thiourea, tryptophane, benzoate and mannitol, and not by heparin. [ABSTRACT FROM AUTHOR]

Activated macrophages from Corynebacterium parvum-treated mice are cytotoxic to non-antibody-coated tumour cells and have an augmented respiratory burst potential when compared to resident macrophages. We have investigated the possible involvement of the respiratory burst as an effector mechanism in this type of tumour killing. Scavengers of toxic metabolites of oxygen such as catalase, superoxide dismutase, 2,3-dihydroxybenzoate, ethanol, and cytochrome c did not inhibit macrophage cytotoxicity in this system. To investigate whether or not neoplastic cells stimulate the macrophage respiratory burst, we exposed activated macrophages to viable tumour cells and monitored macrophage superoxide anion production, chemiluminescence, and hexose monophosphate shunt activity. None of these indicators of the macrophage respiratory burst was stimulated by the tumour cells towards which the macrophages were cytotoxic. The data suggest that the respiratory burst is not utilized as an effector mechanism in the non-antibody-mediated macrophage tumour cytotoxicity reaction. [ABSTRACT FROM AUTHOR]

The naturally-occurring antibody-independent cellular cytotoxic activity (NOCC) of normal circulating human monocytes and neutrophils was investigated employing a number of erythrocytes and the K-562 cell line as target cells simultaneously. The identity of the effector cell(s) was shown to be dependent upon or be a function of the type of target cell selected for the assay system. A number of erythrocyte targets (rabbit, horse, sheep and ox erythrocytes) were lysed to varying degrees by neutrophils and monocytes and not by lymphocytes. Irrespective of the red blood cell (RBC) target, the effector monocyte invariably possessed receptors for both C′3 and the Fc of IgG. In contrast, the cytotoxic cells using the K-562 target cell were lymphocytes. Monocytes and neutrophils were inactive. The cytotoxic-enhancing activity in normal human serum exhibits specific and non-specific properties which suggests that more than one factor is involved. With respect to the monocyte cytotoxic cells, only the rabbit erythrocytes could totally absorb the serum factor in a specific fashion. Absorption of the serum with horse, sheep or ox erythrocytes resulted in a significant loss of potentiating activity with respect to all of the erythrocyte targets but a more marked loss of activity using the absorbing erythrocytes as targets. With respect to the polymorphonuclear leucocyte effector cells, only the rabbit RBC were capable of specifically absorbing out the cytotoxic-enhancing factor present in the normal human serum. Absorption of the serum with sheep, horse or ox RBC resulted in total cross-absorption of the enhancing factor. Chicken and human RBC, which do not serve as targets for the NOCC assay, could not absorb out the cytotoxic-enhancing factor with respect to any of the target erythrocytes. The composition of the soluble serum factor(s) is under current investigation but it is not an immunoglobulin since pure serum albumin can substitute for normal serum in the NOCC assay. The mechanism of erythrocyte lysis by the cytotoxic monocyte was investigated. Mononuclear cells were incubated with target cell monolayers and with target cells under optimal rosetting conditions. No inter- action between the effector and target cells could be detected. The monocytes did not adhere to the target cell monolayer nor did they form rosettes with the target cells. Thus, the results fail to corroborate or support the assumption that the cytotoxic activity of the monocyte is dependent upon conventionally-detectable receptors. Erythrophagocytosis was not observed to any significant degree under the assay conditions used. Therefore, the nature of the interaction between the cytotoxic monocyte and the erythroid target cell which results in lysis of the target cell remains to be elucidated. [ABSTRACT FROM AUTHOR]

Effects of thymectomy at various times after birth on cytotoxicity, delayed footpad reaction and antibody production were examined in 6 week old mice immunized intraperitoneally with chicken erythrocytes. Cytotoxicity and antibody production were abolished by thymectomy within 3 days after birth. Mice thymectomized at 5 days of age or later showed appreciable degrees of cytotoxicity and antibody production. On the other hand, a delayed footpad reaction could be elicited even in mice thymectomized within 24 h after birth, although the degree of the reaction was reduced by such a treatment. Therefore, T cells responsible for delayed hypersensitivity were less thymus-dependent and required the presence of the thymus for a shorter period in their development that those responsible for cytotoxicity or antibody production. [ABSTRACT FROM AUTHOR]

