Characterization of Effector Cells Mediating IgG and IgM Antibody-Dependent Cellular Cytotoxicity

Spleen effector cells for IgG- and IgM-induced antibody-dependent cellular cytotoxicity (ADCC) were characterized with respect to density and cell surface markers by using sheep erythrocytes (SRBC) coated with hybridoma-derived monoclonal anti-SRBC IgG or anti-SRBC IgM antibodies as targets. While basically the same effector cells are cytolytic for IgG and IgM antibody-coated SRBC, they differ with respect to their relative killing capacity for IgG- versus IgM-coated target cells. On the basis of physical and biochemical properties three populations with cytolytic capacity could be separated: (I) A light fraction of large cells had high cytolytic potential for both IgG- and IgM-coated SRBC. The cells were negative for the Fc receptor for IgG (Fc gamma-R-) and the C3-receptor (C3-R-), they carried the receptor for Helix pomatia A agglutinin (HP-A+), and reactivity was strongly reduced after treatment with anti-Thy-1 and complement (C). (II) High activity was also observed with a medium-dense fraction, preferably lysing IgG antibody-coated cells. The cells were Fc gamma-R+, partly C3-R+, mostly HP-A-, and only a minor portion of the cells were Thy-1+. (III) A dense fraction, displaying on a per cell basis low cytolytic potential, was more active in IgM than IgG ADCC. The cells were Fc gamma-R+, HP-A+ and Thy-1+. All three effector cell populations were non-adherent, non-phagocytic, and surface immunoglobulin-negative (s-Ig-).     

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 radioresistant. Sedimentation velocity analysis further established that these effector cells were restricted to rapidly sedimenting fractions ( s > 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 were mainly restricted to slowly sedimenting fractions ( s < 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.     

Studies of Antibody-Dependent Cytotoxicity in a Plaque Assay: Evidence of Human Monccyte-Like Effector Cells

Two different methods for evaluating 'in vitro' cytotoxicity against antibody-coated target cells mediated by mononuclear leukocytes were compared. One was a plaque assay for identification of the cytotoxic cell and the other the classical chromium release assay for antibody-dependent cytotoxicity (ADCC). A marked decrease in plaque-forming cells (PFC) was observed in a cell suspension depleted of peroxidase-positive cells and cells with membrane-bound Ig (B lymphocytes) by fractionation on a nylon fiber column. In contrast, the ADCC activity was considerably increased by these depletions. A similar effect was obtained by removal of phagocytic cells with iron. These results, together with the observations after depletion of E-RFC (T lymphocytes) or EA-RFC (Fc-receptor-bearing cells), suggest that the PFC in the assay system used were of monocytic origin and differnet from the cells responsible for the ADCC.     

Natural Killer Cell Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells in Human Pregnancy

ABSTRACT: Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a ⁵¹Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.     

A Comparison of the Effector Cells Involved in Cell-Mediated Lympholysis and Antibody-Dependent Cell-Mediated Cytotoxicity in Man

Some physical and mechanistic properties of cytotoxic lymphocytes and antibody-dependent killer cells were compared. Cytotoxic lymphocytes were measured by the cell-mediated lympholysis (CML) technique after sensitization of human peripheral blood leukocytes (PBL) in unidirectional mixed leukocyte culture (MLC). Killer cells were measured before and after MLC in an antibody-dependent cell-mediated cytotoxicity (ADCMC) assay using 51-Cr-labeled mouse mastocytoma cells as targets. Quantitative analysis of the cytotoxic activity of populations separated by velocity sedimentation at 1 g indicated that cytotoxic lymphocytes in MLC were a relatively heterogeneous population of rapidly sedimenting cells that corresponded morphologically to lymphoblasts. Further studies in which PBL were separated before MLC suggested that such cytotoxic lymphocytes were derived from small lymphocyte progenitors. Analysis of ADCMC in separated populations showed that most killer cells in PBL had the sedimentation properties of small lymphocytes. After stimulation of PBL in MLC, increased ADCMC activity was detected, and the increased activity was associated with a population of rapidly sedimenting cells, Comparison of the sensitivity of cytotoxic lymphocytes and killer cells to inhibition by EDTA, cytochalasin B, and dimethyl sulfoxide showed no significant differences between the two types of effector cells.     

