Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma

Serum IgG fractions from a large and homogeneous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the 'non-selective' CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.     

Biological activity in the human system of isotype variants of oligosaccharide-Y-specific murine monoclonal antibodies

Summary The capacity of isotype variants of BR55-2, an anti-tumor monoclonal antibody directed against Y oligosaccharide, to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in the human system was evaluated using freshly isolated peripheral blood mononuclear cells, lymphocytes, monocytes and complement. The ADCC activities of the BR55-2 IgG3 isotype and its switch variants (IgG1, IgG2b, and IgG2a) with human monocytes were high for all isotypes, whereas the activity of all isotypes was lower with freshly isolated lymphocytes, IgG1 being the least effective. The CDC on the other hand was strong with IgG3, IgG2b and IgG2a and negative with the IgG1 variant. The IgG3 and IgG2a isotypes were selected for further development. Their strong ADCC and CDC activity against mammary carcinoma, colon carcinoma and small-cell lung tumor cell lines was confirmed quantitatively using proteins highly purified from tissue-culture supernatants. Significant variations in ADCC and CDC competence was observed among human donors.This work was supported by grants CA 10 815, CA 25 874 and CA 21124 from the National Institutes of Health     

Cytolytic activity of murine anti-dog lymphoma monoclonal antibodies with canine effector cells and complement

Peripheral blood leukocytes (PBL), nonadherent lymphocytes, and adherent monocytes separated from freshly isolated blood of 15 dogs were analyzed for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in combination with murine anti-tumor monoclonal antibodies (MAbs). Canine monocytes isolated from most donors by adherence to gelatin-fibronectin-coated plastic surface presented high ADCC activity against the canine lymphoma 17-71 tumor cell line in combination with antilymphoma MAbs 231 (IgG2a) and 234-2a (IgG2a). Canine lymphocytes generally showed lower ADCC activity than total PBL or monocytes. Canine PBL effector cells showed high ADCC activity against the human colorectal carcinoma SW948 cell line using the Y-6-specific MAb isotype switch variants 55-2 IgG3, 55-2 IgG1, 55-2 IgG2b, and 55-2 IgG2a. Analysis of the role of murine MAb isotypes on ADCC activity against tumors by canine cells using anti-human tumor class-switch variant MAbs and a panel of anti-canine lymphoma MAbs of different IgG subclass revealed the highest ADCC activity with MAbs of the IgG2a and IgG3 subclasses. IgG2a antilymphoma MAbs were also able to lyse tumor cells in complement-dependent cytotoxicity (CDC) assay. These results suggest the potential value of MAbs of IgG3 and IgG2a subclasses in immunotherapy against canine lymphoma.     

Chimeric B72.3 mouse/human (IgG1) antibody directs the lysis of tumor cells by lymphokine-activated killer cells

Summary Chimeric mouse/human B72.3 (cB72.3) antibodies having a human IgG1 (1) or IgG4 (4) constant region were compared to the native murine IgG1 B72.3 (nB72.3) monoclonal antibody (mAb) for their ability to participate with human effector cells in antibody-dependent cellular cytotoxicity (ADCC). Because the TAG-72 antigen recognized by B72.3 is poorly expressed on tissuecultured tumor cell lines, the xenografted OVCAR-3 human ovarian carcinoma ascites was used as a cytotoxicity target. The lytic activity of the cB72.3(1) mAb with peripheral blood lymphocytes was 1.5- to 50-fold greater than that of the nB72.3 mAb and usually the cB72.3(4) mAb. However, lymphocytes from some donors had similar ADCC activity with either the cB72.3(1) or cB72.3(4) mAb. The cB72.3(1) and the murine anti-colon carcinoma CO17-1A mAb had comparable activity in mediating ADCC against the OVCAR-3 tumor. Exposure of lymphoid cells to interleukin-2 (IL-2) (100–500 U/ml) for 24 h to generate lymphokine-activated killer (LAK) cells augmented ADCC mediated by the cB72.3(1) mAb 2- to 22-fold. By contrast, LAK cells from most donors expressed weak non-specific cytotoxicity against OVCAR-3 ascites tumor cells. The cB72.3(1), and to a lesser extent, the cB72.3(4) chimera also participated with monocytes in mediating ADCC, but the antibody-dependent lytic potency of monocytic effectors was much weaker than that of IL-2-activated lymphoid cells. These studies show that the cB72.3(1) mAb has appreciable ADCC-mediating properties, suggesting a potential role for its incorporation into treatment strategies utilizing adoptive killer cell and/or lymphokine therapy. Offprint requests to: J. Schlom, to whom reprint requests should be sent at 9000 Rockville Pike, Building 10, Room 8B07, Bethesda, MD 20892, USA     

