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.     

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 effect of various cytokines on the in vitro induction of antibody-dependent cellular cytotoxicity in murine cells. Enhancement of IL-2-induced antibody-dependent cellular cytotoxicity activity by IL-1 and tumor necrosis factor-alpha

We have previously demonstrated that incubation with IL-2 can induce ADCC activity in murine cells and that this activity was mediated by asialo GM1+, FcR+ cells. In the present study we show that the cytokines IFN-alpha and IFN-gamma, TNF-alpha, and IL-1 alpha are unable to induce antibody-dependent cellular cytotoxicity (ADCC) in murine cells; however, TNF-alpha and IL-1 alpha could substantially augment the ADCC induced by IL-2. IL-1 increased the IL-2-induced ADCC activity in a dose-dependent fashion and in cells isolated from the thymus and spleen. The precursors of the ADCC induced by the combination of IL-1 and IL-2 were asialo GM1+ cells, similar to the precursor cells of IL-2-induced ADCC. The effect of IL-1 and TNF on ADCC was not the result of an increase in the FcR density on the cell surface or the result of an increase in the number of FcR+ cells although IL-1 increased the recovery of viable cells in culture. The main effect of IL-1 and TNF was the enhancement of the lytic ability of the IL-2 cultured cells as indicated by increased intra-cellular benzyloxycarbonyl L-lysine thiobenzylester-esterase activity. These results suggest that lymphokines such as IL-1 and TNF may synergize with IL-2 in the induction of ADCC and could thus potentially be useful for the immunotherapy of established tumors when combined with the administration of specific anti-tumor antibodies.     

Differences in antibody-dependent cellular cytotoxicity against tumor cells in in vitro differentiating mononuclear phagocytes from bone marrows of normal and inflamed mice

Mononuclear phagocytes, differentiated in vitro from bone marrow cells of mice inflamed in vivo with either Corynebacterium parvum or thioglycollate, expressed a higher activity in antibody-dependent cellular cytotoxicity (ADCC) against tumor cells, than those of normal mice. A good correlation between the cytolytic activity and chemiluminescence activity of the different mononuclear phagocyte populations was observed. The ADCC activity of BMDMP from normal mice was inhibited by exogenous prostaglandin E₂ (PGE₂) to a higher extent than that of BMDMP of inflamed mice. When the three BMDMP populations were cultured in the presence of aspirin (without exogenously added PGE₂), the ADCC was significantly increased. The three populations gave identical high values. This suggests that the differential ADCC activity of BMDMP from normal and inflamed mice is due to their differential response to endogenous prostaglandins. PGE₂ showed also a differential effect on the mononuclear phagocyte-forming capacity of bone marrow macrophage precursor cells from normal and inflamed mice. Bone marrow precursor cells from inflamed mice showed a higher resistance to the suppressive effect of PGE₂ (10^?5M) on mononuclear phagocyte-forming capacity than those of normal mice which were totally suppressed. It is concluded that the observed differential properties of the three bone marrow-derived mononuclear phagocyte populations originate at the level of bone marrow precursor cells and that, therefore, the similar functional differences observed in inflammation-induced peritoneal macrophage populations, observed by our and other groups, stem at least partly from differences at the level of bone marrow precursor cells.     

Deposition of reactive oxygen metabolites onto and within living tumor cells during neutrophil-mediated antibody-dependent cellular cytotoxicity

In this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5′ (or 6′) carboxyl-2′,7′-dichlorodihy-drofluorescein (H₂-CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative. H₂-CDCF-conjugated IgG prepared from the succinimidyl ester derivative was used to opsonize targets. In vitro studies have shown that H₂-CDCF becomes fluorescent upon exposure to reactive oxygen metabolites, including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposited on tumor cell membranes during neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC). This deposition process is catalase sensitive. The role of reactive oxygen metabolites produced by neutrophils in triggering the oxidation of H₂-CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were also labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 and dihydrotetramethyl-rosamine within tumor cells. Using two-color automated epifluorescence micros-copy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (<12) sec. These studies show that reactive oxygen metabolites are deposited both onto and into tumor cells during ADCC, wherein both compartments could become vulnerable to oxidant-mediated damage. © 1993 Wiley-Liss, Inc.     

