Lymphocyte-mediated immune cytotoxicity in dogs infected with virulent canine distemper virus.

Immune lymphocyte-mediated cytotoxicity (ILMC) was evaluated in dogs after intranasal exposure to one of the following three virulent strains of canine distemper virus: Cornell A75/17, Ohio R252, and Snyder Hill. Cytotoxicity was tested with peripheral blood lymphocytes as effector cells and primary dog testicle cells that were matched for histocompatibility as target cells. A strong correlation was found between ILMC and the course of the infection. Dogs that succumbed to encephalitis with any of the strains had little or no ILMC, whereas dogs that recovered had the highest activity. In the intermediate range were dogs with a delayed or reduced ILMC which developed persistent but subclinical central nervous system infections. A significant difference in onset, peak, and duration of ILMC was observed in dogs infected with different strains of canine distemper virus. ILMC responses began at 14 days postinfection (p.i.), reached a peak at 21 to 28 days p.i., and returned to preinoculation levels by 63 to 70 days p.i. in canine distemper virus A75/17- and R252-infected dogs. In contrast, ILMC in canine distemper virus Snyder Hill-infected dogs began at 10 days p.i., peaked by 14 to 17 days p.i., and approached preinoculation levels by 28 days p.i. Antiviral immunity as measured by ILMC appears to be a critical factor in determining the outcome in canine distemper virus-infected hosts. Furthermore, for certain viral biotypes, a delayed ILMC response correlated with persistent infection of the central nervous system.     

Antibody-dependent cytotoxicity mediated by natural killer cells is enhanced by castanospermine-induced alterations of IgG glycosylation

Inhibitors of glycosylation and carbohydrate processing were used to probe the functional consequences of specific, differential alterations in glycosylation of monoclonal IgG secreted by hybridoma clones. Neither the absence of glycosylation nor the presence of atypical oligosaccharides significantly influenced binding of the monoclonal antibody to the cell surface antigen recognized. However, lymphocyte-mediated antibody-dependent cytotoxicity was enhanced significantly, as compared to native (unmodified) IgG-sensitized target cells, when target cells were sensitized with IgG bearing the a typical oligosaccharides induced metabolically by castanospermine. N-methyldeoxynojirimycin, deoxymannojirimycin or monesin, but not by swainsonine. The enhanced cytotoxicity was mediated by natural killer cells but not by monocytes or interferon-activated polymorphonuclear leukocytes. By contrast, antibodydependent cytotoxicity mediated by activated polymorphonuclear leukocytes against target cells sensitized with the IgG glycosylation phenotypes induced by swainsonine and tunicamycin. but not by castanospermine, was decreased in comparison to cytotoxicity against target cells sensitized with native IgG.

The enhanced lymphocyte-mediated cytotoxicity was Fc receptor-dependent.

A panel of monoclonal antibodies directed against different human tumor target cells was used to demonstrate that the castanospermine-induced IgG phenotype generally enhanced antibody-dependent tumoricidal activity mediated by natural killer cells. However, differences in lymphocyte response to an alteration in IgG glycosylation were observed.     

Cytomegalovirus-specific cytotoxicity mediated by non-T lymphocytes from peripheral blood of normal volunteers.

The cytotoxicity of circulating mononuclear cells from normal volunteers was determined using human leukocyte antigen (HLA)-typed, low-passaged human skin fibroblasts infected with cytomegalovirus as target cells. Peripheral blood lymphocytes from both seropositive and seronegative individuals possessed virus specific cytotoxicity. Although all target cells used were susceptible to virus specific lysis, lymphocytes from some individuals were more active against some target cells than others. This differential cytotoxicity did not follow a consistent pattern of HLA restriction. Some variations in cytotoxic activity were noted on sequential studies of individual volunteers. Studies of fractionated lymphocytes from selected immune and nonimmune individuals demonstrated that cytotoxicity of lymphocytes from both groups was mediated by nonadherent, Fc receptor bearing cells which did not form rosettes with sheep erythrocytes. Repeated washing sometimes decreased cytotoxicity of lymphocytes from immune individuals, and addition of serum containing antibody to cytomegalovirus enhanced cytotoxicity, suggesting antibody dependence. It is concluded that cytotoxic lymphocytes from nonimmune volunteers possessed characteristics of natural killer cells, whereas those from immune volunteers probably consisted of both natural killer cells and antibody-dependent killer cells.     

