Human recombinant interleukin-6 enhances antibody-dependent cellular cytotoxicity of human tumor cells mediated by human peripheral blood mononuclear cells

Summary The effects of human recombinant interleukin-6 (hrIL-6) on antibody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC) were investigated. Human PMNC were preincubated for 24 h with various concentrations of hrIL-6 and were used as effector cells in a 4-h⁵¹Cr-release assay. The ability of hrIL-6 to augment ADCC was measured using anti-colorectal carcinoma mAbs D612, 17.1A and 31.1 (each directed against a distinct tumor antigen) and using three human colorectal carcinoma cell lines, LS-174T, WiDr and HT-29, as targets. A significant increase in ADCC activity was observed after PMNC were preincubated in 100–400 U/ml but not in lower concentrations of hrIL-6. Variations in activities of PMNC among donors were observed. Non-specific mAb showed no effect in augmenting ADCC activity. hrIL-6 treatment did not augment non-specific (non-mAb-mediated) cytotoxicity. The enhancement of ADCC activity was blocked by the addition of an antibody against hrIL-6 but not by an antibody to the IL-2 receptor (capable of blocking the induction of lymphokine-activated killer cell cytotoxicity by IL-2), suggesting that hrIL-6 augmentation of ADCC activity may not be mediated through IL-2. These results demonstrate that hrIL-6 augments ADCC activity of human PMNC using mAbs to human tumor antigens and human tumor cells as targets, suggesting a potential role for IL-6 in combination with anti-cancer antibodies for cancer immunotherapy.     

Within peripheral blood mononuclear cells, antibody-dependent cellular cytotoxicity of rituximab-opsonized Daudi cells is promoted by NK cells and inhibited by monocytes due to shaving

Treatment of chronic lymphocytic leukemia patients with anti-CD20 mAb rituximab (RTX) leads to substantial CD20 loss on circulating malignant B cells soon after completion of the RTX infusion. This CD20 loss, which we term shaving, can compromise the therapeutic efficacy of RTX, and in vitro models reveal that shaving is mediated by effector cells which express Fc gammaRI. THP-1 monocytes and PBMC promote shaving, but PBMC also kill antibody-opsonized cells by antibody-dependent cellular cytotoxicity (ADCC), a reaction generally considered to be due to NK cells. We hypothesized that within PBMC, monocytes and NK cells would have substantially different and competing activities with respect ADCC or shaving, thereby either enhancing or inhibiting the therapeutic action of RTX. We measured ADCC and RTX removal from RTX-opsonized Daudi cells promoted by PBMC, or mediated by NK cells and monocytes. NK cells take up RTX and CD20 from RTX-opsonized B cells, and mediate ADCC. PBMC depleted of NK cells show little ADCC activity, whereas PBMC depleted of monocytes have greater ADCC than the PBMC. Pre-treatment of RTX-opsonized B cells with THP-1 cells or monocytes suppresses NK cell-mediated ADCC, and blockade of Fc gammaRI on monocytes or THP-1 cells abrogates their ability to suppress ADCC. Our results indicate NK cells are the principal cells in PBMC that kill RTX-opsonized B cells, and that monocytes can suppress ADCC by promoting shaving. These results suggest that RTX-based immunotherapy of cancer may be enhanced based on paradigms which include infusion of compatible NK cells and inhibition of monocyte shaving activity.     

Induction of cytotoxicity in human peripheral blood mononuclear cells by monoclonal antibody OKT3

It has been previously reported from this laboratory that incubation of PBMC with OKT3 generates potent cytotoxic lymphocytes that can be targeted by using antibody heteroconjugates consisting of anti-target cell antibody and OKT3. In the present study these conjugates were used to explore the kinetics of induction of cytotoxicity in PBMC and the subpopulations of lymphocytes involved. It was found that in addition to conjugate-dependent cytotoxicity, a considerable amount of conjugate-independent cytotoxicity was generated during OKT3 stimulation. Although the conjugate-dependent activity resided in the CD8+ population, the conjugate-independent cytotoxicity was found to be a function of CD4-/CD8- natural killer-like cells. Being largely CD3-, those cells were most likely activated by lymphokines produced by OKT3-stimulated CD3+ cells. They were capable of killing not only tumor cells but also autologous lymphocytes. The CD4+ cells of some donors were found to exhibit low but clearly demonstrable cytotoxicity. Induction of cytotoxicity was characterized as an early event in T cell activation, correlating with the kinetics of RNA synthesis. Cytotoxicity, interleukin 2 receptor expression, and DNA synthesis declined after 3 days of activation with OKT3, indicating the existence of as yet undefined regulatory mechanisms.     

