Human neutrophil--mediated destruction of antibody sensitized herpes simplex virus type I infected cells

Human peripheral blood polymorphonuclear neutrophils (PMN) were tested for their ability to act as effector cells in antibody-dependent cell cytotoxicity (ADCC) against Herpes simplex virus (HSV) infected target cells sensitized with anti-HSV serum. The PMN from all 29 individuals tested could mediate ADCC in the presence of a standard human anit-HSV serum. Since PMN are prominent cells early in herpes lesions, it was hypothesized that because ADCC could represent an in vitro model for antiviral recovery, perhaps the efficacy of PMN at mediating ADCC might be impaired in those subjects to frequent recrudescent herpes. However, evidence for the hypothesis was not obtained since the PMN from individuals with frequent, infrequent, or unrecorded herpes labialis all showed approximately the same activity at mediating ADCC. Alternative ways in which PMN could be involved in antiviral recovery were discussed.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

The genetic deficiency of leukocyte surface glycoprotein Mac-1, LFA-1, p150,95 in humans is associated with defective antibody-dependent cellular cytotoxicity in vitro and defective protection against herpes simplex virus infection in vivo

The role of the Mac-1, LFA-1, p150,95 leukocyte glycoprotein family in mediating antiviral host defense was investigated by utilizing mononuclear cells (MC) obtained from eight patients with a genetic deficiency of Mac-1, LFA-1, and p150,95, and normal MC incubated with subunit-specific monoclonal antibodies (MAb) directed against these glycoproteins. As shown with an in vitro chromium-release cytotoxicity assay to herpes simplex virus (HSV)-infected Chang liver target cells, MC of these patients with the severe phenotype or normal MC preincubated with a combination of MAb against Mac-1 glycoprotein subunits were deficient in antibody-dependent cellular cytotoxicity (ADCC). When used individually, MAb directed at LFA-1-alpha or -beta also inhibited ADCC and natural killer cytotoxicity (NKC). In a single cell agarose assay, MC of Mac-1-deficient patients formed fewer effector-target cell conjugates in the presence of specific anti-HSV antibody. To investigate the in vitro contributions of these glycoproteins to cytotoxic host defense mechanisms, two in vivo adoptive transfer models were explored in which neonatal mice are protected against a lethal HSV challenge by normal human MC plus anti-HSV antibody (in vivo ADCC) or human interferon-alpha (NKC stimulated in vivo). In each model, MC from patients with "severe" or "moderate" phenotypes of Mac-1 deficiency, or normal MC incubated with a combination of anti-LFA-alpha, Mac-1-alpha, p150,95-alpha plus -beta MAb failed to protect neonatal mice against lethal HSV infection. These studies further indicate requirements for adhesion-dependent mechanisms in the mediation of MC-ADCC, and suggest that Mac-1-dependent cellular adhesive properties are necessary for normal cytotoxic functions in vivo in experimental models of human ADCC or interferon-stimulated NKC. These findings, in addition to the recognized occurrence of severe or even lethal viral infections in some Mac-1-deficient patients, suggest that glycoproteins of the Mac-1 family may be important determinants of antiviral host defense.     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. II. Identification as a K cell.

Studies were carried out to determine whether the mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity (ADCC) to herpes simplex virus (HSV)-infected target cells has surface Fc receptors which participate in the reaction. The F (ab')2 fragment of human IgG antibody was inactive both in ADCC and in complement-mediated cytolysis, but retained the capacity to neutralize infectious virus, to agglutinate erythrocytes coated with viral antigens, and to bind to the surface of virus-infected cells. Treatment of sensitized virus-infected target cells with staphylococcus protein A, which has affinity for the Fc epitope of IgG, strongly reduced their susceptibility to lysis by ADCC in a dose-dependent relationship. These findings indicate that the Fc portion of IgG antibody to the virus is necessary for cytotoxicity. Treatment of blood mononuclear cells with either heat-aggregated gamma-globulin or HSV immune complexes inhibited effector cell activity. The presence of "third party" cellular immune complexes also strongly inhibited ADCC. Adsorption of mononuclear cells to plastic surfaces coated with soluble third party immune complexes resulted in a significant reduction in effector cell activity. These findings demonstrate that the ADCC effector cell possesses surface Fc receptors which are utilized in the ADCC reaction. The presence of Fc receptors on the surface of the effector cell indicates that it is a K cell rather than a null cell.     