The nature of cell-dependent antibody (CDA) and the mechanism of inhibition of antibody-dependent cellular cytotoxicity (ADCC) were studied in the ADCC assay system in which culture cells of methylcholanthrene-induced rat fibrosarcoma (KMT-50) were used as target cells, xenogeneic antiserum (rabbit anti-KMT-50) as the CDA, and human peripheral blood leucocytes (PBL) as effector cells, respectively. By using protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-KMT-50 serum, CDA was shown to bind protein A. Complement dependent-cytotoxicity (CDC), however, was demonstrated in both the adsorbed fraction (eluate) and the non-adsorbed fraction (effluent) to protein A from the same affinity column chromatography. These data confirmed that CDA was IgG with an intact Fc portion. Inhibition of ADCC occurred by pre-treatment of effector cells with rabbit anti-effector (human PBL) serum even with extremely small amounts of antiserum. Such inhibition was demonstrated with the eluate but not with the effluent from protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-effector serum. F(ab')2 fragments of the same eluate (IgG) did not inhibit the ADCC activity. These data showed that the inhibition of ADCC was induced by the blocking of Fc receptors of effector cells with the Fc portions of IgG in anti-effector serum. The data obtained indicate the usefulness of protein A in separation and analysis of CDA and in investigation of the inhibitory mechanisms of ADCC. [ABSTRACT FROM AUTHOR]

Three antibody populations were raised in rabbits against surface antigens on guinea-pig L2C leukemic lymphocytes: against idiotypic determinants on the λ chain of the surface immunoglobulin, against C region determinants on the λ chain, and against the surface antigens recognized by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L2C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules. [ABSTRACT FROM AUTHOR]

Experiments are described in which the inactivation of macrophage activity by quartz particles is used to investigate the role of macrophages in antibody-dependent cell-mediated cytotoxicity in the rat. The results confirm previous findings which indicated that macrophages play no significant role in assays using cell line cells as targets. On the other hand, previous suggestions that macrophages are active against antibody-coated chick erythrocytes cannot be substantiated. In fact, it is shown that macrophages can play a protective role, and that inhibiting macrophage activity in peritoneal exudates leads to a spectacular increase in antibody-dependent lysis of chick erythrocytes. It is suggested that the confusion surrounding the role of macrophages in this assay has resulted from the failure to recognize that adherence techniques such as carbonyl-iron treatment of cell suspensions can result in substantial depletion of non-phagocytic adherent cells. [ABSTRACT FROM AUTHOR]

The present study characterizes the effect of two nitric oxide (NO) donors, S-nitrosoglutathione (OSNO) and sodium nitroprusside (SNP), on the ability of neutrophils to perform different responses triggered by immune complexes (IC). Pretreatment of neutrophils with either GSNO or SNP exerted a biphasic action on antibody-dependent cellular cytotoxicity (ADCC) performed against erythrocytes (E) coated with IgG antibodies (IgG-E), depending on the amount of lgG employed. While with high amounts of antibodies ADCC was markedly inhibited, at low amounts of antibodies it was significantly increased. Both effects were prevented by haemoglobin, a NO scavenger. Moreover, these effects were reproduced by the cell-permeable analogue of cGMP, dibutyryl cGMP (Bt2cGMP). Other neutrophil functions triggered by lgG-E were also examined. It was found that NO donors did not affect either the phagocytosis of IgG-E or the emission of chemiluminescence (CL). Finally, neutrophil functions triggered by soluble IC (sIC) and precipitating IC (pIC) were analysed. It was observed that NO donors did not modify either cytotoxicity performed towards non-sensitized target cells or CL emission. The significance of these results is discussed. [ABSTRACT FROM AUTHOR]

Investigations of the cytotoxic activity of T cells induced following one or two intraperitoneal doses of live Salmonella revealed that cytotoxicity was restricted to the Lyt-2+ T-cell subset and was enhanced following secondary infection with Salmonella. Initial studies using the lectin-dependent cellular cytotoxicity (LDCC) assay detected Lyt-2+ cytotoxic T cells in peritoneal cell suspensions of S. enteritidis 11RX (11RX)-infected mice, with the peak of activity occurring 5 days after infection. This did not correlate with the proliferative activity of these cells, which peaked 10-12 days after infection. Secondary challenge with 11RX or S. typhimurium C5 (C5) induced a rapid increase in the cytotoxic activity of Lyt-2+ peritoneal T cells and was detected even 21 days later. The antigen specificity of some of these cells was confirmed in cytotoxicity assays using P815 tumour cells infected with 11RX organisms as targets. No cytotoxic activity was detected in the spleen cell suspensions of infected (and normal) mice unless the cells were first activated by in vitro culture with concanavalin A (Con A). Both types of activated spleen cells showed LDCC but Salmonella-specific cytotoxic Lyt-2+ T cells were detected only in spleen cell (SC) cultures prepared from mice challenged with a second dose of Salmonella. [ABSTRACT FROM AUTHOR]