Target cell directed NK inactivation. Concomitant loss of NK and antibody-dependent cellular cytotoxicity activities

We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct.     

Monocyte antibody-dependent cellular cytotoxicity in splenectomized subjects

Monocyte antibody-dependent cellular cytotoxicity (ADCC) was determined in normal subjects by using human A1 erythrocytes and immune human anti-A1 antiserum. The experimental data were fitted to a mathematical model and the values for maximal cytotoxicity and monocyte numbers required to produce 15% specific cytotoxicity (MD15) were subsequently computed and compared with the values for these two parameters estimated from the dose response curves. The values for both were significantly associated, and the mathematical model permitted precise quantitation of cytotoxicity in instances where this was impossible by standard methods. Maximal monocyte ADCC was significantly reduced in a group of splenectomized subjects, whereas the MD15 was increased. These findings emphasize the possible influences of the method of quantitation of ADCC and provide one explanation for the apparent conflict of published data.     

Monocyte antibody-dependent cellular cytotoxicity in cancer patients

Summary Antibody-dependent cellular cytotoxicity (ADCC) mediated by peripheral blood monocytes was determined in 120 patients who had gastrointestinal tract (GIT), lung and breast cancer, melanoma, or Hodgkin's and non-Hodgkin's lymphoma. Results were expressed in terms of maximum cytotoxicity and cytotoxicity at E : T=1 : 10 and were compared with the results obtained in 63 normal subjects. There was a significant decrease in maximal cytotoxicity for both the GIT cancer and the melanoma patient groups, but not for any of the other groups. These differences were not confirmed when results were expressed at low effector: target cell ratios, e.g., cytotoxicity at E : T=1 : 10. The relationship between monocyte ADCC and disease extent was examined in those groups with sufficient numbers. Monocyte ADCC was higher in patients with GIT cancer of limited extent than in patients with extensive GIT cancer and in the control group.     

Polymorphonuclear leukocytes in antibody-dependent cellular cytotoxicity.

Human polymorphonuclear leukocytes (PMN) lyse antibody-coated target cells in an immunologically specific fashion--antibody-dependent cellular cytotoxicity (ADCC). PMN-mediated cytolysis is independent of complement, de novo protein synthesis, and DNA replication. Cytolysis is rapid, detectable at low PMN:target cell ratios, and exceeds lymphocyte-mediated ADCC. The interaction appears to be mediated via an Fc receptor and is inhibited by aggregated gamma-globulin. A role for PMN in host tumor cell immunity is discussed.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil mediated antibody-dependent cellular cytotoxicity

The human lymphokine, leukocyte inhibitory factor (LIF), was investigated for its effect on neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) for K562 targets. Highly purified LIF (0.5 to 2 U/ml) induced a significant dose-dependent potentiation of neutrophil ADCC by up to 54.9% (p less than 0.001). Higher concentrations of LIF inhibited cytotoxicity. The degree of cytotoxicity was found to correlate (r = 0.99) with the increased secretion of superoxide after neutrophil-target cell interaction. Anaerobic conditions inhibited cytotoxicity mediated by both control and LIF-treated neutrophils. The latter observation lends support to the concept that enhanced ADCC was mediated through increased superoxide production and not through the induction of a separate pathway. Increased superoxide production may have resulted from an upregulation of the transduction mechanism leading to neutrophil stimulation through the Fc receptor. In addition, we demonstrated an increased capacity of the neutrophil to adhere to its target (average 3.3:1 effector:target ratio in untreated cells to 4.8:1 after treatment with LIF), and this may also have been responsible for the increase in the respiratory burst and subsequent enhanced ADCC. These observations provide potential support for an in vivo role for LIF in tumor immunity.     

The role of apoptosis in antibody-dependent cellular cytotoxicity

Apoptosis in three lymphoma cell lines has been studied following cytotoxicity induced in vitro by normal human blood lymphocytes utilizing either natural killer (NK) or antibody-dependent cellular cytotoxic (ADCC) mechanisms. Guinea-pig L₂C leukaemic lymphocytes, but not the human cell lines Daudi and Jurkat, revealed a degree of time- and temperature-dependent apoptotic death upon simple culture in vitro. NK cytotoxicity at low effector: target ratios (E: T) induced both release of⁵¹Cr and apoptosis. However NK cytotoxicity at higher E : T, and ADCC at all E : T, increased the level of⁵¹Cr release while reducing the level of apoptosis. The findings were consistent with the apoptotic process being cut short by intervention of necrotic death. The same characteristics accompanied ADCC whether the effectors were recruited by Fc regions of antibody coating the targets, or by bispecific antibodies attaching one arm to the targets and the other to Fc receptors type III on effectors. This finding, and the high level of cytotoxicity elicited by the bispecific method, confirm the belief that NK cells, in addition to exerting NK cytotoxicity, represent the principal effectors for ADCC among blood mononuclear cells. Our results suggest that NK cells have both apoptotic and necrotic mechanisms available for killing their targets, but use only the latter for ADCC.     

TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.     

Natural killer and antibody-dependent cellular cytotoxicity in cervical carcinoma patients

Summary Natural killer (NK) cell activity and antibody dependent cell-mediated cytotoxicity (ADCC) was measured in 62 untreated cervical carcinoma patients and 25 normal healthy women, using a short-term chromium release assay. A significant reduction in NK and ADCC activity was observed in disseminated disease than in localized disease, when compared with normal donors. The majority of the patients received radiotherapy and both NK and ADCC activity recovered after therapy. Furthermore, interferon- was demonstrated to augment NK activity of peripheral blood mononuclear cells from healthy donors as well as patients. Also large granular lymphocytes separated on Percoll density gradient were the same in number in both the populations studied, although in cervical cancer there seemed to be a defect in killing activity.     

Evidence that natural cytotoxicity and antibody-dependent cellular cytotoxicity are mediated in humans by the same effector cell populations.

The present study strongly suggests that, in humans, natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) are mediated by the same effector cell population. This is supported by two different experimental approaches. First, competition for NK effector cells was accompanied by simultaneous inhibition of ADCC activity. Target cells sensitive to NK activity were capable of inhibiting specifically an ADCC assay in cold target competition experiments. Second, specific removal of NK cells on monolayers formed by target cells sensitive to NK activity caused simultaneous depletion of ADCC effector cells. In association with the removal on the monolayers of effector cells for ADCC as well as NK activity, we also found a significant depletion of cells bearing Fc gamma receptors.     

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

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

Anti-renal cell carcinoma chimeric antibody G250: cytokine enhancement of in vitro antibody-dependent cellular cytotoxicity

The chimeric monoclonal antibody cG250 targets the G250 antigen, a transmembrane protein which is expressed on renal carcinoma cells and is identical to the MN/CAIX antigen. In vitro studies have previously demonstrated that cG250 induces antibody-dependent cellular cytotoxicity (ADCC) of G250-positive targets. In order to investigate the upregulation of ADCC mediated by cG250, ADCC was examined using effector cells cultured in the presence or absence of the cytokines interferon-gamma (IFN-gamma), interferon-alpha isoforms IFN-alpha (2a) and IFN-alpha (2b) and interleukin-2 (IL-2), and the time course of effects over a 7-day period was determined. Renal cell carcinoma lines expressing high (SK-RC-52) and low (SK-RC-09) G250 antigen levels were used as target cells, and freshly isolated peripheral blood mononuclear cells (PBMC) from a healthy donor were used as the effector cells. PBMC were incubated with the respective cytokine at a range of concentrations or with a media alone control for a period of 7 days. The ADCC activity mediated by cG250 or control isotype matched huA33 with the different PBMC treatment groups was assessed in triplicate daily. Corresponding lymphokine activated killing (LAK) activity was measured concurrently for each treatment group. Chimeric G250 specifically recognised G250 antigen on high and low expressing cell lines SK-RC-52 and SK-RC-09, and mediated specific in vitro ADCC of both lines. In the absence of cytokine stimulation, the specific ADCC of cG250 declined rapidly within three days. IL-2 strongly enhanced and maintained cG250-mediated ADCC activity and K562 cytotoxicity when applied to PBMC in culture for seven days. IFN-gamma also enhanced the ADCC of cG250 throughout the study period, but was not as effective as the IL-2 treatment, and the SK-RC-09 line displayed lower specific cytotoxicity than the SK-RC-52 cell line. In contrast, IFN-alpha 2a and 2b increased cG250-mediated ADCC and K562 cytotoxicity for only three days of the study period. The potent and sustained immune effector activity observed with cG250 and cytokines in this in vitro study suggests that the combination immunotherapy of cG250 with cytokines such as IL-2 shows promise in the treatment of renal cell carcinoma (RCC).     

