Antibody-dependent Cellular Cytotoxicity Reactions Research Articles

Serum anti-immunoglobulins in multiple myeloma and benign monoclonal hyperglobulinemia

Anti-immunoglobulins with specificity to human native IgG and (Fab′) 2 were detected in 69 sera of patients with multiple myeloma and 71 sera from 22 patients with benign monoclonal hyperglobulinemia with titers distinctly higher than those found in two control groups (healthy persons, sarcoidosis patients). Without further differentiation, no correlation of anti-immunoglobulin titers to the clinical status of the disease was observed. Chromatographic separation of sera on Sephadex G-200 revealed three molecular species of anti-immunoglobulins: type I, MW ca. 900,000; type II, MW 160,000; type III, MW < 100,000. Elevated titers of type III anti-immunoglobulins were observed only in multiple myeloma. Using immunoadsorbent techniques type III anti-immunoglobulins were identified as (Fab′) 2 fragments. Lacking idiotypic specificity, type III anti-immunoglobulins were shown to react with allotypic as well as idiotypic immunoglobulins on the surface of PBL B-lymphocytes. In addition, they inhibited significantly antibody-dependent cellular cytotoxicity reactions in vitro using IGR 3 melanoma cells as targets, and preparations of peripheral blood lymphocytes from healthy individuals as effector cells. In contrast, no significant inhibition of cytotoxic activity was observed using type II (IgG) anti-immunoglobulins from benign monoclonal hyperlgobulinemia patients' sera in equal concentrations.

Early Damage of Herpes-Infected Cells by Antibody-Dependent Cellular Cytotoxicity: Relative Roles of Virus-Specified Cell-Surface Antigens and Input Virus

Abstract Previous studies in our laboratory indicated that tissue culture cells acutely infected with type 1 herpes simplex virus (HSV) could be recognized at 2 hr post-infection (PI) by antibody-dependent cellular cytotoxicity (ADCC) and damaged by ADCC at 3 hr PI. Since the design of these studies did not preculde the possibility that damage occurs even earlier in the infectious cycle, we performed the present series of experiments to assess this possibility as well as to gauge the relative contributions of virus-specified cell-surface antigens and input virus in rendering target cells susceptible to cytotoxic attack. When present during both the first 2 hr of infection as well as during the following 4-hr ADCC assay period, both actinomycin D (1 µg/ml), an inhibitor of RNA synthesis, and cycloheximide (5 µg/ml), an inhibitor of protein synthesis, strongly reduced the cytotoxic reaction (by 70 and 90%, respectively). The inhibitory effects of these agents indicate that the bulk, but not all, of the ADCC reaction was directed at target cells synthesizing virus-specified surface antigens. Between 10 and 30% of the injured cells were attacked because of input virus that had not penetrated the target cell. This was shown by a) the small portion of ADCC that resisted actinomycin D and cycloheximide, b) the biphasic ADCC kinetic reaction curve of target cells exposed to antibody and effector cells immediately after virus adsorption, and c) the small but reproducible cytotoxic reaction against target cells exposed to virus inactivated by ultraviolet light. A small portion of the infected cell population could be injured by ADCC within the first hour after viral adsorption due to input virus.

Leukocyte cytotoxicity in a persistent virus infection: presence of direct cytotoxicity but absence of antibody-dependent cellular cytotoxicity in horses infected with equine infectious anemia virus

Antibody-dependent cellular cytotoxicity and direct cytotoxicity assays were performed with equine infectious anemia virus-infected target cells, equine leukocytes, and equine anti-equine infectious anemia virus antibody to determine whether these mechanisms play a role in controlling viral replication in equine infectious anemia. Direct cytotoxicity was observed by using peripheral blood mononuclear cells from 7 of 10 infected horses. Antibody-dependent cellular cytotoxicity was not observed. The antibody-dependent cellular cytotoxicity reaction in horses was then studied by using sheep erythrocytes and trinitrophenylated sheep erythrocytes as target cells. Lysis of these target cells was mediated by neutrophils, monocytes, and lymphocytes. The reaction was activated by antibody of the immunoglobulin G class but not by immunoglobulin G(T). Furthermore, immunoglobulin G(T) efficiently inhibited immunoglobulin G in this function.

Development of antibodies reactive in antibody-dependent cellular cytotoxicity in infectious mononucleosis

Serial sera from patients with infectious mononucleosis were examined for the emergence of antibodies reactive in antibody-dependent cellular cytotoxicity tests, using Epstein-Barr virus-superinfected Raji cells as targets. For this specific purpose, the antibody-dependent cellular cytotoxicity test proved to be of limited sensitivity because only relatively high serum dilutions can be tested dependably, due to prozone effects at low serum concentrations, and because antibody-dependent cellular cytotoxicity reactions at the 5% level are not always statistically significant. Under the conditions of the test, antibody-dependent cellular cytotoxicity-reactive antibodies were not measurable, or only barely measurable, in early-acute-phase sera, but they became detectable during convalescence and increased thereafter, gradually over many months to the range of titers seen in healthy persons after long-past-primary Epstein-Barr virus infections. The percentages of antibody-dependent cellular cytotoxicity ultimately attained were on the order of 20% in most patients and healthy individuals, but in others did not exceed 10%. The likely identity of the antibodies reactive in the test with antibodies to late Epstein-Barr virus-determined cell membrane antigens has been discussed.