Two distinct mechanisms of cytotoxicity by porcine alveolar macrophages in antibody-dependent and immobilized immune complex-dependent cellular cytotoxicity

The mechanisms of cytotoxicity by porcine pulmonary alveolar macrophages (PAM) involved in antibody-dependent cellular cytotoxicity (ADCC) and immobilized immune complex-dependent cellular cytotoxicity (IIC-DCC) were investigated. The results indicate that IIC-DCC was inhibited by both catalase and thioglycollate broth whereas these peroxide scavengers had no effect on ADCC in an 18-hr chromium-release assay. Furthermore, it was found that when the PAM and red blood cell targets were cross-linked with PHA, catalase still completely eliminated IIC-DCC and had no effect on ADCC, which suggests that catalase is able to penetrate the lytic site when the effector and targets are cross-linked as in ADCC. The presence of cytochalasin B, which inhibits internalization of immune complexes by PAM and presumably prevents intracellular killing, also had no effect on the differential susceptibility of IIC-DCC and ADCC to catalase. Finally, it is shown that the nonspecific cytotoxicity generated by exposing PAM to immune complexes in suspension in conjunction with cytochalasin B, so that the immune complex-bound Fc receptor (FcR) cannot be internalized, also was susceptible to catalase. These data show that the lytic mechanism involved in the nonspecific cytotoxicity generated by exposing PAM to immobilized immune complexes or immune complexes in suspension in conjunction with cytochalasin B, both of which prevent the internalization of immune complex-bound FcR, is mediated solely by peroxide whereas the lytic mechanism involved in ADCC operates, at least partially, through a peroxide-independent mechanism.     

Interferon enhances the antibody-dependent cellular cytotoxicity (ADCC) of human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMN) from healthy volunteers were tested for ADCC activity against both erythrocyte and tumor targets with and without the addition of human leukocyte interferon (IFN). It was demonstrated that IFN within 30 to 60 min enhanced the reaction in a dose-dependent manner with minimal IFN doses ranging from 1 to 100 units. Formal proof that the augmenting agent was IFN was obtained by using pure IFN proteins in combination with both mock-IFN preparations, which showed no enhancing activity, and anti-IFN antisera, which inhibited the action of the completely purified IFN proteins. In the light of data demonstrating that the IFN effect was most pronounced when the IgG antibodies in the ADCC reaction were present in suboptimal amounts, it is hypothesized that IFN may play a special role in the early nonspecific immune response against non-self antigens.     

In vitro enhancement of natural and antibody-dependent lymphocyte-mediated cytotoxicity against tumor target cells by interferon.

Interferon derived from virus-infected human leukocytes or fibroblasts was found to enhance spontaneous and antibody-dependent lymphocyte cytotoxicity against human target cell lines in vitro. The greater enhancement occurred with spontaneous lymphocyte cytotoxicity. Interferon exerted its effect directly on lymphocytes; no effect on target cells was seen. The mechanism of enhancement was unclear: It did not reflect antibody production or lymphocyte proliferation. Enhancement appeared to be immunologically nonspecific, but clarification of this effect awaits further study.     

Polymorphonuclear leukocyte-mediated, antibody-dependent, cellular cytotoxicity against tumor cells: dependence on oxygen and the respiratory burst.