Micro-scission of YAC tumor cells during antibody-dependent cellular cytotoxicity mediated by human neutrophils

The cellular events accompanying neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) directed against YAC erythroleukemic target cells have been studied by time-lapse fluorescence-intensified microscopy. The YAC plasma membrane and cytosol were labeled with the fluorescent probes diC18Icc and eosin Y, respectively. Fluorescently labeled and IgG-opsonized YAC cells were incubated at 37 degrees C while observed by optical microscopy. During temporal studies of neutrophil-YAC conjugates, the cytosol of YAC cells accumulated in tubular and spherical compartments of the neutrophils' vacuolar apparatuses. To distinguish between several possible mechanisms of target cytosol uptake, diC18Icc-labeled YAC cells were observed during identical conditions. The membrane label diC18Icc was found to accumulate within neutrophils in an identical fashion. At roughly 30 min, 25 and 38% of neutrophils in apparent conjugates had internalized tumor cell cytosol or plasma membrane, respectively, within a vesicular compartment. The IgG-dependent uptake of eosin Y and diC18Icc by neutrophils was diminished by exposure to 2.5 mM sodium azide. When cells were exposed to 5.5 mM sodium azide, 1 mM iodoacetamide, or 4 degrees C, conjugate formation and uptake of eosin Y or diC18Icc were abolished. An artifactual accumulation of eosin Y or diC18Icc in neutrophils was further ruled out by control studies. Non-specific exchanges of eosin Y and diC18Icc labels of YAC cells with tannic acid-treated red blood cells (RBCs) and normal neutrophils were studied. Since hemoglobin binds tightly to eosin Y, RBCs can easily detect eosin Y leakage. No exchange of eosin Y or diC18Icc from YAC cells into bound tannic acid-treated erythrocytes was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. I. Identification of the population of effector cells.

Mononuclear cells (MC) from human blood were fractionated by a variety of physical and immunologic techniques, and the cellular subpopulations generated were assessed for their capacity to lyse herpes simplex virus (HSV)-infected target cells in the presence of IgG antibody to HSV. Latex phagocytosis and surface marker studies were performed in parallel in order to identify the major effector cells by their phagocytic properties and their possession of surface immunoglobulin and receptors for either sheep erythrocytes, C3, or the Fc fragment of IgG. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of plastic-adherent or carbonyl iron-adherent MC, indicating that the major effector cell is not a classical monocyte. Similar results were obtained after removal of more than 90% of the T cells by depletion of rosette-forming cells. Likewise, effector cell activity was generally unchanged when more than 95% of the B cells were removed by filtering MC on nylon wool columns. Effector cell function was also found to be normal in three patients with B cell-deficient X-linked agammaglobulinemia. These observations strongly suggest that the effector cells are not T cells or B cells. A 4- to 5-fold enrichment in effector cells, however, was consistently found in a subpopulation, consisting of 5% of the unfractionated MC, that was dramatically enriched both for nonphagocytic cells with only Fc receptor (K cells) and for nonphagocytic cells with no detectable surface markers (null cells). Since, as is demonstrated in the accompanying report, effector surface Fc receptors play a critical role in the mediation of antibody-dependent cellular cytotoxicity directed at HSV-infected target cells, the major mononuclear effector cell in human blood is a K cell.     

The role of antibody in cellular cytotoxicity to culture human colon carcinoma cells.

The effect of autologous serum in cellular cytotoxicity was compared with the level of antibody towards the same target cells. In cancer patients, a highly significant correlation (P < 0.001) argues that enhancement of cytotoxicity by autologous serum is due to high antibody levels, and conversely an inhibition of cytotoxicity is associated with low antibody. Moreover, some antibody was detected in virtually all cancer patient and normal donor sera. It is suggested that antibody may be responsible for cytotoxicity conventionally ascribed to “natural killer” cells.     

Synergistic cytotoxicity. I. Characterization of a heat labile plasma fraction that induces nonspecific cytotoxicity by human mononuclear cells.