Generation of virus-specific cytolytic activity in human peripheral lymphocytes after vaccination with vaccinia virus and measles virus

Human peripheral blood lymphocytes (PBL) harvested after vaccination with vaccinia or measles virus showed a specific activity against virusinfected target cells. This activity peaked on day 7 and was specific for the target cells infected with the virus used for the vaccination. The cytotoxic activity was not related to HLA markers. The cells involved in the cytolytic process were lymphocytes bearing Fc receptors. In addition, the cytotoxic activity was abrogated by more than 90% by rabbit Fab'2 anti-human IgG. It is therefore likely that two subpopulations of lymphocytes are involved: an antibody-secreting cell providing specific antiviral antibody and an effector cell bearing Fc receptor (K cells). Finally, these experiments suggest that antibody-dependent cell cytotoxicity may play a major role in the recovery from virus infection in man.     

Monoclonal antibodies against leucoagglutinin-reactive human T lymphocyte surface components. Two antibodies which inhibit cell-mediated cytotoxicity at a post-binding stage

Two out of 20 monoclonal antibodies (IgM, kappa), mAb 3192 and mAb K3G, raised against leucoagglutinin-reactive components on human T cells, effectively blocked lymphocyte-mediated cytotoxicity in vitro. No antigenic polypeptide reactive with these antibodies has been identified thus far. However, they have previously been shown to react specifically with certain neutral glycolipids obtained from spleen. Both mAb inhibited the cytotoxicity of natural killer (NK) cells against K562 cells, antibody-dependent cellular cytotoxicity (ADCC) towards antibody-coated bovine erythrocytes and cytotoxic T lymphocyte activity against allogeneic target cells. In both NK and ADCC, preincubation of the lymphocytes with different antibody concentrations resulted in a dose-dependent reduction of cytotoxicity. In contrast, preincubation of the target cells had no effect indicating that the mAb inhibited cytotoxicity at the effector cell level. When studied at the single-cell level, the mAb did not alter the number of lymphocytes forming conjugates with K562 but significantly reduced the frequency of conjugates containing dead target cells. Addition of the mAb to preformed conjugates resulted in a dose-dependent reduction in the proportion of conjugates containing dead target cells. Furthermore, mAb 3192 did not reduce the number of lymphocytes forming rosettes with bovine erythrocytes, indicating that inhibition of ADCC was not due to blocking of the effector cell-target cell interaction mediated by the Fc receptor of the effector cells. Taken together, these results suggest that the mAb inhibited cytotoxicity by interfering with a post-binding step common for the different cytotoxicity systems.     

Effect of Fc-Receptor Modulation on Mumps-Virus-dependent Lymphocyte-mediated Cytotoxicity in Vitro

The natural cytotoxicity of peripheral blood lymphocytes (PBL) from normal human donors to a variety of tissue culture target cells increases upon brief exposure of lymphocytes to mumps virus. The effector cells operative in this system have Fc receptors for IgG (FcR), since cytotoxicity was abolished when FcR ⁺ cells were removed by passage of the lymphocytes over immune-complex columns. When PBL were treated with immune complexes for 16 h at 37°C, their FcR activity was sharply decreased (modulation), as indicated by a significantly reduced capacity of the treated cells to display antibody-dependent cytotoxicity (ADCC). Modulation had variable effects on natural cytotoxicity. In contrast, the virus-dependent cytotoxicity above the natural cytotoxicity remained essentially unchanged, indicating that a functionally intact FcR is not required in this system for carrying out cytolysis.     