Interleukin-10 Down-Regulates Oxidative Metabolism and Antibody-Dependent Cellular Cytotoxicity of Human Neutrophils

The authors investigated the ability of interleukin-10 (IL-10) to modulate some constitutive or interferon-γ (IFN-γ)-enhanced activities of human neutrophils. An 18 h culture of neutrophils with IL-10 dose-dependently down-regulated their capacity to produce O₂⁻ and lucigenin-amplified chemiluminescence in response to n-formyl-methionyl-leucylphenyl-alanine (FMLP). Furthermore, treatment of neutrophils with IL-10 decreased in a dose-dependent fashion, their capacity to lyse antibody-coated sheep erythrocytes. Membrane expression of FcγRI, FcγRII, FcγRIII, CR1, CR3 and FcγR- and CR-mediated phagocytosis were not modified by the cytokine. Culture of neutrophils with IFN-γ (100 U/ml) did not modify their FcγR- and CR-mediated phagocytosis, but significantly up-regulated FcγRI and CR3 membrane expression as well as their oxidative metabolism and antibody-dependent cellular cytotoxicity (ADCC). When IL-10 and IFN-γ were added simultaneously to neutrophil culture, IL-10 dose-dependently reduced IFN-γ-induced increase of CR3 expression, O₂⁻ production (in response to both FMLP and phorbol 12-myristate 13-acetate, or PMA) and ADCC, but did not change FcγRI expression on phagocytes. These results demonstrate that IL-10 is a significant neutrophil deactivator and provide new information on the role of IL-10 in the regulation of neutrophil-mediated inflammatory processes.     

Simultaneous Determination of the Concentration and Lytic Activity of Effector Cells that Mediate Natural and Antibody-Dependent Cytotoxicity

Cellular cytotoxicity reactions can be studied in a manner analogous to that used to measure enzyme activity. This approach yields two parameters; V_max the maximal rate of target cell lysis that can be achieved by the lymphocyte preparation tested, and K _M^app, the apparent Michaelis constant. By analogy to many enzyme-catalysed reactions, K _M^app values for cytotoxicity reactions have generally been interpreted in terms of dissociation constants for the interaction of receptor sites on effector cells with antigens on the target cells, In this paper we demonstrate that experimentally determined K _M^app values for natural or antibody-dependent cytotoxicity reactions are approximately equal to the concentration of NK or K. effector cells in the lymphocyte preparation tested. This result makes possible the simultaneous determination of both effector cell frequency and lytic activity in a given lymphocyte preparation.     

Comparative Study of Antibody-Dependent and Direct Lymphocyte-Mediated Cytotoxicity In Vitro After Alloimmunization in the Human

Two major types of cell-mediated cytotoxicity in vitro after alloimmunization in the human have been described–antibody-dependent lymphocyte-mediated cytotoxicity (ADLC) and direct immune-lymphocyte-mediated cytotoxicity (DEC) The characteristics of these two in vitro systems are compared. The cytolysis occurred as a linear function of time in both cases The effector cell dose response relationship was in both systems well described by a linear correlation between the logarithm of the effector cell dose and the cytotoxic activity. Anti-immunoglobulins inhibited ADLC but not DLC. The reactivity in DLC was potentiated by the addition of isoantibody directed against target cells.     