Interaction of monocytes with tumor cells coated with complement with or without antibody

Raji, a human B lymphoblastoid cell line has the ability to activate the complement cascade by alternate pathway mechanisms with subsequent fixation of C3 to receptors on the Raji cell membrane. Using this property, we examined the role that complement plays in mediating a cytolytic event between human peripheral blood monocytes and Raji cells coated with C3b, antibody, or both. Presence of C3 was confirmed by immune adherence. IgG bound to the Raji membrane was quantitated using I¹²⁵ Staphylococcal protein A assay. The presence of alternate pathway-activated C3 on Raji cells failed to produce monocyte-mediated cytotoxicity. These same target cells subsequently coated with antibody concentration ranging from 200 to >600,000 SPA molecules per Raji cell produced neither enhancement nor inhibition of antibody-dependent, cell-mediated cytotoxicity (ADCC). ADCC was enhanced by complement when complement activation and binding of C3 to the cell surface occurred by classical pathway mechanisms. ADCC of 32% ± 3.2 occurred with undiluted antiserum (625,000 SPA molecules bound/Raji cell) with enhancement to 52% ± 1.1 in the presence of C3. IgG inhibition of ADCC was unaffected by the presence of membrane-bound C3.     

Ultrastructure of antibody dependent cellular cytotoxicity in HSV 1 infected and uninfected Chang liver cells

The binding and ultrastructural features of antibody dependent cellular cytotoxicity (ADCC) mediated by human peripheral blood lymphocytes were studied in herpes simplex virus type I (HSV-1) infected Chang liver (CL) cells plus human anti-HSV-1 serum, and in uninfected CL cells plus guinea pig anti-CL antiserum. Non-cytolytic controls included target cells treated with normal serum in place of sensitized targets and heat shocked lymphocytes instead of normal lymphocytes. By transmission electron microscopy, target cell membranes were either broadly indented by effector cells or locally invaginated by means of effector cell filopodia. In neither case did the indentation appear to break the plasma membrane of the target. Control preparations showed only non-indented areas of simple membrane contact. By scanning electron microscopy, the effector lymphocytes in both the active ADCC and normal serum control preparations had a sparse distribution of short microvilli over their surfaces. The majority of heat shock control lymphocytes appeared normal, but 12-20% demonstrated surface patches devoid of microvilli. The hypothesis that ADCC may involve a three-step process is discussed.     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

The direct antiviral cytotoxicity by bovine lymphocytes is not restricted by genetic incompatibility of lymphocytes and target cells.

Experiments were designed to determine if in the ox a requirement for genetic compatibility between antiviral cytotoxic cells and target cells was needed for cytotoxicity to occur. Unrelated bovine animals were immunized with vaccinia or IBR virus (a herpesvirus), and PBL were collected at various times for measurement of their cytotoxicity against autologous or heterologous uninfected or virus-infected fibroblasts. In all instances, cytotoxicity was expressed against heterologous as well as autologous targets and in most cases there was no evidence of enhanced killing of autologous cells. The cytotoxicity was shown to be direct, presumably T cell mediated, and was not attributable to ADCC. The likelihood of the animals under investigation sharing histocompatibility antigens was considered extremely remote but was not formally excluded.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

Human monocyte-macrophage-mediated antibody-dependent cytotoxicity to herpes simplex virus-infected cells.