Cell-mediated cytotoxicity following influenza infection and vaccination in humans

Cell-mediated cytotoxic activity in circulating mononuclear cells from 31 volunteers challenged with live influenza A/Victoria virus, and 22 volunteers vaccinated with inactivated influenza vaccine, was examined employing target cells infected with several viruses by means of a ⁵¹ Cr release assay. Effectors from infected volunteers, and from volunteers who manifested four-fold rises in serum HAI antibody after vaccination, demonstrated significantly elevated levels of cytotoxicity against targets infected with the homologous virus. Elevated cytotoxicity was seen by days 3 and 4 after challenge or vaccination and returned to baseline levels by day 9 to 10. In infected volunteers, cytotoxic activity was broadly directed, rising against targets infected with an antigenically distinct virus within the same influenza type (A), against targets infected with a serologically unrelated virus of a different influenza type (B), and also against cells infected with Newcastle disease virus, a paramyxovirus from another species. However, elevated levels of cytotoxicity were not observed when targets infected with herpes simplex virus, a member of an entirely different virus group, or when uninfected target cells were employed. In vaccinated volunteers, the rise in cytotoxicity was more restricted than after infection, since elevated cytotoxic activity was seen only against cells infected with the homologous virus and not against influenza B-infected cells. Fractionation of mononuclear cell populations indicated that effector cell activity is associated with T-cell depleted fractions and can only partially be reduced by depletion of adherent cells. The rapid development, short duration, and broadly directed specificity of this cytotoxic response suggest that it may be involved in early events following acute influenza infection in humans.     

Cell-mediated immunity in rubella assayed by cytotoxicity of supernatants from rubella virus-stimulated human lymphocyte cultures.

Rubella virus-stimulated lymphocytes from rubella-seropositive donors produced in the culture medium cytotoxic activity with preferential action against rubella-infected over uninfected target cells. The ability of lymphocytes to produce the cytotoxic activity upon stimulation by rubella virus correlated with the humoral rubella-immunity status, i.e. no such cytotoxic activity developed in the supernatants of lymphocyte cultures of rubella-seronegative donors. Stimulation of lymphocytes from seropositive donors by rubella virus was also detected by thymidine incorporation, but the correlation of lymphocyte responsiveness to the humoral rubella antibody status was not so clear as in the cytotoxicity assay. Conversion of lymphocytes from unresponsive to responsive to rubella virus following natural rubella infection and after rubella vaccination was demonstrated using both methods. Following vaccination rubella-specific cell-mediated immunity first became demonstrable at 14 days. The responsiveness of lymphocytes to phytohaemagglutinin (PHA) after rubella vaccination was followed by studying thymidine uptake and the ability of lymphocytes to produce lymphootoxin. By both tests marked suppression of PHA response occurred at days 3 and 7 after vaccination.     

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC.     

Immune mechanisms against canine distemper. II. Role of antibody in antigen modulation and prevention of intercellular and extracellular spread of canine distemper virus.

Specific antibody was shown to be highly effective in neutralizing extracellular canine distemper virus (CDV) as well as preventing the intercellular spread of this virus. Thus, relatively low levels of antibody neutralized 1 x 10(5) TCID50 of extracellular CDV and the development of plaques or CPE in Hep-2 and Vero cells respectively could be prevented even when up to 5% of the cells were infected. This inhibition of CPE and virus spread was most pronounced when antibody was added early but could still limit the degree of CPE if added as late as 48 h post-infection. This anti-viral activity was observed in different cell types including canine macrophages, cells normally infected with CDV in vivo. Prolonged exposure of infected target cells to high concentrations of antibody led to redistribution of surface viral antigens and their subsequent disappearance. The possible role of antibody in the defence against, and/or recovery from CDV and the mechanism(s) by which antibody may aid in recovery are discussed.     

Immune mechanisms against canine distemper. I. Identification of K cell against canine distemper virus infected target cells in vitro.