Deficient Antibody-Dependent Cellular Cytotoxicity against Human Immunodeficiency Virus (HIV)-Expressing Target Cells in Perinatal HIV Infection

Peripheral blood mononuclear cells (PBMC) of human immunodeficiency virus (HIV)-infected children, age-matched HIV-seronegative controls, and HIV-infected asymptomatic and symptomatic adults were compared for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell-mediated cytotoxicity against target cells expressing HIV or herpes simplex virus (HSV) antigens. Target cells consisted of CD4 lymphocytes purified from PBMC of HIV-seronegative adults and incubated with the IIIB strain of HIV, HUT78 cells chronically infected with IIIB, and HSV-infected human fibroblasts. PBMC of asymptomatic HIV-infected adults were generally able to lyse CD4 cells expressing HIV antigens. Direct correlation was found between the magnitude of lysis and absolute CD4 cell counts in these individuals. In contrast to these results, PBMC from HIV-infected children were generally unable to lyse IIIB-expressing CD4 cells, regardless of the children’s clinical status, age, or absolute CD4 cell counts. Cells from HIV-seronegative adults and children did not directly lyse these target cells either but, in contrast to cells of HIV-seropositive children, were able to mediate cell lysis when serum from an HIV-seropositive adult was added. However, effector cells from these HIV-infected children were able to mediate both ADCC against HSV-infected fibroblasts and NK cell-mediated cytotoxicity against IIIB-infected HUT78 cells. Reduced ability of PBMC from vertically HIV-infected children to mediate ADCC against HIV antigen-expressing CD4 cells may contribute to rapid progression to AIDS.     

Selective Inhibition of Human Natural Killing and Antibody-Dependent Cellular Cytotoxicity by a Polyanion

A high molecular polyanion, Liquoid, was found to inhibit at nontoxic concentrations (12-50 micrograms/ml) the natural killing (NK) and the antibody-dependent cellular cytotoxic (ADCC) activity of human peripheral blood mononuclear cells selectively. Whereas NK of the K 562 target cell was slightly or not at all affected, the spontaneous lysis of PDe-B-1, an EBV-transformed B-cell line, was strongly inhibited or even completely abolished. ADCC activity could only be inhibited by Liquoid if the target cells were mycoplasma-free, while the polyanion had no effect when mycoplasma-contaminated target cells were used. Liquoid did not alter the target binding capacity of the NK effector cells and did not activate monocytes or induce other suppressive cells. Alpha interferon, but neither beta nor gamma interferon, was able to neutralize the NK reduction. These results suggest that Liquoid inhibits a target cell-related, selective process in the post-binding stage of NK cell lysis.     

Sex-Associated Differences in the Antibody-Dependent Cellular Cytotoxicity Antibody Response to Measles Vaccines

In some countries, excessive non-measles-related mortality has been observed among female recipients of high-titer measles vaccines. We determined if differences in the immune response to measles vaccines underlie the excessive female mortality by measuring the measles virus (MV)-specific antibody-dependent cellular cytotoxicity (ADCC) antibody response in 65 3-year-old Gambian children immunized with Edmonston-Zagreb medium-titer (EZ) or Schwarz standard vaccines during infancy. Among the 20 females and 22 males with undetectable anti-MV antibodies at the time of immunization, females had significantly lower ADCC than males (median cytotoxicities of 1/100 serum dilutions = 8.4 and 12%, respectively; P = 0.04). This sex-associated difference was present only among the six female and seven male recipients of EZ vaccine (median cytotoxicities = 5.1 and 19.0%, respectively; P = 0.02). There were no significant sex-associated differences in neutralizing antibody activity. Decreased ADCC antibody activity may contribute to the lower survival rate observed in females receiving high-titer measles vaccination.     

''Nonspecific''Blocking of Human Ovarian Carcinoma-Associated Cellular Cytotoxicity In Vitro

The blocking ability of heat-aggregated human polyclonal IgG and complexes formed by preservative-free tetanus toxoid (TT) and human anti-tetanus serum was investigated on the cell-mediated tumor-specific cytotoxicity reaction of ovarian carcinoma patients against cultured ovarian carcinoma cells. Aggregated IgG effectively blocked the cytotoxic reaction when the purified effector cells were prein-cubated with various concentrations of this material, whereas no blocking activity was detected when target cells were preincubated and washed before addition of cytotoxic lymphocytes. Optimal concentration was about 1.6 μg/ml, and higher or lower concentrations were less effective. Anti-TT/TT immunocomplexes behaved similarly and could effectively block at the effector cell but not at the target cell level. The results suggest that the effector cells in human ovarian carcinoma-associated cellular cytotoxicity are Fc-receptor-carrying cells and that their cytotoxic activity can be 'nonspecifically' blocked by saturating these sites with aggregated IgG or unrelated immunocomplexes.     