Experiments were done to determine 1) whether the respiratory burst of superoxide anion (O2-) production in polymorphonuclear leukocytes (PMN) is triggered during antibody-dependent killing of tumor cells and 2) whether O2- production is essential for cytotoxicity. Three parameters of the respiratory burst (1-14C-glucose oxidation, oxygen consumption, and O2- release) were increased 2.5- to 7.3-fold during killing of antibody-primed tumor cells by human PMN. Added catalase and superoxide dismutase did not inhibit lysis, possibly because these enzymes were unable to diffuse into the inter-plasma-membrane space between killer and target cells. Evidence for an O2- requirement for cytotoxicity was the fact that concentrations of amobarbital or phenylbutazone sufficient to inhibit the cyanide-insensitive respiration of PMN also inhibited cytotoxicity. Also, hypoxic conditions inhibited cytotoxicity from 29 to 73%. The requirement for oxygen was most likely related to O2- generation and not mitochondrial respiration since cyanide and azide, which inhibit mitochondrial respiration, increased cytotoxicity.     

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.     

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).     

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

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

Natural killing and antibody-dependent cellular cytotoxicity: characterization of effector cells by E-rosetting and monoclonal antibodies

Recent investigations have indicated that the OKM1 hybridoma monoclonal antibody reactive with cells of the myelomonocytic series identifies a subpopulation of human peripheral blood mononuclear cells (PBMNC) which mediate natural and antibody-dependent cellular cytotoxicity (ADCC). However, it was not clear whether this OKM1+ group was heterogeneous with regard to cytotoxic function or the presence of receptors for sheep erythrocytes. Thus, the purpose of the present study was to further define the phenotype of the ADCC effector cell and natural killer (NK) cell using a combination of reactivity with hybridoma antibodies and separation of subsets by sheep erythrocyte rosette (E+) formation. Furthermore, the phenotypes of the NK population were assessed directly by performing two-color immunofluorescent staining on tumor cell conjugates. These studies led to the following conclusions: (1) that NK activity is mediated by both E+ OKM1+ and E- OKM1+ cells; the E+ OKT3+ cell possessed essentially no ADCC or NK activity; (2) that E+ OKM1+ cells mediated more NK activity on a per cell basis than E- OKM1+ cells; this was verified by separating OKM1+ cells on a cell sorter into E+ and E- with the OKT11 monoclonal antibody (anti-E-receptor antibody); (3) that E+ OKM1+ cells mediated both ADCC and NK activity; (4) that the phenotypes of PBMNC forming tumor cell conjugates were (a) OKM1+ (both E-receptor positive and negative) and (b) OKM1- E-receptor positive.     

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.     

Development of immunomonitoring of antibody-dependent cellular cytotoxicity against neuroblastoma cells using whole blood

Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a ‘real-time’ ex vivo whole-blood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement-dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.     

A subset of CD16-natural killer cells without antibody-dependent cellular cytotoxicity function

The low affinity IgG receptor, CD16 (Fc gamma RIII), is expressed on almost all peripheral blood natural killer (NK) cells. A small subset of CD3- CD16- CD56+ NK cells, representing less than 1% of peripheral blood lymphocytes, expands during in vivo IL-2 treatment. To analyze this CD16- NK cell subset in more detail, NK clones have been generated. One of them (TNK2) has been used to study the function of these cells in more detail. It is demonstrated that TNK2 exerts normal NK activity and displays large granular lymphocyte morphology. Since this clone lacks CD16 expression, antibody-dependent cellular cytotoxicity cannot be exerted. CD16 monoclonal antibodies fail to induce cytotoxic activity against NK-resistant target cells. These studies reveal that the lack of CD16 detection is not due to the modulation or the stage of activation of these NK cells. TNK2 is representative of this small subset of peripheral blood NK cells, expanded during IL-2 treatment, which does not express Fc gamma RIII and therefore cannot perform antibody-dependent cellular cytotoxicity.     