Canine peripheral blood lymphocytes, polymorphonuclear leucocytes (PMN) and monocytes (macrophages) were obtained by various cell separation techniques and were tested for their cytotoxic capacity against antibody-sensitized canine distemper virus (CDV) infected Vero cells by an in vitro chromium release assay. Canine lymphocytes were found to destroy CDV infected target cells effectively, while neither PMN nor monocytes (macrophages) could do so. The active lymphocyte was characterized by various rosetting techniques to be a non-T and a non-B lymphocyte. These cells bear no surface immunoglobulin (SIg-) but possessed both Fc receptors (Fc+) and complement receptors (EAC+) suggesting that these cells are neither classical T nor B cells. The possible roles of this K cell in the resistance against canine distemper are discussed.     

Scanning and transmission electron microscopy study of antibody-dependent lymphocyte-mediated cytotoxicity on measles virus-infected cells.

The structural events related to antibody-dependent lymphocyte-mediated cytotoxicity (ADLC) have been studied on measles virus-infected cells using human peripheral blood lymphocytes (PBL) and anti-measles virus serum. The first event in ADLC was a recognition process occurring with 15 min after contact between the infected cells and lymphocytes. Plasma membrane and microvilli of adsorbed PBL were specifically attached to virus-induced ridges over nucleocapsids and to viral buds. After 30 min, a fraction of adsorbed PBL (K cells) changed shape and extended long filipodia toward the target cells which, in turn, showed long villi contacting the PBL. At 4 h, when cytotoxicity as measured by chromium release was maximum, K cells had flattened and numerous blebs and ruffles formed on their surface. The K-cell alterations varied in intensity with the type of measles-infected target cell, but frequently the K cells appeared irreversibly damaged. T- and non-T-cell fractions were separated, and in situ erythrocyte rosettes were used as markers for subpopulations which were easily recognized by scanning electron microscopy. Most of the cytotoxic K cells were identified as non-T cells carrying Fc receptors for immunoglobulin G. However, a small subpopulation of cells bearing both sheep erythrocyte and Fc receptors was also found to be involved in ADLC by chromium release assay as well as by electron microscopy. Some of these interacting T cells extended a long uropod on the target cell, but their intracellular structure remained unaltered through ADLC, in contrast with the other T cells and the non-t killer cells. This suggests that perhaps some T killer cells might remain functional after the cytotoxic interaction with a target cell.     

The immune response against foot-and-mouth disease virus: influence of the T lymphocyte growth factors IL-1 and IL-2 on the murine humoral response in vivo.

Recombinant and pure "natural" IL-1 and IL-2 were compared with the muramyl dipeptide (MDP) component of Freund's adjuvant for their capacity to enhance the humoral immune response against foot-and-mouth disease (FMD) virus antigen. Using a dose of this antigen which alone did not give a detectable immune response, anti-FMD virus antibody was measured at 14 and 28 days post-vaccination. Although IL-1 could enhance the response against the virus antigen, in particular when administered 24 h before the vaccine, this was not as strong as that obtained when MDP was adjuvant. In contrast, IL-2 was at least as efficient as MDP when applied concomitantly with the antigen. If the IL-2 treatment preceded the vaccination by 24 h, a diminution in the magnitude of the response was seen; however, this was countered by the fact that 10 times less IL-2 was required, compared with concomitant cytokine/vaccine administration, in order to have the maximum effect. When both IL-1 and IL-2 were used together, an even greater enhancement of the immune response against FMD virus antigen was observed, but only when given concomitantly with the antigen. These results demonstrate the relevance of T lymphocyte growth factors to the immune response against FMD virus, and how current immunological and biotechnological knowledge could be applied to the improvement of adjuvant systems in a chemically and biologically defined manner.     

Interference of hepatitis B virus surface antigen with natural killer cell function.