Spontaneous lymphocyte-mediated cytotoxicity and antibody-dependent cell cytotoxicity of human effector cells

Antigen dependent cellular cytotoxity (ADCC) and non-killer cell activities of haematological healthy donors were investigated in the 51Cr release test. Attempts of cell fraction reveal that lymphocytes are active as killer and non-killer cells. Granulocytes were efficient effector cells of antigen dependent cellular cytotoxity (ADCC), however, they had no natural-killer activity. In testing leukocyte fractions of 11 donors, killer cell would only be found in the non-T-fraction. In contrast to that, three types could be observed in the distribution on non-killer cells: Distribution on T-lymphocyte fraction (27.3%), distribution on non-T-lymphocyte fraction (9.1%) and approximately equal distribution on T- and non-T-lymphocyte fraction 63.7%). Without any treatment patients with acute lymphocytic leukemia showed an antigen dependent cellular cytotoxity and non-killer activity only in exceptional cases. Normal activities were reached in remission, with chemotherapy having a depressive effect on non-killer activity.     

Inhibition of antibody-dependent cellular cytotoxicity by autologous lymph node cells.

A population of lymph node cells that lack the usual T, B, or K cell markers was found to inhibit autologous spleen cells from mediating antibody-dependent cellular cytotoxicity (ADCC) to antibody-coated chicken erythrocytes. Inhibitor cells were not susceptible to treatment with anti-Thy 1.2 or anti-Ig and C; they did not adhere to Sephadex G-10, to nylon wool, or to monolayers of sheep erythrocytes (E) or erythrocytes plus 7S antibody (EA). After a brief (4-min) exposure to 45 degrees C, the ability to inhibit was lost whereas other cellular responses remained intact. ADCC mediated by nonadherent splenic effector cells (presumptive K cells) was highly susceptible to inhibition. Possible mechanisms for and implications of lymphocyte-mediated inhibition of ADCC are discussed.     

Adiponectin is a negative regulator of NK cell cytotoxicity

NK cells are a key component of innate immune systems, and their activity is regulated by cytokines and hormones. Adiponectin, which is secreted from white adipose tissues, plays important roles in various diseases, including hypertension, cardiovascular diseases, inflammatory disorders, and cancer. In this study the effect of adiponectin on NK cell activity was investigated. Adiponectin was found to suppress the IL-2-enhanced cytotoxic activity of NK cells without affecting basal NK cell cytotoxicity and to inhibit IL-2-induced NF-kappaB activation via activation of the AMP-activated protein kinase, indicating that it suppresses IL-2-enhanced NK cell cytotoxicity through the AMP-activated protein kinase-mediated inhibition of NF-kappaB activation. IFN-gamma enhances NK cell cytotoxicity by causing an increase in the levels of expression of TRAIL and Fas ligand. The production of IFN-gamma, one of the NF-kappaB target genes in NK cells, was also found to be suppressed by adiponectin, accompanied by the subsequent down-regulation of IFN-gamma-inducible TRAIL and Fas ligand expression. These results clearly demonstrate that adiponectin is a potent negative regulator of IL-2-induced NK cell activation and thus may act as an in vivo regulator of anti-inflammatory functions.     

Analysis of effector cells in human antibody-dependent cellular cytotoxicity with murine monoclonal antibodies

We examined purified human large granular lymphocytes, peripheral monocytes, and T cells for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) with murine monoclonal antibodies. We also evaluated the effects of pretreatment of cells with interleukin 2 and interferon to augment ADCC activity. MB3.6, a murine monoclonal antibody directed against the GD3 ganglioside, induced high levels of ADCC. This ADCC was mediated predominantly, if not completely, by human killer cells (large granular lymphocytes) whereas other effector cell populations demonstrated no significant cytotoxic activity in 6- or 18-hr assays. The IgG2a an anti-melanoma antibody 9.2.27 generated low or no ADCC with most normal donors or melanoma patients. IL 2 was a very potent booster of ADCC activity. Interferon alpha also was effective, whereas interferon gamma did not augment but rather inhibited reactivity. We tested a large panel of antibodies of various isotype against colon carcinoma cells and found that gamma-3 isotype antibodies more frequently generated ADCC and produced higher levels of cytotoxic activity than did IgG1 or IgG2 antibodies. It appears that a variety of parameters can affect ADCC reactions, including the type of effector cell and its level of activation, the isotype of the antibody, and properties of the target cell line such as its susceptibility to lysis.