Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Antibody-dependent cell-mediated cytotoxicity using a murine monoclonal antibody against human colorectal cancer in cancer patients

Summary Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by a murine monoclonal antibody against human colerectal carcinoma, antibody 19–9, with human effector cells was tested in 33 patients with various carcinomas, 16 patients with benign lesions, and 13 normal controls, using a 12-h ⁵¹Cr release assay using human colorectal cancer cells as targets. Peripheral blood mononuclear cells (PBM) from these groups of patients and normal controls achieved moderate levels of target cell lysis in the presence of the monoclonal antibody at the high effector to target cell ratio of 200:1. The ADCC activity of PBM in cancer patients was significantly higher than that in either normal persons or patients with benign lesions. Since the ADCC was shown to be mainly mediated by adherent monocytes in the PBM, ADCC activity of monocytes from cancer patients was compared to those from control groups at an effector to target cell ratio of 30:1. The results also showed that the lytic capacity of monocytes was significantly higher in cancer patients than that in the control populations.     

Tissue distribution and molecular profile of a differentiation antigen detected by a monoclonal antibody (345.134S) produced against human melanoma cells

Summary The mouse IgG2a monoclonal antibody (MoAb) 345.134S, secreted by a hybridoma derived from a mouse immunized with cultured human melanoma cells, reacts with an 85,000-dalton glycopolypeptide which is disulfide-bridged to a 30,000-dalton polypeptide having little if any covalently attached carbohydrate. The 115,000-dalton complex is peripheral rather than integral in its association with the plasma cell membrane. Indirect immunofluorescence of cryostat thin sections of human tissues with the MoAb 345.134S showed (1) strong staining of the sebaceous glands and basal layer of normal hyperpigmented skin; (2) weak staining of the basal layer of normal pigmented skin and epithelial cells of the gastrointestinal tract, parotid, renal proximal tubules, thyroid, and urinary bladder; and (3) no staining of melanocytes, mammary gland, lung, brain cortex, or liver. The staining pattern of tissues from a 20-week-old fetus is similar to that of tissues from adults. The MoAb 345.134S stained some cases of virtually all tumors tested, including some derived from normal tissues non-reactive with the antibody; intensity of staining of tumors was in general much greater than in normal tissues. The expression of the antigen detected by MoAb 345.134S in a panel of cultured human tumor cells did not correlate with the expression of other tumor-associated antigens or with HLA-A,B or Ia-like antigens. The MoAb 345.134S can mediate complement- and cell-dependent lysis of cultured human tumor cells. The lack of correlation between the extent of immune lysis and the expression of the antigen detected by MoAb 345.134S as well as the effect of puromycin on antibody-mediated cell-dependent lysis indicated that factors other than antigen density play a significant role in the outcome of immune lytic reactions mediated by this monoclonal antibody. Abbreviations used: MAA, melanoma-associated antigens; MoAb, Monoclonal antibody; ₂-, ₂-microglobulin; PBL, peripheral blood lymphocytes; IIF, indirect immunofluoresence; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; NP40, nonidet P40; ADCC, antibody-dependent cell-mediated cytotoxicity     

Antibody-dependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2

The incubation of murine splenocytes in recombinant interleukin 2 (RIL 2) gives rise to lymphokine-activated killer (LAK) cells that can lyse fresh, NK-resistant tumor cells but not normal cells in 4-hr 51Cr-release assays. Lysis by this IL 2-activated cell population was enhanced up to 100-fold by prior reaction of target cells with specific antisera reactive with antigens on the target cells. This antibody-dependent cellular cytotoxicity (ADCC) also resulted in lysis of fresh normal target cells, which are not usually susceptible to LAK lysis. The ADCC was evident after 24 hr of incubation of splenocytes in RIL 2, but peak lytic activity was reached after 3 to 4 days of incubation. The concentrations of RIL 2 needed for the in vitro activation of the effectors in order to attain maximal ADCC was between 100 and 3000 U/ml and parallel the IL 2 concentrations required to generate LAK cells. ADCC mediated by IL 2-activated splenocytes was completely blocked by anti-FcR monoclonal antibodies. Although antisera directed against MHC antigens were used in most experiments, anti-B16 monoclonal antibodies have also shown the ability to induce ADCC mediated by RIL 2-activated syngeneic and allogeneic cells. Treatment of the precursor splenocyte populations with anti-asialo GM1 and complement eliminated the direct LAK activity and the antibody-dependent cytotoxicity, suggesting that both direct and indirect tumor cell lysis may be caused by the same effector cell. ADCC mediated by LAK cell populations represents another possible mechanism for the in vivo therapeutic effects of these cells.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Cytolytic T lymphocytes: an overview of their characteristics