The influence of purified hepatitis B virus surface antigen (HBsAg) preparations or of supernatants derived from PLC/PRF/5 cell line (which produces HBsAg) on human natural killer (NK) activity was examined. Lymphocytes pre-incubated with HBsAg and subsequently washed showed a significant decrease in NK cytotoxicity against K-562 target cells. This effect was reversible and dose-dependent. In addition, pre-incubation with either HBsAg or PLC/PRF/5 supernatants inhibited in a reversible manner lymphocyte--K-562 conjugates and the binding of B73.1 monoclonal antibody (MoAb), which recognizes Fc receptors on NK cells. This effect was not observed with HNK-1, T3, T4, T6, T8 and T11 MoAb. HBsAg was non-toxic to lymphocytes, and ineffective with K-562 target cells. Beta-interferon did not modify HBsAg-mediated inhibition, when added either before or during the contact with HBsAg. Moreover, no modification was observed when neutrophils (at various neutrophil:lymphocyte ratios) were added, even though HBsAg is known to stimulate neutrophils to produce oxygen radicals which may modulate NK activity. We speculate that HBsAg produces these effects by reacting into receptor sites (possibly Fc receptor sites) on NK cell membrane. The overall significance of our results in relation to hepatitis and hepatocellular carcinoma is discussed.     

Anti-class II antibodies in AIDS patients and AIDS-risk groups

The specificity of anti-lymphocyte antibodies was evaluated in AIDS patients and in individuals at risk of AIDS [R-AIDS: male homosexuals (Ho) and haemophiliacs (He)]. Antibodies capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against non-T cells and lymphoblastoid cell lines (P3HR-1K and Raji) were detected in AIDS patients and in R-AIDS with positive or negative human immune deficiency virus (HIV) serology. Anti-class II antigen specificity was revealed by experiments in which class II antigens on target cells were blocked with monoclonal anti-class II antibody (DA6,231) and the cytotoxic reaction induced by patient's sera was abolished. In contrast, ADCC was not impaired by preincubating the target cells with anti-class I monoclonal antibody (W6/32). Prevalence of antibodies to non-T cells was confirmed by standard C-mediated microlymphocytotoxicity. However, with this technique anti-T lymphocyte cytotoxicity was also observed in three AIDS patients with haemophilia. R-AIDS peripheral blood mononuclear cells (PBMC) were also cytotoxic against autologous non-T cells, and lysis was slightly increased by sensitization of the target cells with autologous serum. In addition to ADCC and C-mediated cytotoxicity, the specificity of anti-lymphocyte antibodies was assayed by their ability to interfere the binding of fluorescein-labelled anti-class II (HLA-DR) and anti-class I (W6/32) monoclonal antibodies to PBMC, non-T cells, P3HR-1K and Raji. Anti-class II specificity was confirmed, and antibody titres tended to be higher in Ho than in He R-AIDS, using non-T cells and Raji as targets. Higher titres of anti-class II antibodies in the Ho group could play a role in the different susceptibility of HIV-infected Ho when compared to HIV (+) He to develop AIDS.     

Specificity in the immunosuppression induced by avian reticuloendotheliosis virus.

Several parameters of the cellular and humoral immune responses of chickens infected with reticuloendotheliosis virus (REV-T), an avian defective acute leukemia virus, or with its helper virus, reticuloendotheliosis-associated virus (REV-A), were evaluated. Spleen cells from chickens infected with REV-T (REV-A) or REV-A exhibited depressed mixed lymphocyte and mitogen responses in vitro. Allograft rejection was delayed by 6 to 14 days in birds infected with REV-A. The specific antitumor cell immune response was also studied by a 51Cr-release cytotoxicity assay. Lymphocytes from chickens infected with low numbers of the REV-T-transformed cells exhibited significant levels of cytolytic reactivity against the 51Cr-labeled REV-T tumor cells in vitro. The mitogen response of lymphocytes from these injected birds was similar to that of uninjected chickens. In contrast, lymphocytes from chickens injected with higher numbers of REV-T-transformed cells exhibited suppressed mitogen reactivity and failed to develop detectable levels of cytotoxic activity directed against the REV-T tumor cells. These results suggest that the general depression of cellular immune competence which occurs during REV-T (REV-A) infection could contribute to the development of this acute leukemia by inhibiting the proliferation of cytotoxic cells directed against the tumor cell antigens. The cytotoxic effect observed after the injection of chickens with non-immunosuppressive levels of REV-T-transformed cells appears to be specific for the REV-T tumor cell antigens since cells transformed by Marek's disease virus or avian erythroblastosis virus were not lysed. In marked contrast, birds whose cellular immune responses were suppressed by infection with REV-A were capable of producing a humoral immune response to viral antigens. Detectable levels of viral antibody, however, did not appear until 12 to 15 days after REV-A infection. Since REV-T (REV-A) induced an acute leukemia resulting in death within 7 to 14 days, it appears unlikely that the ability of chickens to make antiviral antibody influences the development of lethal reticuloendotheliosis.     