The mechanism by which non-immune mononuclear cells recognize invading foreign material and are activated for cytotoxic attack was studied in a model system employing human mononuclear cells, fresh plasma, and 51Cr-labeled xenogeneic target erythrocytes. In these experiments, fresh antibody-depleted plasma or mononuclear leukocytes alone were poorly cytotoxic to xenogenic erythrocytes. However, these target cells were rapidly lysed when both fresh antibody-depleted plasma and mononuclear cells were present in the assay. The plasma factor could not be removed by extensive absorption with the target cells, was present in plasma from hypogammaglobulinemic patients, was heat labile, and was sensitive to incubation with zymosan and cobra venom factor. The "antigen" specificity of this reaction was directed by the serum factor inasmuch as target cells autologous to the effector cells could be killed in the presence of antibody-depleted xenogeneic plasma, but not autologous plasma. These data suggest that an important mechanism for the recognition of "foreigness" by non-immune mononuclear cells is via interaction with a plasma component, possibly a factor related to serum complement.     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. II. Identification as a K cell.

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 gamma-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.     

Antibody-dependent cytotoxicity of human and mouse mononuclear cells against Trypanosoma cruzi epimastigotes

Human peripheral mononuclear cells were cytotoxic to antibody-sensitized Trypanosoma cruzi epimastigotes. The cytotoxic effect depended on the concentration of effector cells and antiserum, and was progressive until 17 hr of incubation at 28 °C. After 3 hr of incubation the highest specific activity was achieved at a 50:1 effector to target cell ratio. A nonspecific cytotoxic effect in the absence of antiserum was observed at a 100:1 parasite to cell ratio or after 17 hr of incubation. When the human mononuclear cell population was depleted of adherent cells by Sephadex G-10 filtration or adsorption to glass, the cytotoxic effect was greatly reduced. Similar results were obtained using mouse spleen cells, indicating that only the adherent cells were cytotoxic to sensitized T. cruzi in both systems. When human mononuclear cells were incubated with amobarbital, cyanide, azide, or aminotriazole, an inhibition of cytotoxicity against sensitized T. cruzi was observed, suggesting that oxygen reduction products and myeloperoxidase were involved in the destruction of sensitized T. cruzi epimastigotes by normal human mononuclear cells.     

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.     

Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd⁺⁺-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd⁺⁺ cytotoxic effect and deccreased the LD_50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd⁺⁺. Thus the results demostrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd⁺⁺ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd⁺⁺ cytotoxic response. Comparison of reduced glutathione levels and of Cd⁺⁺ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd⁺⁺ resistance.Abbreviations BSO DL-buthionine-(R,S)-sulfoximine - DEM diethyl maleate - DMSO dimethyl sulfoxide - GSH reduced glutathione - MT metallothionein     

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.     

Antibody-dependent cellular cytotoxicity and natural killer cytotoxicity of peritoneal cells from nude mice to herpes simplex virus-infected cells

Nude BALB/c mice (athymic) were more susceptible to fatal herpes simplex virus (HSV) than normal BALB/c mice (P = 0.002). The peritoneal cells of nude mice mediated levels of antibody-dependent cellular cytotoxicity (ADCC) of equal or greater magnitude than cells from normal BALB/c, heterozygote nu/+, or C57BL/6 mice. Unstimulated natural killer cytotoxicity of peritoneal cells from nude mice was higher (P less than 0.05) than that mediated by cells from C57BL/6 mice. Nude mice failed to make anti-HSV ADCC antibody 6 to 14 days post HSV inoculation, at times when nu/+, BALB/c, and C57BL/6 mice produced antibody. Passive reconstitution of nude mice with high titer intraperitoneal anti-HSV immune globulin provided circulating anti-HSV ADCC antibody and significant protection against lethal HSV infection.     

Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

Lack of cross-reaction in antibody-dependent cellular cytotoxicity between human immunodeficiency virus (HIV) and HIV-related West African strains

Sera from individuals infected with human immunodeficiency virus (HIV) and HIV-related West African viruses can mediate high-titered, virus-specific antibody-dependent cellular cytotoxicity (ADCC) in all stages of infection. No cross-reactive ADCC can be detected between HIV and HIV-related West African strains LAV-2, HTLV-IV, and SBL-6669. Because these two groups of viruses have antigenically distinct envelope glycoproteins, ADCC-mediating antibodies are most likely directed against envelope antigens. For HIV-specific ADCC, this was further confirmed by using sera reacting with HIV envelope but negative for antibodies against viral core antigens.     

