Characterization of the human peripheral blood effector cells mediating antibody-dependent cell-mediated cytotoxicity against respiratory syncytial virus

Peripheral blood lymphocytes were separated into several subpopulations and evaluated for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against respiratory syncytial virus (RSV)-infected HeLa cells. Using erythrocyte rosetting methods, nylon wool filtration, and cytolysis with OKT-3 monoclonal antibody, two lymphocyte subpopulations were shown to mediate RSV-ADCC; non-T, non-B, and IgG-Fc receptor-bearing lymphocytes and E-rosetting cells with IgGFc receptors (T gamma cells). Removal of phagocytic cells did not alter ADCC activity. Monoclonal antibody to human NK and K cells, HNK-1, recognized these two lymphocyte effector subpopulations.     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. III. Ultrastructural studies.

On the basis of electron microscopic observations, four types of cells apparently cytotoxic for antibody-coated chicken erythrocytes were identified in non-immune mouse spleen; monocytes, polymorphonuclear leucocytes, immature granulocytes and a type of lymphoid cell. Both phagocytosis and extracellular lysis of target cells were observed. Monocytes and polymorphonuclears were able to interact with the target cell by both mechanisms while the intermediary granulocytes and lymphoid cells were only capable of extracellular lysis. It is argued that these observations provide a morphological basis for the previous classification of antibody-dependent cytotoxic cells into myeloid and lymphoid cells (Greenberg et al., 1973b, 1975).     

Apparent direct cellular cytotoxicity mediated via cytophilic antibody. Multiple Fc receptor bearing effector cell populations mediating cytophilic antibody induced cytotoxicity

Guinea-pigs immunized with chicken red blood cells (CRBC) developed cytotoxic effector cells in peripheral blood, spleen, lymph nodes, bone marrow and peritoneal exudate cells. Although it appeared that direct cytotoxicity was the mechanism of killing in this model, the true mechanism of cytotoxicity was in fact cytophilic antibody firmly bound to the effector cell rendering it specifically cytotoxic to the CRBC targets. Using multiple cell separation procedures, we demonstrated at least three distinct effector cell populations capable of mediating cytotoxicity in this model: a monocyte-macrophage, a non-phagocytic lymphocyte and a neutrophil, all bearing Fc receptors for Ig. Cell free eluates produced from immune effector cells were capable of rendering non-immune cells of all three Fc receptor bearing leucocyte classes cytotoxic.It is noteworthy that several techniques commonly employed to deplete effector cell populations were shown also to remove cytophilic antibody from the surface of these effector cells. If this had not been recognized, the cytophilic antibody component of the system would have been overlooked and erroneous conclusions would have been made as to which cell populations were functioning as effectors.Recent clinical studies have demonstrated a direct cytotoxicity by K lymphocytes—the usual effector cells in antibody dependent cellular cytotoxicity. The present study suggests that in at least some of these cases true direct cytotoxicity may not be the mechanism of killing and that K cells bearing cytophilic antibody may in fact be the effector cell operating by antibody dependent cellular cytotoxicity.     

Antibody dependent cellular cytotoxicity against coronavirus 229E-infected cells.

An antibody dependent cellular cytotoxic (ADCC) reaction was elicited using human mixed lymphocyte cultures against coronavirus 229E-infected C-16 cells. The response was assayed in microtitre plates by radioactive chromium release. Optimal killing of target cells was achieved using cells labelled 22 h after infection. A prozone was present and the optimal dilution of sera was invariably 1:200. The majority (70%) of sera tested gave a positive ADCC response. These included all sera judged positive by ELISA, but also some which were judged negative. Two experiments using purified IgG from serum which was ADCC and ELISA positive suggested that the response is mediated by an IgG antibody and that the prozone is due to a separate serum component.     

Cell-mediated cytotoxicity. Characterization of the effector cells.

Isolated human mononuclear cells were fractionated according to their membrane characteristics or physical properties. Adherent cells were depleted by filtration through glass columns; phagocytic cells were removed by iron treatment and cell subpopulations capable of forming rosettes with sheep erythrocytes (E), erythrocyte-antibody-complement (EAC) and chicken erythrocyte-antibody complexes (CEA) were separated by centrifugation of Ficoll-Hypaque gradients. The functional activity of the cell subpopulations obtained was assayed by testing PHA-induced cytoxicity (PIC), antibody-dependent cytoxicity (ADCC) and blast transformation by PHA. The results of this study demonstrate that: (1) cells reacting in PIC and ADCC assays are different, adherent and phagocytic cells being necessary for full expression of PIC and not for ADCC; (2) PHA induces direct blast transformation of purified E-RFC in the absence of PIC cytotoxic cells; (3) cell populations specifically enriched in E or EAC rosette-forming cells are not cytotoxic neither in the PHA nor in antibody mediated cytotoxic assays; (4) cells participating in ADCC can be selectively purified by centrifugation of CEA rosettes.     