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.     

Cell-mediated cytotoxicity in carcinoma of the human urinary bladder

Summary Lymphocytes from patients with tumors of the bladder or other unrelated tissues or with non-malignant genitourinary (GU) conditions and from normal subjects were tested in a microcytotoxicity assay against long- and short-term cultures (T24 and BT) derived from bladder carcinoma and several other target cell types, to determine the validity of the hypothesis that cell-mediated cytotoxicity (CMC) in bladder cancer patients is a specific disease-related phenomenon. At the effector cell level, lymphocytes from bladder cancer patients displayed uniformly greater cytotoxicity for T24 and BT cells than those from normal donors. This proclivity was shared by the lymphocytes of patients with non-GU cancers but not of those with non-malignant GU disorders. At the target cell level, CMC was observed less frequently against non-bladder tumor targets than against T24 and BT cells and the CMC of bladder cancer patients did not differ significantly from that of the other groups. The pattern of CMC observed against the bladder tumor-derived targets was thus one of target cell sensitivity rather than tumor-specificity and disease-related only to the extent that the CMC of patients with cancer was greater overall than of healthy subjects. Abrogation of CMC by passage of lymphocytes through immunoglobulin-coated columns indicated that the effector cells were principally of non-T type, bearing a superficial resemblance to those in normal individuals which induce non-disease related CMC.     

Titration of natural and disease-related cytotoxicity of lymphocytes from bladder cancer patients

Summary A controlled investigation of lymphocyte-mediated cytotoxicity has been carried out in patients with transitional cell carcinoma (TCC) of the urinary bladder, three long-term established cell lines with comparable sensitivities to the natural cytotoxicity (NC) being used as targets, namely HU 456 derived from human TCC, HU 609 from normal human urothelium, and SAOS 2 from a human osteosarcoma. The 44-h incubation microcytotoxicity assay (MA) was used with blinded semiautomatic visual counting of target cells. The cytotoxicity was determined by titration with a serial dilution of lymphocyte suspensions 1:2, five different concentrations being used in each experiment. Fifty tests were performed with lymphocytes from 48 TCC patients. As controls, 65 patients with diseases other than TCC were tested simultaneously. The average cytotoxicity of lymphocytes from TCC patients against HU 456 was only slightly and insignificantly higher than that of the control patients. However, a marked decrease of the NC against the control cells HU 609 and SAOS 2 was noted with TCC patient lymphocytes.After correction of the data for the depression of background NC, increased tumor-specific cytotoxicity (TSC) and tumor type-specific cytotoxicity (TTSC) were demonstrated in TCC patients with noninvasive malignant tumors (grade 2 or 3), whereas no increased specific cytotoxicity was found in TCC patients with invasive grade 2 or 3 tumors. Neither was any increased specific cytotoxicity found in patients with very well-differentiated grade tumors. The presence of specific reactivity in patients with noninvasive TCC in contrast to invasive TCC is supposed to indicate growth-controlling function of the cellular immune reaction.Tests were also performed with lymphocytes from 35 patients in the postoperative phase without any history of TCC. Surprisingly, this group revealed a significantly elevated TSC but not increased TTSC when compared with the 65 untreated control patients, thus indicating that serious reservations must be made in the interpretation of the cytotoxocity assay.     