Effect of histamine and histamine antagonists on natural and antibody-dependent cellular cytotoxicity of human lymphocytes in vitro

The in vitro effect of histamine and its antagonists, cimetidine and clemastine fumarate, on natural killer (NK) and antibody-dependent cellular Cytotoxicity (ADCC) activities of human lymphocytes was investigated. The histamine 1 (H1) antagonist, clemastine fumarate, and the histamine 2 (H2) antagonist, cimetidine, but not histamine alone, inhibited the NK and ADCC activities of lymphocytes when added directly to the mixture of effector and target cells in a ⁵¹Cr-release assay. This inhibition was proportional to the concentration of drugs added and was observed at various effector to target ratios against several targets. H1 and H2 antagonists also inhibited NK activities of T cells as well as Percoll-separated, NK-enriched effector cells. The inhibition was significantly reversed by histamine. In target binding assays, clemastine fumarate and cimetidine also decreased the target binding capacity of effector lymphocytes. Further, PBL precultured with histamine (10^?3–10^?4M) for 24 hr showed a significant decrease in their NK and ADCC activities. In coculture experiments, PBL precultured with histamine suppressed the NK activity of normal autologous effector lymphocytes. PBL precultured with histamine showed an increased number of OKT8⁺ cells, as estimated using monoclonal antibodies. The suppression of Cytotoxicity was not due to either direct toxicity, steric hindrance, crowding, or cell death, but by functionally viable suppressor cells. An immunoregulatory role for histamine in NK and ADCC reactions is proposed.     

Identification of target antigen for antibody-dependent cellular cytotoxicity on cells carrying Epstein-Barr virus genome

The target antigen for antibody-dependent-cellular cytotoxicity (ADCC) on Epstein-Barr virus-(EBV) carrying lymphoblastoid cells expressing EBV-specific membrane antigen (MA) were examined with human serum antibody and adult human peripheral lymphocytes as effector cells. These studies confirmed that anti-MA-positive but not MA-negative sera were reactive in the ADCC. The ADCC reaction was positive with cells in which the MA consisted of late (LMA) and early (EMA) components. These included 1) MA-positive cells prepared by EBV antigen-adsorption, 2) cells carrying de novo-synthesized MA without adsorbed MA, and 3) EBV-producer cells expressing MA spontaneously. In all these preparations, the target cells were lysed roughly in parallel with the frequency of MA-positive cells. Inhibition of LMA synthesis in EBV-superinfected cells by phosphonoacetate (PA) reduced ADCC sensitivity significantly and to a far greater extent than MA synthesis as measured by immunofluorescence. This suggests that a target for ADCC is the PA-sensitive LMA. No ADCC reaction occurred with the cell preparation comprised of a high percentage of MA-positive cells induced by 5-iodo-2'-deoxyuridine, which is believed to be EMA only. These results strongly suggest that the target antigen for ADCC in EBV-positive cells is a late MA but not early MA.     

Influence of cytokines on HIV-specific antibody-dependent cellular cytotoxicity activation profile of natural killer cells

There is growing interest in HIV-specific antibody-dependent cellular cytotoxicity (ADCC) as an effective immune response to prevent or control HIV infection. ADCC relies on innate immune effector cells, particularly NK cells, to mediate control of virus-infected cells. The activation of NK cells (i.e., expression of cytokines and/or degranulation) by ADCC antibodies in serum is likely subject to the influence of other factors that are also present. We observed that the HIV-specific ADCC antibodies, within serum samples from a panel of HIV-infected individuals induced divergent activation profiles of NK cells from the same donor. Some serum samples primarily induced NK cell cytokine expression (i.e., IFNγ), some primarily initiated NK cell expression of a degranulation marker (CD107a) and others initiated a similar magnitude of responses across both effector functions. We therefore evaluated a number of HIV-relevant soluble factors for their influence on the activation of NK cells by HIV-specific ADCC antibodies. Key findings were that the cytokines IL-15 and IL-10 consistently enhanced the ability of NK cells to respond to HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate "educated" KIR3DL1(+) NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate "uneducated" KIR3DL1(+) NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies in vitro. Manipulating the immunological environment to enhance the potency of NK cell-mediated HIV-specific ADCC effector functions could be a promising immunotherapy or vaccine strategy.     

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.