Antibody dependent cellular cytotoxicity against coronavirus 229E-infected cells

An antibody dependent cellular cytotoxic (ADCC) reaction was elicited using human mixed lymphocyte cultures against coronavirus 229E-infected C-16 cells. The response was assayed in microtitre plates by radioactive chromium release. Optimal killing of target cells was achieved using cells labelled 22 h after infection. A prozone was present and the optimal dilution of sera was invariably 1:200. The majority (70%) of sera tested gave a positive ADCC response. These included all sera judged positive by ELISA, but also some which were judged negative. Two experiments using purified IgG from serum which was ADCC and ELISA positive suggested that the response is mediated by an IgG antibody and that the prozone is due to a separate serum component.     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. II. The mouse nonadherent K cell

A cell of lymphoid morphology capable of killing antibody-coated chicken erythrocytes was isolated from nonimmune mouse spleen using a combination of carbonyl iron treatment and glass bead column passage. This nonphagocytic effector cell, which is referred to as the nonadherent K (killer) cell, is distinguished from the non-phagocytic myeloid K cell described earlier (Greenberg, A.H., Shen, L. and Roitt, I.M., Clin. Exp. Immunol. 1973. 15: 251) by its relatively weak surface adherence properties and low concentration within the mouse spleen. The cell is further characterized by its relatively large size, lack of theta or immunoglobulin determinants, the presence of Mg⁺⁺-independent complement receptors, affinity for aggregated IgG₂ myeloma proteins, inhibition by cytochalasin B and good survival in cell culture. The possible lineage of the cell is discussed.     

Antibody-dependent cell-mediated cytotoxicity: heterogeneity of effector cells in human peripheral blood.

We have compared antibody-dependent cell-mediated cytotoxicity (ADCMC) of human peripheral blood leukocytes (PBL) in three model systems. target cells were 51Cr-labeled mouse mastocytoma cells, chicken erythrocytes (CRBC), and human erythrocytes (HRBC) coated with appropriate heterologous or isologous antisera. Effector cells were characterized on the basis of their adherence, phagocytosis, radiosensitivity, and sedimentation velocity(s) at 1 g. In predominantly mononuclear (Ficoll-Isopaque-purified) PBL preparations (MPBL) HRBC were lysed by an adherent, phagocytic population of cells that was markedly radio-resistant. Sedimentation velocity analysis further established that these effector cells were restricted to rapidly sedimenting fractions (s greater than 4.5 mm/hr). On the other hand, mastocytoma cells were lysed by a population of MPBL that was nonadherent, nonphagocytic, and relatively radiosensitive. These cells mainly restricted to slowly sedimenting fractions (s greater than 4.5 mm/hr) following 1 g velocity sedimentation. CRBC appeared to be susceptible to lysis by both types of mononuclear effector cell. In some experiments, enriched populations of polymorphonuclear leukocytes (PMN) were isolated. These cells were found to lyse both HRBC and CRBC very efficiently, whereas mastocytoma cells were lysed very little if at all by the same effector populations. Taken together, these results suggest that antibody-coated mastocytoma cells are lysed uniquely by effector cells in human peripheral blood with the physical properties of lymphocytes, whereas antibody-coated HRBC are lysed by both monocytes and PMN, but not by lymphocytes. Antibody-coated CRBC would appear to be lysed by all of the three effector cell types tested.     

Antibody-dependent cellular cytotoxicity against tumor cells. II. The promonocyte identified as effector cell.

Macrophage precursor cells were cultured from the bone marrow of mice in a liquid culture system in the presence of conditioned medium. To separate their different maturation stages, they were passed through a discontinuous Ficoll density gradient, treated with iron plus magnet or passed through glass bead columns. The different maturation stages have been tested for their function in antibody-dependent cellular cytotoxicity (ADCC) against tumor target cells and in lymphokine-induced macrophage-mediated cytotoxicity. It is shown that the promonocyte, a nonadherent, nonphagocytic precursor cell, is a highly potent cytotoxic effector cell against antibody-coated tumor targets but is totally inactive as an effector cell in lymphokine-induced macrophage-mediated cytotoxicity. In contrast, in the lymphokine-induced macrophage-mediated cytotoxicity the cytotoxic effector cell is a mature macrophage. Thus, ADCC seems to be a function of the macrophage precursor promonocytes, whereas lymphokine-induced cytotoxicity is performed by mature macrophages. The relationship of promonocytes and killer (k) cells in ADCC against tumor targets is discussed.     