Human peripheral blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus- (HSV) infected target cells consist of both adherent (MA) and nonadherent (MNA) effector cell populations. These two cell populations can be distinguished by their different phagocytic properties and morphologic appearance, their requirement for antibody in the ADCC reaction, and the rapidity with which they lyse target cells in the presence of immune serum. The MA cells are predominantly phagocytic and have the morphologic characteristics of monocyte-macrophages, whereas the MNA cells are nonphagocytic and appear to be small to medium-sized lymphocytes. Optimal expression of ADCC by MA cells requires higher concentrations of immune serum than does MNA cell-mediated ADCC. MA-mediated cell killing is first detectable by 8 hr and reaches completion after 24 hr of incubation. In contrast, MNA-mediated ADCC produces target cell damage by 2 hr and reaches completion at 8 hr of incubation. Unlike MNA effector cells, the MA effector cells are profoundly inhibited after preincubation with either latex or silica particles. The HSV immune status of the donor had no effect on the ability of either cell population to mediate ADCC. These data demonstrate the participation of both nonadherent mononuclear cells, presumably K cells, and monocyte-macrophages, in ADCC directed against HSV-infected target cells.     

Polymorph-mediated antibody-dependent cytoxicity--modulation of activity by drugs and immune interferon.

Bovine polymorphonuclear leukocytes (PMN) mediated antibody-dependent cell cytotoxicity (ADCC) against erythrocyte and herpes virus-infected target cells. The extent of cytotoxicity was not affected by drugs that inhibited DNA, RNA, or protein synthesis. The effect did not occur in the absence of divalent cations, was suppressed by pretreatment of PMN with silica and cytochalasin B, and was subject to the bidirectional control by cyclic nucleotides; drugs decreasing cyclic AMP or elevating cyclic GMP levels enhanced ADCC. The ADCC phenomena was also enhanced by supernates containing immune interferon activity from antigen-stimulated-immune lymphocyte-macrophage cultures. The possibility that immune interferon(s) might be causing the elevation of ADCC and the relevance of this observation in terms of the part interferon might play in modulating recovery from herpes virus infections was discussed.     

Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII.

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.     

Potentiating effect of mizoribine on the anti-herpes virus activity of acyclovir

Pharmacological induction of low deoxyribonucleoside triphosphate (dNTP) levels in virus-infected cells could result in an increased antiviral effectiveness of some selective antiviral nucleoside analogues. That could be exploited as a new combined strategy in the treatment of herpes virus infections. From this point of view the alteration of antiherpes activity of acyclovir (ACV) in combination with mizoribine (N'-[beta-D-ribofuranosyl]-5-hydroxyimidazole-4-carboxamide) (MZR), an inhibitor which lowers the intracellular pool of dGTP, was studied. MZR applied alone at non-toxic concentrations had no effect on herpes simplex virus type 1 (HSV-1) replication in human embryonic skin-muscle fibroblasts (HESMF). The combination of MZR and ACV acts synergistically, as measured by the virus yield assay in the above mentioned system. The potentiating effect of MZR on the anti-HSV-1 activity of ACV was reversed by guanosine (Guo). In this case dGTP could be considered as the "key metabolite" responsible for the higher effectivity of the combination of drugs.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.     

Evaluation of the antibody-dependent cytotoxic capabilities of individual human monocytes. Role of Fc gamma RI and Fc gamma RII and the effects of cytokines at the single cell level.