Inhibition of Spontaneous and Antibody-Dependent Cellular Cytotoxicity by Sera and Isolated Antiglobulin Preparations from Rheumatoid Arthritis Patients

Using sera from patients with rheumatoid arthritis and antiglobulin preparations obtained from such sera, inhibition of human lymphocyte cytotoxicity in antibody-dependent (ADCC) and spontaneous cell-mediated cytotoxicity (SCMC) reactions against an allogeneic melanoma cell line (IGR3) has been demonstrated. Fractionation of effector cell preparations indicated that Fc-receptor-bearing lymphocytes were operative in both reactions. Removal of phagocytic and adherent cells from the effector cell population resulted in more pronounced inhibition of SCMC and ADCC reactions by rheumatoid sera. The results indicate that antiglobulin preparations from human sera containing rheumatoid factor activity can effectively block the cytotoxic activity of Fc-receptor-bearing effector lymphocytes (K cells) in vitro. On analogy with the inhibition of ADCC and SCMC reactions by immune complexes and aggregated IgG, antiglobulin complexes present in the antiglobulin preparations are responsible for this effect.     

Natural Killer Cytotoxicity and Antibody-Dependent Cell Cytotoxicity to Herpes Simplex Virus-Infected Target Cells in Murine Pregnancy

ABSTRACT: In vitro natural killer cytotoxicity (NKC) and antibody-dependent cell cytotoxicity (ADCC) activity against herpes simplex virus (HSV)-infected cells were evaluated in a pregnant murine model (C₅₇B1₆inbred strain). Virgin (n = 16) and pregnant (late gestation) mice (n = 15) were infected intraperitoneally with HSV, type 1. After 18 hr, a 0.5-ml aliquot of the peritoneal wash was frozen for virus plaque assay, and the cells were cultured in the ⁵¹chromium release assay for NKC and ADCC. %NKC (mean ± S.E.) to HSV-infected targets was significantly suppressed (P < 0.05) in pregnant mice, 10.3% ± 1.9, compared to that of virgin mice, 32.5% ± 2.5. This suppression was abrogated with HSV-specific antisera (%ADCC); 53.9% ± 4.4 (pregnant) compared to 49.1% ± 3.6 (virgin). The diminished NKC activity in pregnant mice was reflected in an increased mean number of virus particles in the peritoneal wash, 266 + 66 PFU/ml, compared to 38 ± 11 PFU/ml in virgin mice (P < 0.05). We concluded that NKC, but not ADCC, to HSV-infected targets was suppressed and that HSV elimination was impaired in pregnant mice.     

Natural and Antibody-Dependent Cellular Cytotoxicity in Tumour-Adherent T Lymphocytes: Expression of Fc Receptors and of a Human T-Subgroup-Specific Antigen

Adherence of human lymphocytes to allogeneic tumour cell monolayers was found to depend on the presence of monocytes. Adherent lymphocytes could lie separated from tumour cells by treatment with lidocaine followed by nylon wool passage. Tumour-adherent cells (70% E-RFC, 45% Fcγ-R, 23% Fcμ-R, 5% monocytes) exhibited enriched natural killer (NK) activity not only against the tumour cell line used for isolation but also against seven other targets. When T cells were isolated subsequently as E-rosettes by density gradient centrifugation through Percoll, the enrichment in cytotoxicity was even more pronounced. Tumour-adherent T cells were severalfold enriched in both N K and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, this enrichment was not paralleled by a concomitant increase in the number of Tγ cells (tumour-adherent T cells: 17% Tγ, 40% Tμ: tumour-nonadherent T cells: 12% Tγ, 60% Tμ). Marked differences could be observed by staining with a monoclonal antibody that was raised against human leukaemic Tγ cells of high NK and ADCC activity. This antibody (T8–11) stained 60% of tumour-adherent T cells. 20% of nonadherent T cells and 29% of T-cell controls. These results indicate that the spontaneous cyloloxic activity of human T cells resides within a small population, most of which are characterized by a specific surface antigen but not by conventional Fcγ receptors.     