Immunological and Functional Characteristics of Peripheral Blood and Synovial Fluid Lymphocytes from Patients with Rheumatoid Arthritis

Spontaneous (SCMC) and antibody dependent cellular cytotoxicity (ADCC); mitogenic responsiveness (PHA, Con A, PPD, dextran and pokeweed) as well as lymphocyte subpopulations (E-, EA-, EAC-rosettes, S-Ig) were studied simultaneously in peripheral blood (PBL) and synovial fluid lymphocytes (SFL) of fifteen patients with rheumatoid arthritis. Marked differences were observed in the cytotoxic activity of SFL and PBL. Whereas SCMC activity of SFL was always significantly elevated above the cytotoxic levels of PBL, the reverse was true for the ADCC reaction; here, 50% of the patients showed a decreased cytotoxicity of SFL compared to PBL. Synovial fluid neutrophils (SFN) were found to be inactive in both cytotoxic assays. No differences were found in ADCC activity of PBL between normal controls and RA patients. In SCMC assays a significantly increased activity of control PBL was only observed at L/T ratios of 100:1. Overnight incubation of PBL from RA patients and normal controls resulted in a marked decrease in SCMC and, to a smaller extent, in ADCC activity. SFL from three out of four patients lost less SCMC activity after overnight incubation than the corresponding PBL. In one patient even an increased activity in both cytotoxic systems was obtained. Regarding lymphocyte populations, T-cells were significantly decreased in PBL of RA patients. With the exception of a significantly lowered percentage of C3 receptor positive cells in SFL, no significant differences were recorded in the lymphocyte distribution between the patients' PBL and SFL. In the RA patients, the response to T-cell mitogens was significantly depressed in SFL while PPD and pokeweed reactivity was equal to that of PBL.

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.

The Mononuclear Cell in Human Blood Which Mediates Antibody-Dependent Cellular Cytotoxicity to Virusinfected Target Cells

Abstract 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 γ-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.

Murine neuroblastoma cured in vivo by an antibody-dependent cellular cytotoxicity reaction.

CYTOLYSIS of antibody-coated tumour cells mediated by reticuloendothelial (RE) or bone marrow cells is a phenomenon of unknown biological relevance1–4. The specificity of the reaction lies in the antibody5,6; probably most or all cells able to recognise the Fc fragment of the antibody molecule can mediate this reaction, including lymphocytes and monocytes6,7, circulating cultured transformed lymphoblasts8, polymorphonuclear leukocytes9 and malignant reticulum cells10. In tumour systems the contribution of this reaction is difficult to distinguish in vivo from other cytolytic phenomena, including complement-dependent antibody-mediated cytotoxicity, T-cell cytotoxicity or cell killing exerted by “armed” macrophages11,12, all of which may be active in various degrees in different experimental circumstances. But antibody-dependent cellular cytotoxicity (ADCC), as this phenomenon has been termed13, seems to be of potential therapeutic interest because both its magnitude and specificity depend on specific antibody levels, and antibody levels may be potentially more manipulable in man than are the cellular elements of a host anti-tumour response. In addition, there is preliminary evidence that fixed tissue macrophages may be active in eliminating circulating metastatic cells by this mechanism14. It was therefore interesting to determine whether functional ADCC could be demonstrated in vivo independent of other cytolytic mechanisms since its demonstration has so far been limited to in vitro assays.

The Effector Cells in Human Peripheral Blood Mediating Mitogen-Induced Cellular Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity

The identity of the effector cells in human peripheral blood capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cell populations consisting of purified polymorphonuclear leukocytes, macrophages, lymphocytes, and cell surface immunoglobulin (sIg)-negative and sIg-positive lymphocyte subpopulations obtained by Sephadex anti-Fab immunoabsorbent column fractionation techniques. Chicken erythrocytes (CRBC) and Chang liver cells were used as target cells in both cytotoxicity assays. With CRBC targets MICC was mediated by polymorphonuclear leukocytes, macrophages, sIg-positive lymphocytes (B cells), and sIg-negative lymphocytes. On the contrary, with Chang liver cells as targets, MICC was mediated only by lymphocytes, and effector cells occurred exclusively in sIg-negative lymphocyte subpopulations containing thymus-derived lymphocytes (T cells). Further purification of sIg-negative lymphocyte subpopulations on antigen-antibody coated plastic surfaces yielded a nonadherent T lymphocyte population depleted of Fc receptor-bearing lymphocytes that was capable of mediating MICC against both CRBC and Chang cell targets. With use of CRBC targets, ADCC was mediated by polymorphonuclear leukocytes, macrophages, and sIg-negative lymphocyte subpopulations. However, with Chang cell targets, ADCC was mediated only by lymphocytes, and effector cells were present only in sIg-negative lymphocyte subpopulations. SIg-positive lymphocytes (B cells) and T lymphocytes were not effective in mediating ADCC against either CRBC or Chang cell targets. These studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions is of critical imporatnce in defining the effector cell(s) capable of mediating cytotoxicity.

The nature of the effector cells mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC)

The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions.

Cytotoxic effector cell function in organized gut-associated lymphoid tissue (GALT)

Lymphocytes from the organized gut-associated lymphoid tissues (GALT) of adult guinea pigs were examined for surface markers characteristic of T and B lymphocytes and for their capacity to function as effector cells in mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) reactions. GALT lymphocytes formed rosettes with rabbit erythrocytes, a T-cell marker, and underwent proliferative responses in vitro in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). GALT lymphocytes were cytotoxic in vitro for erythrocyte and DBA mastocytoma targets in the presence of PHA. A population of GALT lymphocytes bound aggregated γ-globulin; however, they functioned poorly in ADCC reactions. Thus, organized GALT in the guinea pig contains lymphocytes capable of functioning in T-cell-dependent MICC reactions but either lacks the effector cell population which mediates ADCC or contains an effector cell which functions poorly in ADCC.