Antibody dependent cell mediated cytotoxicity against anti-D sensitized human erythrocytes. Influence of various effector cells and enzyme treatment of the target cells

Antibody dependent cell mediated cytotoxicity (ADCC) was measured against anti-D sensitized erythrocytes using various mononuclear cell populations from peripheral blood in a chromium release assay. Adherent effector cells gave stronger cytotoxicity than unseparated and non-adherent cells, and were less dependent on pretreatment of the target cells with papain. However, close correlations were found between the cytotoxicity obtained with the various effector cell populations, indicating that either of them might be used for the purpose of establishing in vitro methods to determine the clinical significance of erythrocyte antibodies. The optimal choice of effector cells is discussed.     

Inhibition of whole blood antibody dependent cellular cytotoxicity by heat aggregated human IgG

Inhibition of the whole blood antibody dependent cellular cytotoxicity (ADCC) of a lymphoblastoid cell line by heat aggregated human IgG (HAI) is described. Optimal sensitising antibody and effector cell concentrations were established to permit the detection of 0.1 μg/ml HAI. Conditions which avoid the ADCC inhibitory effects of normal human sera were defined.Twelve paired normal human sera were stored at ?70°C for prolonged (> 3 years) and shorter (<6 months) periods of time. Sensitised sheep red cells were used to determine complement depletion in serum dilutions heated at 40°C, 45°C, 50°C and 56°C. Concentrations of human IgG (1 000–0.1 μg/ml) were prepared in foetal calf serum before heating at 63°C for 30 min to aggregate the IgG.ADCC inhibitory activity, frequently characteristic of normal human serum was minimised (<10%) when sera were stored at ?70°C and heated to 50°C for 30 min to inactivate serum complement. This assay provides an economical, reproducible and sensitive test for the detection of circulating IgG complexes.     

Characterization of the human effector cell causing antibody-mediated cytotoxicity

Human peripheral mononuclear cells were used as effector cells in antibody-mediated cytotoxicity. Chicken and sheep erythrocytes coated with antibody were used as target cells.

The nature of the effector cell was characterized by different in vitro methods of cell separation. It is concluded that both monocytes and lymphocytes are cytotoxic in this system. The lymphocyte appears to have a receptor for the Fc part of immunoglobulin but lacks detectable surface immunoglobulin.

     

In vitro effect of immune complex positive human sera on antibody dependent cellular cytotoxicity

The effect of immune complex positive and negative (control) sera on the antibody dependent cytotoxicity of normal human lymphocytes was studied in a xenogeneic assay in vitro. Significant inhibitions were obtained with the sera of immune complex positive patients as compared with age and sex matched controls. The possible significance of these findings in autoimmune diseases and malignancies is discussed.     

Two T lymphocyte populations as effector cells fro IgG dependent cytotoxicity.

The characteristics and proportions of human lymphocyte populations capable of lysing IgG coated target cells were studied. Monolayer of target erythrocytes coated with rabbit IgG were employed to detect and isolate the lytic effector lymphocytes. Normal peripheral blood effector lymphocytes were shown to express IgG receptors, and a total of four cell types possesing different surface markers were identified. An average of 68% (range 51-80%) of the effector lymphocytes had T lymphocytes antigen and were capable of SRBC rosette formation, and 32% of the lymphocytes lacked T lymphocyte markers. Two populations of these T lymphocytes were identified: one possesses SRBC and IgG receptors, and the other possesses SRBC, IgG and complement receptors. The proportion of the former was estimated as 53.2% and the latter as 14.8% of the total effector cell population. Two other cell types were distinguished among the non-T lymphocytes. One type of lymphocytes bear IgG receptors (21.6%) and the other has both IgG and complement receptors (10.4%). The cytolytic capacity of the two T lymphocyte subpopulations is suggested to be an important reaction of the immune reactive T lymphocytes during the immune response.     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. I. Phagocytic and non-phagocytic effector cell activity against erythrocyte and tumour target cells in a 51Cr release cytotoxicity assay and [125I]IUdR growth inhibition assay.