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.     

Functional polymorphisms of Fc receptors in human monocyte-mediated cytotoxicity towards erythrocytes induced by murine isotype switch variants

Human monocytes can be triggered to antibody-dependent cell-mediated cytotoxicity (ADCC) by murine antibodies. In this study, a series of H chain isotype switch variant antibodies against glycophorin A on human RBC was used to study the influence of isotype on the induction of ADCC. Furthermore, it was studied whether the functional heterogeneity in responsiveness to IgG1 and IgG2b anti-CD3 antibodies, as found among different donors in T cell proliferation induction experiments, was reflected in ADCC. Whereas IgG2a induced ADCC to the same extent in monocytes from all donors, IgG1 showed a heterogeneous pattern, which corresponded to the heterogeneity in T cell proliferation studies. IgG1 anti-CD3 nonresponder monocytes could, however, be induced to ADCC by IgG1 antiglycophorin, although they needed a much higher antibody density on the target cell than did responder monocytes. IgG2b antiglycophorin at a high density induced ADCC in monocytes from all donors irrespective of responsiveness to IgG2b anti-CD3, whereas IgE and IgA antiglycophorin were barely effective in monocytes from all donors. By specific blocking with mAb, the FcR that were involved in ADCC directed by the various isotypes were characterized. ADCC by IgG2a was predominantly mediated by FcRI and could be specifically enhanced by culturing the monocytes with rIFN-gamma. ADCC by IgG1 was predominantly mediated through FcRII in both anti-CD3 responder and nonresponder monocytes. FcRII was also involved in ADCC by IgG2b, although other receptors seemed to contribute significantly to ADCC. When FcRII or FcRI were blocked, IgG1 and IgG2a could also functionally interact with FcRI and FcRII, respectively, provided that the target cells were sensitized to a high degree. These findings indicate that FcRI and both forms of FcRII can mediate cytotoxicity and that the specificity of human FcR for murine isotypes is relative.     

Cell-mediated lysis of tumor targets directed by murine monoclonal antibodies of IgM and all IgG isotypes

Seven murine monoclonal antibodies to antigens expressed on T lymphoma targets were tested for directing antibody-dependent cellular cytotoxicity (ADCC). Peptone-induced peritoneal exudate macrophages, the LPS-stimulated RAW264.10 cell line, and human blood nonadherent mononuclear leukocytes were used as effector cells. All six IgG monoclonals tested, representing the four murine IgG isotypes and directed against four antigens (Thy-1.2, H-2k, Ly-2.1, Ly-9.2), were all active in ADCC. In contrast, an IgM anti-Thy-1.2 showed no activity despite very high C-cytotoxic titers. Thus, there does not seem to be any restriction among IgG classes for directing ADCC to tumor targets mediated by murine macrophages or human K cells.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.     

Granulocyte-monocyte-colony-stimulating factor augments the cytotoxic capacity of lymphocytes and monocytes in antibody-dependent cellular cytotoxicity

Summary Human peripheral blood mononuclear cells (lymphocytes and monocytes) were preincubated for 0–24 h with human recombinant granulocyte-monocyte-colony-stimulating factor (GM-CSF) and used as effector cells in an 18 h antibody-dependent cellular cytotoxicity (ADCC) assay with SW948 (a human colorectal carcinoma cell line) as target cells and mAb 17-1A. A significant increase in the lytic capability was noted after 0.5–2 h of preactivation while longer preincubation times did not significantly increase the lytic potential. GM-CSF at 0.01 g/ml induced the best tumor cell lysis while higher concentrations were inhibitory. GM-CSF pretreatment induced a statistically significant increase in the lytic capacity of both monocytes and lymphocytes in ADCC as well as in the spontaneous cytotoxicity.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Alterations in antibody-dependent cellular cytotoxicity during the course of HIV-1 infection. Humoral and cellular defects