Antibody-dependent cellular protection against herpes simplex virus dissemination as revealed by viral plauqe and infectivity assays.

Mouse nonimmune peripheral blood lymphocytes (PBL) plus antibody to herpes simplex virus inhibited virus dissemination in herpes simplex virus-infected 3T3 cell culture as revealed by development of viral plaque, size of immunofluorescent foci, appearance of polycaryocytes, and viral infectivity appearing in the culture. These nonimmune PBL did not act alone in inibiting virus dissemination, but did act synergistically in combination with antibody. The ratio of PBL to target monolayer cells needed to produce this effect was 20. Splenic lymphocytes had weak activity, whereas thymocytes were without effect, even in the presence of antibody. Neither interferon nor lymphotoxin was detected in this lymphocyte-mediated response. These findings support previous observations, based on cytotoxicity assays, that antibody-dependent cellular immune processes could be important in control of and recovery from herpesvirus infection.     

Target cell-dependent T cell-mediated lysis of vaccinia virus-infected cells

Vaccinia virus specific cytotoxicity against infected target cells was observed in vitro. Spleen lymphocytes from normal and immunized mice of the inbred strains C3H and DBA/2 were incubated with vaccinia virus-infected and non-infected ⁵¹Cr-labeled mastocytoma P-815-X2 cells and L-929 fibroblasts, which were used as targets. Cytotoxic lymphocytes could be isolated from the mice as early as 2 days after infection with vaccinia virus. The highest cytotoxic effect was obtained with lymphocytes taken 6 days after infection. The degree of lysi was correlated with the ratio of immune lymphocytes to target cells. Specific blocking of target cell lysis resulted after addition of anti-vaccinia antibody from different sources. The effector cells could be characterized as T cells by elimination of macrophages and B cells. Target cell killing was only possible in a syngeneic system; allogeneic infected target cells were not lysed significantly.     

Antibody-dependent cellular cytotoxicity (ADCC) against Epstein-Barr virus-determined antigens II. Induction of ADCC by FC-FC receptor bridging.

Four different methods to test antibody-dependent cellular cytotoxicity (ADCC) against Epstein-Barr virus (EBV) determined antigens were analysed. It was found that optimal ADCC was obtained if viral antigens were present during the cytotoxicity reaction and that killing probably was mediated by EBV-related immune-complexes forming Fc-Fc receptor bridges between Fc receptor-bearing effector cells and Fc receptor-bearing target cells. Only lymphoid target cells with a high expression of Fc receptors were found to be susceptible in this particular system.     

Lymphocyte cytotoxicity to influenza virus-infected cells: response to vaccination and virus infection

Peripheral blood leukocytes obtained from volunteers at various times following influenza vaccine or live influenza virus infection were assayed for cytotoxicity against influenza virus-infected cells. Cytotoxicity was highest on days 3 and 7 following vaccination with commercial A/Port Chalmers/1/73 inactivated influenza virus vaccine. Maximal cytotoxicity was found 9 days after infection induced by intranasal inoculation of a strain of A/Scotland/840/74 influenza virus. Individuals naturally infected with A/Victoria/3/75 were also found to have elevated cytotoxicity approximately 1 week after onset of illness. Cytotoxicity levels decreased toward base line approximately 30 days after the virus exposure. The effector mechanism(s) responsible for the early, transient elevation in specific immune release to influenza virus-infected cells may be different from the antibody-dependent cell cytotoxicity demonstrated in the peripheral blood leukocytes from volunteers who had a remote experience with influenza virus.