The heterocytotoxicity of human serum. II. Role of natural antibody and of the classical and alternative complement pathways.

Mouse lymphoid cells and tumor cell lines were employed as target cells for the investigation of the mechanism of heterocytotoxicity in human serum samples. It was shown that the heterocytotoxic effects were due to two differing mechanisms. Cytotoxicity was mediated in part, by activation of the alternative complement pathway on target cell membrane, a process which was antibody-independent. A second mechanism of cytotoxicity was induced by natural antibodies to a target cell, which probably mediated activation of the classical complement pathway. These data may shed light on the frequently observed cytotoxicity in mammalian sera for various target cells.     

Differential recognition of tumor-derived and in vitro Epstein-Barr virus-transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T-lymphocyte clones

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.     

Polymorphonuclear neutrophil-mediated antibody-dependent cell cytotoxicity of herpesvirus-infected cells: ultrastructural studies

Bovine polymorphonuclear neutrophils (PMN) can mediate antibody-dependent cell cytotoxicity (ADCC) of herpesvirus-infected cells. Since cytotoxicity occurs only in the presence of PMN and specific antiviral antibody, but not until viral membrane antigens are expressed on the target cell, it is concluded that antibody must recognize viral membrane antigens before cytotoxicity can occur. Cytotoxicity also requires very close contact between the target cell and the PMN cell. These interactions occur as early as 1 h after incubating antibody, infected cells, and PMN, but the actual lysis and release of intracellular components occur over an extended period. It was assumed that degranulation was not involved in the initiation of cytotoxicity, but was involved in the final stage of destruction. The mechanism of lysis is proposed to involve the interaction of PMN membranes with target cell membranes with subsequent reorganization and activation of the PMN plasma membrane at points of contact with the target cell. This results in possible production of transmembrane channels which allows for the release of target cell contents.     

Resistance of multidrug-resistant lines to natural killer-like cell-mediated cytotoxicity

Multidrug resistance (MDR) refers to a complex phenotype that describes a number of features characterized primarily by resistance to a wide range of structurally unrelated drugs. In this paper we investigated the relationship between drug resistance and resistance to NK-mediated cytotoxicity. Studies with two independently selected multidrug-resistant cell lines indicated that increased drug resistance was associated with both an increased resistance to NK-mediated cytotoxicity and increased levels of membrane P-glycoprotein expression. This resistance to cytotoxicity appears to result partly from an alteration in the membrane structure of the target cells inasmuch as there was a reduction in effector:target cell recognition. Resistance to NK-mediated cytotoxicity should be included with the numerous pleiotropic changes associated with the multidrug resistance phenotype.     

Induction of nonspecific cytotoxicity by monoclonal anti-T3 antibodies

The effects of monoclonal anti-T3 antibodies on the effector phase of cytotoxic T lymphocytes (CTL) were studied with respect to antigen-specific and antigen-nonspecific lysis of different target cells. Anti-T3 antibodies inhibited the antigen-specific lysis by CTL generated in mixed lymphocyte cultures (MLC), but they concomitantly augmented the nonspecific killing of third-party cells such as the cell lines Daudi, Raji, and K562. This nonspecific cytotoxicity was induced by various anti-T3 antibodies, whereas antibodies reactive with other antigens expressed on the cytotoxic effector cells lacked any such activity. Anti-T3 antibodies induced nonspecific cytotoxicity only when activated T cells, obtained by primary MLC, by repeated restimulation, or after cloning, were used. The antibodies had no effect on unstimulated peripheral T lymphocytes or thymocytes. The inhibition of the antigen-specific lysis and the induction of nonspecific lysis by anti-T3 was dose dependent, and both effects occurred at the same concentration range of anti-T3. F(ab')2 fragments of anti-T3 inhibited the specific lysis but were not able to induce cytotoxic activity, indicating that this induction is an Fc-dependent process. When different target cells were tested, only Fc receptor-positive cells were susceptible for this nonspecific cytotoxicity. Thus, anti-T3 antibodies have a dual effect on effector CTL: they inhibit antigen-specific lysis and concomitantly induce nonspecific lysis in an Fc-dependent way.     

Effect of halothane on lung carcinoma cells A 549

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.     

Apoptotic activity of frog Bombina maxima skin albumin

Albumin, the most abundant protein components of blood plasma, is synthesized and secreted by liver cells in vertebrates. Recently, it was demonstrated that frog Bombina maxima albumin is also expressed in skin. Both B. maxima albumins from skin and serum (BmA-skin and BmA-serum) have similar biochemical characteristics except that the former contains haem b. Present studies showed that BmA-skin exhibited cytotoxic activity on H9 and C8166 cells. Pretreated with hemin to induce erythroid differentiation, K562 cells lost their resistance to cytotoxicity of BmA-skin. After treating cells with BmA-skin for 48 h, 50 percentage cytotoxic concentrations (CC(50)) of BmA-skin on H9, C8166 and hemin-treated K562 cells were 1.31+/-0.09, 1.59+/-0.08 and 2.28+/-0.06 microM, respectively. The cell death induced by BmA-skin was mediated by apoptosis of the tested cell lines, as demonstrated by nuclear morphological changes, DNA fragmentation and DNA hypodiploidy of apoptosis cells. At BmA-skin concentration of 2 microM, 27.3%, 19.7% and 17.8% of H9, C8166 and hemin-treated K562 cells were found to be apoptotic. In contrast, BmA-serum possessed no cytotoxic and apoptosis-inducing activity on all the cell lines tested, even with concentration used up to 15 microM. These results indicated that bound haem b in BmA-skin contributed significantly to its cytotoxic and apoptosis-inducing activity on the cell lines assayed.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. I. Activation by tuberculin and by Staphylococcus filtrate