HIV-1-specific cell-mediated cytotoxicity (CMC) is a form of antibody-dependent cellular cytotoxicity (ADCC) in which HIV-1-specific antibodies arm NK cells directly to become cytotoxic for targets bearing HIV-1 antigenic determinants. This non-MHC-restricted cytotoxic activity is present in early stages of disease and declines markedly with disease progression. To understand the cellular and humoral factors contributing to the reduction in this activity, the conditions under which maximal arming of cells occurs was examined in vitro. With the use of a large patient cohort, a strong positive correlation was found between the capacity of a serum to direct lysis in standard ADCC assays and its ability to arm NK cells. Patients with minimal HIV-1-specific ADCC-directing antibodies exhibited low levels of CMC and were unable to arm normal effector cells in vitro. The lack of sufficient ADCC-directing antibodies was found to be one cause of defective CMC in some patients. Unlike asymptomatics, only a weak positive correlation was found between arming and ADCC with sera from AIDS patients, indicating that a factor other than absolute HIV-1 specific antibody titer was responsible for decreased CMC in this patient population. Another group of patients was found to have diminished CMC despite the presence of antibodies in the serum that were fully capable of arming normal effector cells to become cytotoxic for gp120-expressing targets. When compared with those of normal individuals, lymphocytes from seropositive patients mediated significantly reduced levels of cytotoxicity in ADCC and arming assays with the use of a high titered HIV-1-specific serum. In both assay systems, the magnitude and frequency of dysfunction in antibody-dependent cytolysis was found to be greater among AIDS patients than among asymptomatic individuals. The demonstration of both cellular and humoral defects in the ability of seropositive individuals to manifest ADCC reactivities strongly suggests that HIV-1 infection may significantly compromise the effectiveness of this potentially important cytolytic reactivity in vivo.     

Cytotoxic activities of normal cultured human T cells.

Normal human T cells grown in continued cultures in medium containing conditioned medium (CM) from PHA-stimulated lymphocytes were studied for their ability to manifest three known forms of cell-mediated cytotoxicity: lectin-induced cellular cytotoxicity (LICC), natural killer cell (NK) activity, and antibody-dependent cellular cytotoxicity (ADCC). The cultured T cells (CTC) were very effective mediators of LICC, being cytotoxic even at very low attacker-target cell ratios in the presence of different lectins, and against different types of targets. When tested without the addition of lectin, the CTC demonstrated a low degree of spontaneous cytotoxicity. This spontaneous cytotoxicity might not be due to conventional NK cells however, since the CTC failed to show significant numbers of cells with Fc receptors (FcR) for IgG, and had no detectable ADCC activity. CTC could represent a population enriched in polyclonal activated T cells with low spontaneous cytotoxicity against a variety of allogeneic target cells, which is greatly enhanced by the addition of lectins dur ing the 51Cr release assay.     

Disease-related lymphocyte cytotoxicity in vitro in patients with transitional cell carcinoma of the urinary bladder. Comparisons with other malignancies,acute cystitis,and healthy donors