Cloned T cells have been useful for assessing the lytic potential of distinct T cell subsets and for determining the relative contribution of different effector mechanism involved in the lytic process. Alloreactive CD8⁺ murine T cell clones and cloned murine CD4⁺ T_H1 and T_H2 T cells reactive with nominal antigen (ovalbumin) lysed nucleated target cells bearing antigen or coated with anti-CD3 monoclonal antibody in a short term⁵¹Cr-release assay. These clones were also evaluated for their ability to lyse efficiently sheep erythrocyte (SRBC) target cells coated with anti-CD3 mAb by a mechanism (presumably involving membrane damage) that does not involve nuclear degradation. Three patterns of lysis were observed: CD8⁺ and some CD4⁺ T_H2 effector cells lysed efficiently nucleated target cells and anucleated SRBC coated with anti-CD3 mAb. However, CD4⁺ T_H1 (and a few T_H2) T cells which lysed nucleated target cells bearing antigen or coated with anti-CD3 mAb didnotlyse efficiently the SRBC coated with anti-CD3 mAb. One CD4 bearing T_H2 cell failed to lyse efficiently either nucleated target cells or anucleated SRBC coated with anti-CD3 mAb. These results indicate that both T_H1 and T_H2 clones have lytic capabilities. Furthermore, they suggest that some but not all T_H2 murine T cell clones have lytic characteristics similar to those of conventional CD8⁺ CTL. However, it is not certain how these patterns of lysis of target cellsin vitro relates to the capacity of CTL to lyse such target cellsin vivo.     

Human leukocytes kill varicella-zoster virus-infected fibroblasts in the presence of murine monoclonal antibodies to virus-specific glycoproteins.

Seven murine monoclonal antibodies reacting with major glycoproteins of varicella-zoster virus were tested for functional activity in assays for antibody-dependent cellular cytotoxicity (ADCC) and antibody-plus-complement-mediated lysis. Human peripheral blood mononuclear cells killed varicella-zoster virus-infected fibroblasts in the presence of three of four monoclonal antibodies directed against gp98/62 and a single monoclonal antibody directed against gp118. Neither of two monoclonal antibodies directed against gp66 was able to mediate ADCC. In 18-h assays, adherent effector cells were more active than nonadherent effector cells in mediating ADCC. Adherent cells treated with anti-Leu-11b and complement retained their cytotoxic activity, suggesting that monocytes are responsible for most of the adherent-cell-mediated cytotoxicity. Both immunoglobulin G1 and G2a murine monoclonal antibodies were able to participate in ADCC. Of the two immunoglobulin G2a monoclonal antibodies tested, both of which reacted with gp98/62, only one mediated lysis in the presence of complement. These results indicate that some murine monoclonal antibodies against major glycoproteins of varicella-zoster virus have functional activity in cytotoxicity assays.     

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.     

Studies on the antibody-dependent cell-mediated cytotoxicity (ADCC) of thioglycollate-stimulated and BCG-activated peritoneal macrophages

Peritoneal macrophages (mφ), activated by Bacillus Callmette-Guerin (BCG) or elicited by thioglycollate broth in vivo in C57BL/6J mice, were compared with regard to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The BCG-activated mφ had several times higher ADCC activity than did the TG-mφ against erythroid targets. When human and murine tumor cells were employed as targets in the ADCC assay, the BCG-mφ lysed each of six separate target lines considerably, while the TG-mφ had little and, occasionally no, lytic activity against the six targets. Although F_c-dependent and -independent phagocytosis of the erythroid targets by the TG-mφ exceeded that by the BCG-mφ, this rapid and extensive phagocytosis did not account soley for the decreased lysis of the erythroid targets by the TG-mφ. The results indicate that BCG-activated peritoneal mφ, in comparision with TG-elicited inflammatory mφ, are potent effectors of ADCC against both erythroid and neoplastic target cells.     

Surface receptors and immune activity of purified human circulating mononuclear cells. III. The mechanisms of lysis, by lymphocytes and monocytes, of target cells in the lymphocyte-dependent and monocyte-dependent ADCC, NK, NOCC, and MICC cytotoxic reactions