In this report we present evidence that not all human peripheral blood monocytes mediate antibody-dependent cellular cytotoxicity (ADCC), and that this function may be determined on an individual cell by both the type and level of expression of FcR, and by the state of cellular activation and/or differentiation. Although the diverse range of effector and regulatory functions performed by human monocytes suggests the possibility of distinct subsets, it is not clear whether observed functional heterogeneity reflects the presence of true monocyte subpopulations, or whether this diversity represents a continuum of maturational states present in the peripheral circulation. In an attempt to address this question, we investigated the ability of human monocytes to carry out ADCC at the single cell level, with emphasis on the role of the three FcR for IgG (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) in mediating cytotoxicity. Using a modified plaque assay, 58.3% +/- 4.9 of freshly isolated monocytes mediated ADCC, as evidenced by the formation of lytic plaques in monolayers of ox erythrocyte (oxE) target cells. Significant increases in the number of plaque-forming cells were observed after positive selection by flow microfluorimetry for those monocytes expressing high levels of Fc gamma RI and Rc gamma RII, but not Fc gamma RIII. Bispecific antibodies composed of Fab fragments of anti-oxE antibody covalently coupled to Fab fragments of anti-Fc gamma R antibodies were used to independently evaluate the ability of Fc gamma RI, Fc gamma RII, and Fc gamma RIII to mediate single cell cytotoxicity. Significant increases in the number of plaque-forming cells were observed in the presence of anti-Fc gamma RI x anti-oxE and anti-Fc gamma RII x anti-oxE bispecific antibodies, confirming the efficiency of Fc gamma RI and Fc gamma RII as cytotoxic trigger molecules on human monocytes. Incubation of monocytes with purified rIFN-gamma and granulocyte macrophage-CSF, but not IL-2, IL-3, IL-4, IL-6, or TNF-alpha, also resulted in significant increases in the number of monocytes mediating cytotoxicity, suggesting that cytotoxic ability at the single cell level may be influenced by factors which effect monocyte activation and differentiation, respectively. Overall, these studies demonstrate that freshly isolated human monocytes are heterogeneous in their ability to mediate ADCC, and suggest that this functional diversity arises not from discrete subpopulations of cells, but from a continuum of maturational/activational states present within the peripheral circulation.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Identification of target antigen for antibody-dependent cellular cytotoxicity on cells carrying Epstein-Barr virus genome

The target antigen for antibody-dependent-cellular cytotoxicity (ADCC) on Epstein-Barr virus-(EBV) carrying lymphoblastoid cells expressing EBV-specific membrane antigen (MA) were examined with human serum antibody and adult human peripheral lymphocytes as effector cells. These studies confirmed that anti-MA-positive but not MA-negative sera were reactive in the ADCC. The ADCC reaction was positive with cells in which the MA consisted of late (LMA) and early (EMA) components. These included 1) MA-positive cells prepared by EBV antigen-adsorption, 2) cells carrying de novo-synthesized MA without adsorbed MA, and 3) EBV-producer cells expressing MA spontaneously. In all these preparations, the target cells were lysed roughly in parallel with the frequency of MA-positive cells. Inhibition of LMA synthesis in EBV-superinfected cells by phosphonoacetate (PA) reduced ADCC sensitivity significantly and to a far greater extent than MA synthesis as measured by immunofluorescence. This suggests that a target for ADCC is the PA-sensitive LMA. No ADCC reaction occurred with the cell preparation comprised of a high percentage of MA-positive cells induced by 5-iodo-2'-deoxyuridine, which is believed to be EMA only. These results strongly suggest that the target antigen for ADCC in EBV-positive cells is a late MA but not early MA.     

The role of neutrophils in antiviral defense--in vitro studies on the mechanism of antiviral inhibition.

Highly enriched populations of bovine neutrophils were added, in the presence of antiviral antibody, to herpesvirus-infected bovine cell cultures. A cell dose-dependent reduction in virus-induced cytopathology was observed. The mechanism of inhibition was presumed to be mediated by a subcellular neutrophil product and not the result of either direct cytotoxicity or antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, inhibition of comparable magnitude was observed when neutrophils and virus-infected cells were separated by cell impermeable membranes. In addition, plaque reduction occurred when antiviral immunoglobulins or fragments unable to mediate ADCC were used in the assays. Killed neutrophils and sonicates were unable to mediate plaque inhibition. Speculations were made as to the origin of the virus-inhibitory substances and the role that neutrophils might assume in mediating recovery from virus infection.