Summary Blood lymphocytes from 100 patients with transitional cell carcinoma of the urinary bladder (TCC-bladder) were studied for their cytotoxicity in vitro against a panel of allogeneic tissue culture cell lines. Of the TCC-bladder patients, 45 were untreated for their disease, while 55 had been treated with local radiotherapy up to 12 years before testing. Control lymphocytes were obtained from (1) 45 untreated, age- and sex-matched patients with other neoplastic diseases, mainly urogenital cancers; (2) 19 patients with acute cystitis; and (3) 45 healthy donors. Lymphocytes from individual donors within all five groups were frequently cytotoxic to any one of the target cells. However, the lymphocytes from each of the two TCC-bladder groups were markedly more cytotoxic to two different bladder tumor targets than to control targets derived from normal bladder epithelium, from colon carcinoma, or from malignant melanoma. Similar comparisons made within each of the three control donor groups did not show this. The results indicate that the two bladder tumor targets were not more susceptible to lymphocyte-mediated lysis than the control targets. The mean cytotoxicity displayed by the lymphocytes from both TCC-bladder groups to the bladder tumor targets was significantly higher than that of the cancer control group and that of the healthy donors. No such elevation was seen when the cancer control group or the cystitis patients were compared with healthy donors. Although untreated TCC-patients with a larger tumor burden (stages T3–T4) appeared to be slightly less cytotoxic to all target cells than those with a smaller tumor burden (T1–T2), these differences were not statistically significant. On the other hand, among the treated TCC-patients, in the main those tested more than 1 year and up to 5 years after therapy exhibited a significantly elevated mean cytotoxicity to the bladder tumor targets. Within all five donor groups, the overall cytotoxicity to the bladder tumor targets and the normal bladder targets showed a statistically highly significant correlation. However, while there was no correlation for the untreated TCC-bladder patients and the clinical controls between cytotoxicity to the bladder tumor targets on one hand and non-bladder targets on the other, the cytotoxicity to the bladder tumor targets of the treated TCC-bladder patients was also correlated with that to the colon carcinoma and the melanoma targets. The results indicate that cytotoxicity in both TCC patients and controls reflects recognition by the lymphocytes of a variety of antigens, shared to different degrees by different groups of target cells. Furthermore, in TCC-bladder patients there is a superimposed cytotoxicity, which is related to their disease and which probably reflects reactions against one or several tumor-associated antigens.     

Cell-mediate cytotoxicity in vitro of human lymphocytes against a tissue culture melanoma cell line (igr3).

Purified peripheral blood lymphocytes from 13 healthy donors, 6 melanoma patients and 1 halo nevus patient were tested for cytotoxic activity against an allogeneic melanoma cell line (IGR3) in, at least, one of the following assays: cell-mediated cytotoxicity (ADCC) and microcytotoxicity assays (ma). The lymphocytes were isolated by Ficoll-Triosil gradient centrifugation (fraction F) followed by removal of iron-phagocytosing and adherent cells (fraction FFF) and by subsequent passage through anti-IgG columns (fraction FFF-C). Leukocytes of each fraction were identified by different methods including morphology, rosette-formation, phagocytic activity, and membrane fluorescence. CMC activity paralled ADCC activity at a log lower level of sensitivity. In both assays lymphocytes of fractions F and FFF had the highest activity, whereas in fraction FFF-C cytotoxicity was strongly reduced. In all three lymphocyte fractions CMC and ADCC activity could be blocked by preincubation of the effector cells in aggregated IgG. Furthermore, depletion of E rosette-forming lymphocytes slightly increased ADCC and CMC activity, whereas depletion of EA and EAC rosette-forming lymphocytes strongly decreased it. Our results therefore indicate that in both CMC and ADCC assays, non-adherent, non-phagocytic Fc receptor-bearing lymphocytes ("K" cells) were the active cytotoxic cells. In MA, on the other hand, mononuclear phagocytes seemed to be the most active cell population. So far no significant difference was observed in CMC, ADCC, and MA between control persons and melanoma patients     

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.     

Human large granular lymphocytes express high affinity receptors for murine monoclonal antibodies of the IgG3 subclass

We have evaluated the ability of murine monoclonal antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by human lymphoid cells. Purified large granular lymphocytes (LGL) and interleukin 2-dependent cloned LGL lines having a CD2+/CD16+/CD3- phenotype were tested as effector cells in an ADCC assay by using a family of IgG isotype switch variant anti-Thy-1.1 antibodies against 51Cr-labeled Thy-1.1+ murine SL2 thymoma target cells, a system in which human cells have no spontaneous cytotoxicity. Cytotoxicity was greatest when using the IgG3, followed in rank order by the IgG2a and IgG2b. No cytotoxicity was observed with the IgG1 antibody. Because the antigen-binding regions of the antibodies are identical, the differences in cytotoxicity directly reflect the relative affinity of LGL Fc receptors for each antibody isotype.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.