The abilities of unfractionated mononuclear cells (MNC), monocytes (98-99% pure), and lymphocytes (98-99% pure) to carry out the lysis of target cells in the ADCC, NK, NOCC, and MICC assays were compared. Lymphocytes by themselves were able to lyse the CRBC (ADCC), K-562 (NK), and RRBC (MICC) target cells. The monocytes were very effective in the lysis of the CRBC (MICC) target cells. However, the lysis of two other target cells--RRBC (NOCC) and HRBC (ADCC)--required the simultaneous presence of both lymphocytes and monocytes in order to effect optimal lysis. Soluble factor(s) secreted by the cytotoxic cells capable of lysing the target cells were detected only in the NK assay. The activity of the soluble cytotoxic factor (NKCF) was only 25-40% of that exhibited by the cytotoxic NK cells and it was secreted by the cytotoxic cells after 48 hr of culture and not 24 hr of culture which is the usual assay condition. The NKCF was cytotoxic only to the NK target cells and not to the target cells used in the ADCC, NOCC, and MICC cytotoxic assays. Different classes of lymphocytes were cytotoxic in the monocyte-independent assays [ADCC (CRBC), NK (K-562), and MICC (RRBC)]. The null lymphocytes and the T lymphocytes were the primary cytotoxic cells in the ADCC and MICC assays, respectively, whereas the T, B, and null cells were almost equally cytotoxic in the NK assay. With respect to the monocyte-dependent assays [ADCC (HRBC), NOCC (RRBC), and MICC (CRBC)], the cytotoxic activity of any one class of lymphocytes failed to approach that of the unfractionated MNC. The T cells were the most cytotoxic; the B cells exhibited limited cytotoxic activity in only the ADCC assay and the null cells showed no cytotoxic activity. However, the combination of T and non-T cells and, to a lesser extent, T and B cells, exhibited much greater cytotoxic activity than the individual cells and together were as cytotoxic as the unfractionated MNC. It is concluded that, depending upon the selection of the target cells, lysis in the ADCC, NK, NOCC, and MICC assays may be effected by lymphocytes only, by monocytes only, by both monocytes and lymphocytes, or as a result of lymphocyte-monocyte collaboration. In the latter instance more than one class of lymphocytes must be present in order for maximum cytotoxic activity to be expressed.     

Definition of a secondary target cell trigger during natural killer cell cytotoxicity: possible role of phospholipase A2

Phospholipase A2 (PA-2) is known to be involved in many calcium-dependent cellular processes and inhibitors of PA-2 have been shown to inhibit natural killer cell-mediated cytotoxicity (NK CMC). Since the trigger stage is calcium dependent, it was postulated that this effector cell-associated enzyme may play a role in early calcium-dependent processes. To define how PA-2 might be involved in NK lysis, the effect of both PA-2 inhibitors and exogenous PA-2 on the stages of NK lysis was examined. PA-2 inhibitors, quinacrine and p-bromophenacyl bromide, inhibited NK CMC at the effector cell level, but affected neither initial target-effector cell binding nor dissociated conjugates during the length of the NK assay, suggesting that they block post-binding lytic events. A calcium pulse assay showed that PA-2 inhibitors inhibit only moderately when added after calcium and only within the first 15 min, demonstrating that these inhibitors blocked very early post-binding lytic events. Because this very early post-binding inhibitory effect was consistent with effects upon the NK trigger mechanism, the effect of exogenous PA-2 on NK lysis was tested. Pretreatment of K562 target cells but not pretreatment of peripheral blood lymphocytes (PBL) with 20 units/ml PA-2 enhanced lysis by two to eight-fold (based upon lytic units), showing its enhancing effect to be at the target cell level. Single cell assays using effector cells purified by indirect panning with monoclonal antibody NKH-1 showed that only the number of killer cells was increased. Calcium pulse assays showed that enhancement of lysis was maximum 15 min after addition of calcium and decreased rapidly thereafter, demonstrating its effect at an early post binding stage. Additionally, PA-2 was shown to overcome inhibition by the monoclonal antibody 13.3, which has been shown to affect the trigger stage of NK lysis (post-binding but prior to calcium dependent events). Thus, it appears that an NK cell-associated PA-2 could function by modulating the target cell surface, revealing a structure which acts as a "secondary" trigger, subsequent to the 13.3 "trigger", requisite for activation of the NK lytic process.     

Augmentation of antileukemia lytic activity by OKT3 monoclonal antibody: synergism of OKT3 and interleukin-2

We have shown that short-term incubation (45 min) of peripheral blood lymphocytes of normal donors with OKT3 monoclonal antibody (MoAb), directed against T-cell-associated antigen CD3, resulted in an acquisition of lytic activity against fresh leukemic cells. Induction of such antileukemia activity was specific for OKT3, since Leu-1 MoAb (directed against another T cell surface molecule, CD5) did not induce a lytic effect. The OKT3-generated antileukemia effect was displayed against various types of leukemia including chronic myelogenous leukemia and acute myelogenous leukemia of various histological subtypes (M1, M2, M5). We furthermore demonstrated that OKT3 MoAb substantially enhanced leukemia killing by interleukin-2 (IL-2)-activated killer cells obtained from peripheral blood of patients with leukemia. Of most importance was the observation that the combined treatment of effector cells with IL-2 and OKT3 MoAb resulted in the highest levels of lysis of both autologous and allogeneic fresh leukemic cells that have been observed in leukemic patients to date. Of importance was to note that OKT3 treatment was effective in induction of cytotoxic activity also in patients whose effector cells were unresponsive to stimulation with IL-2 alone. All of these observations suggest that IL-2-activated and OKT3-MoAb-treated effector cells may represent the most aggressive population of antileukemia-directed killer cells and may play a significant role in the treatment of human leukemia.     