Failure to Detect Complement-Mediated Antibody-Dependent Cytotoxicity Against Human Orbital Tissue Cells in the Serum of Patients with Thyroid-Associated Ophthalmopathy

Antibodies which are cytotoxic to human eye muscle cells and orbital fibroblasts in antibody dependent cell-mediated cytotoxicity (ADCC) are routinely detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In the present study sera from patients with TAO, most of which were shown to be positive in ADCC against eye muscle cell targets, were tested for complement-mediated antibody-dependent cytotoxicity (CMAC) against human and pig eye muscle cells, human (abdominal) skeletal muscle cells and human orbital fibroblasts, in ⁵¹ Cr release assays. Several different assay protocols and complement sources were used and patient and age, sex matched normal sera compared. In preliminary studies tests were always negative when eye muscle cells, other skeletal muscle cells, or orbital fibroblasts were used as targets, regardless of the complement source, or concentration, or assay conditions used. When larger numbers of patients and normals were tested in a single assay mean (± SE) % specific lysis for patients with TAO was not significantly different from that for normals for either eye muscle cells or other skeletal muscle cells and taking the upper limit of normal as mean + 2 SD for the normals, tests were positive in no patient with either target. On the other hand ADCC tests were positive in 57 % of the same sera tested with the same eye muscle cell targets. When human thyroid cells were used as targets, tests were positive in 10 of the 14 patients tested and monoclonal antibodies, in enzyme-linked immunosorbent assay, reactive with eye muscle antigens gave positive lysis of eye muscle cells.

Although eye muscle reactive autoantibodies have been well described in TAO, particularly IgG₃ subclass antibodies against a 64 kDa membrane antigen, their significance in the pathogenesis of the eye muscle component of this disorder is unclear. In contrast to thyroid cells where microsomal, and perhaps other membrane reactive antibodies, are cytotoxic in both ADCC and CMAC, antibodies associated with TAO appear to be cytotoxic only in ADCC. Although the reason for this discrepancy is unknown one possible mechanism is failure of IgG₃ subclass antibodies, which are usually cytotoxic, to activate complement when the target antigen is in too low a density on the cell surface.     

Enhancement of Antibody-Dependent Cellular Cytotoxicity of Neonatal Cells by Interleukin-2 (IL-2) and IL-12

Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HIV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16⁺/CD56⁺ cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.     

The Effects of Cyclophosphamide on Mitogen-Induced and Antibody-Dependent Cellular Cytotoxicity in Guinea Pigs

Abstract

Lymph node cells from normal guinea pigs, when stimulated with the non-specific mitogen PHA were transformed to activated killer cells, capable of lysing ⁵¹Cr labeled mouse fibroblasts. Similarly, these lymphoid cells effected lysis of antibody coated chicken red cells in an antibody-dependent cellular cytotoxicity assay. Following cyclophosphamide, 150 mg/kg IP, the reactivity of an aliquot of lymph node cells to effect either cytotoxic reaction was not diminished. These results indicate that this immunosuppressant does not promote a selective decrease in either lymphoid effector. Rather, they are diminished in parallel with the generalized lympholysis resulting from drug administration. During the recovery phase, lymph node cells showed increased ability to lyse target cells, suggesting a rebound phase of heightened activity. Thymic cells from normal and cyclophosphamide treated animals were poor effectors in either cytotoxic assay. The addition of an equal number of thymocytes to lymph node cells resulted in decreased mitogen-induced cytotoxicity. Thymic inhibitory activity was mediated only by viable cells and the phenomenon did not represent an altered shift in PHA sensitivity. This suppressive activity persisted when thymic cells from cyclophosphamide treated-animals were employed, indicating that inhibitory cells were also not selectively depleted by this drug. In antibody-dependent cellular cytotoxicity assays, thymocytes from normal or cyclophosphamide-treated animals did not alter the cytotoxicity capacity of lymph node cells.