Cytotoxic studies in human newborns: lessened allogeneic cell-induced (augmented) cytotoxicity but strong lymphokine-activated cytotoxicity of cord mononuclear cells

Nonspecific cytotoxic responses such as natural killer activity can be increased in vitro by incubating effector cells with soluble factors or allogeneic cells. We sought to determine if newborn cells, known to be deficient in most cytotoxic responses, including resting NK activity, could develop enhanced cytotoxic responses following incubation with allogeneic cells (augmented cytotoxicity) or with lymphokines (lymphokine-activated cytotoxicity). Cord whole mononuclear cells (WMC) incubated with irradiated Raji cells for 5 days develop lower levels of cytotoxicity toward K562 targets at both a 20:1 effector:target (E:T) ratio (39 +/- 2.7% vs 49 +/- 3.6%) and a 10:1 E:T ratio (29 +/- 2.6% vs 40 +/- 3.6%) than do adult cells. Lessened specific cytotoxicity of cord cells developed toward the sensitizing Raji cells was also observed at both E:T ratios. Attempts to enhance the induced cytotoxicity by incubation with interferon or isoprinosine were unsuccessful. In contrast, lymphokine (i.e., interleukin 2)-activated killer (LAK) cytotoxicity is not deficient in cord WMC. Indeed, the level of LAK cytotoxicity is equivalent to that observed with similarly treated adult cells despite a lower baseline level of cytotoxicity toward the target cells. In the presence of purified IL-2 for 5 days, cord WMC cytotoxicity against K562 cells increased from 12 +/- 2.6 to 71 +/- 4.2% and against Raji cells increased from 9.6 +/- 2.5 to 48 +/- 6.7%. Similarly treated adult cells increased their killing against K562 from 23 +/- 4.2 to 61 +/- 4.5% and against Raji from 12 +/- 3.0 to 36 +/- 5.3%. This substantial lymphokine-activated cytotoxicity of newborn cells suggests the possibility of therapeutic intervention with purified lymphokines in neonatal infections or neoplasms.     

Internalization and degradation of glucagon by human mononuclear cells

The processing of glucagon by circulating human mononuclear cells was examined. Glucagon bound to the membrane with a turnover time of 4.4 minutes per site after 15 minutes of incubation and 8 minutes per site after 90 minutes. The amount of intact intracellular hormone increased by 3-fold by 90 minutes suggesting a slowing of intracellular processing with prolonged incubation. Excess unlabelled insulin also slowed the processing of glucagon at the degradative step with no effect on binding or internalization of glucagon. Subcellular fractionation of the cells showed that most hormone accumulated in the 500xg pellet and in the 100,000xg supernatant. N-ethylmaleimide blocked intracellular glucagon degradation suggesting a role for intracellular sulfhydryl-dependent enzymes. Kinetic analysis of the dissociation of glucagon revealed a second order process with K values of 2.2 X 10(-2) fm-1 min-1 and 1.4 X 10(-2) fm-1 min-1 for dissociation from membranes and from membranes + intracellular sites, respectively. T 1/2 values were 6 min. for membrane dissociation and 9 min for membranes + cells. These findings suggest that glucagon interaction with mononuclear cells has characteristics similar to other receptor bound ligands including internalization processing and metabolism.     

Carbohydrate-specific monoclonal antibodies bind to human granulocytes and stimulate antibody-dependent cellular cytotoxicity

Mouse monoclonal antibodies which specifically recognize human granulocytes are used to study the classification, differentiation, and function of these cells. Mouse monoclonal antibody WEM-G1 specifically binds to human neutrophils and eosinophils. It also affects granulocyte function by stimulating granulocyte-mediated antibody-dependent cellular cytotoxicity. Biochemical studies presented here show that WEM-G1 recognizes the sugar sequence 3-fucosyllactosamine, Gal beta 1-4[Fuc alpha 1-3]GlcNAc. This sequence is present in granulocyte glycolipids and in glycoproteins of average approximate Mr 165,000 and 105,000. WEM-G1 is thus similar to other monoclonal antibodies that recognize this sequence on granulocytes and various other cells. Some of these 3-fucosyllactosamine-specific antibodies affect several other granulocyte functions. Knowledge of the biochemical structure of the WEM-G1 antigen suggested testing granulocyte function with other monoclonal antibodies of similar specificity. Antibodies recognizing both the identical oligosaccharide structure and a related sequence, Gal beta 1-4GlcNAc-R, were also found to stimulate granulocyte-mediated antibody-dependent cellular cytotoxicity.