Both phagocytic and non-phagocytic effector cells were able to kill rabbit antibody-coated chicken erythrocytes (CRBC) while only non-phagocytic effector cells were active against alloantibody-coated SL2 lymphoma. In addition to the variation in susceptibility of erythrocyte and tumour target cells to various effector cell populations, it was found that different tumour cells can vary markedly in their ability to be killed by non-immune spleen cells in the presence of antibody. It is postulated that both the type of antibody and certain characteristics of the cell membrane are important in determining whether target cells are susceptible to antibody-dependent cell-mediated cytotoxicity detected by the 51Cr release assay. It was also demonstrated that alloantibody-coated P-815-Y mastocytoma, which showed very little evidence of cytotoxicity in the 51Cr release assay, was markedly inhibited in its ability to incorporate [125I]IUdR after incubation with antiserum and non-immune spleen cells. This growth inhibition in the absence of cytotoxicity, or cytostasis, is discussed in relation to the potential mechanisms of target cell damage, and in the light of recent observations (Plata, Gomard, LeClerc and Levy, 1974; Newlands and Roitt, 1975) that cytotoxicity and growth inhibition assays detect different effector cell populations in tumour-bearing animals.     

The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity.

A mouse monoclonal antibody, anti-HM1.24 (IgG2a/kappa), binds to a surface antigen preferentially overexpressed on multiple myeloma (MM) cells, and exhibits potent antitumor cell activity against MM cells by antibody-dependent cell-mediated cytotoxicity (ADCC). To develop an antibody-based immunotherapy against MM, a humanized anti-HM1.24 antibody, in which all FRs correspond to naturally processed human FRs, has been successfully constructed with the aid of both the hybrid variable region and two-step design methods. This humanized anti-HM1.24 antibody (IgG1/kappa) is able to effectively induce ADCC against human myeloma KPMM2 and ARH77 cells in the presence of human PBMCs as effectively as a chimeric anti-HM1.24 antibody. The humanized anti-HM1.24 antibody, therefore, could be expected as a potent immunotherapeutic agent for MM patients.     

Aging influence on the E-rosette forming cells, spontaneous cell mediated cytotoxicity, and antibody dependent cellular cytotoxicity in humans

In 131 adults and 107 aging healthy subjects of both sexes, the percentage of the T lymphocytes in peripheral blood and the cytotoxic activities of NK and K cells, were determined. No age-related differences have been found in the number of peripheral blood lymphocytes, the binding of NK cells to the K562 targets, the percentage of E rosette forming cells and the ADCC level. An increased proportion of T suppressor lymphocytes in elderly subjects and a large inter- and intra-individual variation of NK cells activity in both adult and aged subjects were found.     

IgE-dependent cellular adhesion and cytotoxicity to Litomosoides carinii microfilariae--nature of effector cells.

Albino rats immunized with sonicated microfilarial antigen incorporated in Freund's complete adjuvant, produce antibodies that promote cell-mediated adhesion and killing of Litomosoides carinii microfilariae in vitro. Using highly purified cell populations, it has been shown that macrophages and neutrophils are most active in this phenomenon. Eosinophils, while adhering readily to parasites in the presence of the antibody, did not affect the viability of the parasites when observed after 24 hr incubation.     

Lymphocyte-mediated cytotoxicity against tumor cells. III Differentiation of concanavalin A-activated cytotoxic effector cells.

The maturation of selected T cell responses in the lymphoid organs of irradiated CBA mice was followed after adoptive transfer of syngeneic fetal liver cells. Mitogenic responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A) was found to reach control values 3 weeks after reconstitution in the thymus, spleen, and lymph node, of fetal liver repopulated animals. Spleen and lymph node cell reactivity in mixed lymphocyte culture reactions required 6 weeks to reach significant values. However, the ability of spleen cell suspensions to be activated by Con A into cytotoxic effector lymphocytes appeared after only 2 to 3 weeks. It is concluded that two functionally distinct T cell subpopulations exist in the spleen, one which can be activated into cytotoxic effector lymphocytes by Con A, and one which responds to alloantigens by DNA synthesis.     

Characterization of mononuclear effector cells in human blood.

The effector cells responsible for cytotoxic activity induced by phytohaemagglutinin, (PHA) pokeweed mitogen (PWM) target cells complexes with IgG antibody has been investigated using cell separation techniques based on rosette formation and separation through Hypaque-Ficoll mixtures. It was shown the PHA-induced cytotoxicity is predominantly a function of T cells and that Fc receptor-bearing cells are not involved to any major extent. Antibody-dependent killing is conversely a function of Fc receptor-bearing cells among which two subtypes can be distinguished. One of these has receptors for activated complement while the other bears Fc receptors only and has no detectable receptors for complement. PWM appears to induce cytotoxicity in both T- and non-T-cell populations but the major cell type involved appears to be Fc receptor-bearing cells similar to those mediating antibody-dependent killing. It is concluded that PHA and antibody-dependent killing are the two most useful assays for discriminating between the cytotoxic activity of T and non-T cell in clinical studies.