Adjuvant and immunogenic properties of bacterial lipopolysaccharide in IgE and IgG1 antibody formation in mice

The ability of Gram-negative lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in IgE and IgG₁ immune responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG₁ under a variety of experimental conditions. Both isolated LPS and whole heat-killed bacteria were capable of enhancing IgE and IgG₁ antibody formation to a protein antigen, egg albumin (EA). The LPS-induced anti-EA, IgE, and IgG₁ antibody titers exhibited a cycling phenomenon with time. In the presence of LPS, IgE, and IgG₁ antibodies specific for EA did not occur in athymic nude (BALB/c-nu/nu) mice, demonstrating the inability of LPS to substitute for the stringent requirement for T cells in homocytotropic antibody formation.     

Interleukin-4 augments the cytotoxic capacity of lymphocytes and monocytes in antibody-dependent cellular cytotoxicity

Summary Human peripheral blood mononuclear cells (lymphocytes and monocytes) (PBMC) were preincubated for 0–24 h with human recombinant interleukin-4 (IL-4) and used as effector cells in an 18 h antibody-dependent cellular cytotoxicity (ADCC) assay with mAb 17-1A (mouse IgG2A) against SW948 (a human colorectal carcinoma cell line). A statistically significant increase in the lytic capability was noted after 2–24 h of preactivation. IL-4 at 1 ng/ml induced the highest cell lysis while higher and lower concentrations were inferior or had no effect at all. Preactivation for 24 h induced a more effective lytic cell population than 2 h prestimulation: 63 LU (lytic units)/10⁶ cells vs 42 LU/10⁶ cells. Pretreatment with 1 ng/ml IL-4 for 2 h induced a statistically significant increase in the ADCC activity of PBMC (P <0.05), of monocytes (P <0.01) and E-rosette-negative cells (natural killer cells) (P <0.05) compared to non-activated cells. IL-4 did not induce lymphokine-activated killer activity of PBMC against SW948. The spontaneous cytotoxicity against K562 was, however, increased after stimulation with 1 ng/ml IL-4 for 2 h of E-rosette-negative non-adherent cells.     

Production of Monoclonal Antibodies to Guinea Pig Liver Transglutaminase

Monoclonal antibodies are now a powerful tool in biology and medicine. Transglutaminase has been implicated in diverse biological functions, and the characteristics of its catalytic action are suitable for applied enzymology. In this study, we produced hybridoma cells which synthesize monoclonal antibodies against guinea pig liver transglutaminase by fusing mouse myeloma cells with spleen cells of mouse immunized with the enzyme protein. Eight hybridoma clones (coded 2F, 4B, 7C, 8B, 8D, 8E, 9F and 11C) were selected to produce monoclonal antibodies. The subclass of IgG produced by clone 9F was IgG_2a and those from the seven other clones were all IgG₁ The 9F antibody inhibited transglutaminase activity, but the other antibodies did not. A solid-phase antibody-binding assay showed that of these antibodies, 8D antibody has the highest affinity to the antigen. Transglutaminase protein in crude liver extract was identified with Western blotting analysis using 8D antibody as the probe.     

Stabilization and functional studies of high-molecular-weight murine lymphotoxins

High levels of lymphotoxin-like activity (LT) were found in supernatants from secondarily stimulated immune mouse splenocytes activated with concanavalin A (Con A) in vitro. Splenocytes obtained from C57Bl/6 mice immune to the P815 mastocytoma were restimulated in vitro with mitomycin C-treated P815 cells, and then stimulated with Con A. High levels of unstable LT activity are rapidly (2–4 hr) released by these lectin-stimulated splenocytes. The introduction of a crosslinking agent, glutaraldehyde, was found to stabilize this LT activity and allowed us to perform more defined biochemical studies and to examine the functional activities of the LT classes. The lytic activity in these supernatants resided in the high-molecular-weight classes, termed Complex (Cx > 200,000 daltons) and alpha-heavy (α_H 130,000–160,000 daltons). It was found that the Cx and α_H LT classes from the secondarily stimulated immune splenocytes cause lysis of allogeneic target cells, P815 and EL-4, in a 16-hr ⁷⁵Semethionine release assay, and in some cases, this lysis was specific for the sensitizing target cell.     

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.     

Purification of mouse monoclonal anti-HCG IgG1 from ascites fluid by hydroxylapatite chromatography

Summary Mouse monoclonal IgG₁ antibody to human chorionic gonadotropin from ascites fluid was purified by one-step hydroxylapatite chromatography. The purified monoclonal antibody is over 90% yield and essentially free of contaminating mouse IgG found in ascites fluid.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

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.