Normal lymphocytes activated by mitogens such as phytohemagglutinin (PHA), by Staphylococcus filtrate (SF), or lymphocytes from sensitized individuals stimulated by antigen (PPD, etc.) are cytotoxic to tissue culture cells of different origins. In this and the following paper, the results of a detailed quantitative analysis of the specificity of this cytotoxic reaction are presented. Effector cells were human or mouse lymphocytes, activated by PHA, SF, PPD, or serum factors in the culture medium. Cells from established cell lines of human, mouse, hamster, or rabbit origin, or primary human or rat embryonic fibroblasts were used as target cells. Lysis was quantitated by release of ⁵¹Cr from labeled target cells.Purified human blood lymphocytes, activated by PPD, SF, or otherwise, preferentially damaged allogeneic target cells. Lysis of xenogeneic target cells was weak or did not occur. A close correlation was noted between target cell destruction and blastoid transformation of the lymphocytes, but the slope of the regression lines of xenogeneic cytotoxicity was much smaller than that of allogeneic cytotoxicity when plotted as a function of blastoid transformation.Lymph node or spleen cells from CBA mice were stimulated by PPD to transformation and DNA synthesis. CBA lymphocytes also showed an increased degree of blast transformation in medium containing fetal calf serum or certain batches of fresh human serum. Mouse lymphocytes activated in these ways damaged allogeneic L cells but had no effects on xenogeneic Chang cells.These results indicate that lymphocytes activated by various means preferentially damage target cells from their own species. The recognition mechanisms which determine the specificity of the reactions are not known.     

Novel hyaluronic acid (HA) coated drug carriers (HCDCs) for human breast cancer treatment

Hyaluronic acid (HA) coated drug carriers (HCDCs) were successfully synthesized by chemical conjugation method for targeted delivery of doxorubicin (DOX) as a prototype anticancer drug to CD44 expressed human breast cancer cell. From XPS analysis, the HCDCs by conjugation methods demonstrated the superior HA fixation amount and colloidal stability compared with the nanoparticles by nanoprecipitation. The cytotoxicity of the HCDCs formulation accessed by the MTT assay against the higher CD44 expressed cell line (MDA-MB-231) and lower CD44 expressed cell line (ZR-75-1) human breast cancer cell lines demonstrated that the HCDCs formulation exhibited excellent tumoricidal effect and their affinity to cancer cells was predominant. The in vitro drug release profile of the HCDCs showed sustained release behavior and after 14 days, 80% of the encapsulated DOX was released due to a high release rate of DOX from HCDCs. We synthesized that HCDCs have therapeutic potentials of cancer as a target specific fashion by increasing the tumoricidal efficacy of targeted cancer cells while reducing their cytotoxicity of non-targeted cells to minimize the side effect.     

Mechanism of cytotoxicity induced by chimeric mouse human monoclonal antibody IDEC-C2B8 in CD20-expressing lymphoma cell lines

With a new flow cytometric cytotoxicity assay, we examined the mechanism of action of chimeric mouse human anti-CD20 monoclonal antibody IDEC-C2B8. IDEC-C2B8 alone induced direct cytotoxicity in four of eight examined CD20-expressing lymphoma cell lines (RAJI, DAUDI, JOK-1, and WT100) at a concentration above 100 ng/ml. Moreover, after 4 h incubation in human serum, only a moderate complement-dependent cellular cytotoxicity (CDCC) was observed, whereas cytotoxicity increased markedly after 3 days of culture, indicating that combined direct cytotoxicity and CDCC were responsible. IDEC-C2B8 induced an effective antibody-dependent cellular cytotoxicity (ADCC) in seven of eight tested lymphoma cell lines when peripheral blood mononuclear cells were used as effector cells. ADCC was moderately enhanced by cytokine interleukin-2, whereas interleukin-12, interferon-alpha, and GM-CSF had no influence. Interestingly, we could demonstrate a correlation between CD32 expression on lymphoma cell lines and IDEC-C2B8-induced direct cytotoxicity, indicating that crosslinking of CD20 with CD32 may be involved in the mechanism of cytotoxicity. We propose that direct cytotoxicity, CDCC, and ADCC result in the marked elimination of CD20-expressing tumor cells observed after treatment with IDEC-C2B8.     

Multiple mechanisms of target cell disintegration are employed in cytotoxicity reactions mediated by human natural killer cells

The rate of disintegration of target cells subsequent to lytic programming by human peripheral blood natural killer (NK) cells was investigated using a quantitative calcium pulse technique. The rate of this initial calcium-independent target cell disintegration was indicative of a first-order decay process for programmed target cells with a calculated half-life of less than 3 min. This initial, rapid disintegration phase was independent of the overall cytotoxic activity of the lymphocyte preparation tested. Moreover, initial rates of target cell disintegration were comparable for target cell lines that exhibit up to 6-fold differences in overall susceptibility to natural cytotoxicity. In these studies we also consistently observed very slow, calcium-independent disintegration of additional target cells following apparent completion of the rapid disintegration process. Using a 51Cr release assay and K-562 target cells, the kinetics of this slow disintegration process were examined and found to be similar for donors exhibiting up to a 2-fold difference in overall cytotoxic activity and independent of the concentration of programed target cells. Whereas the initial rapid disintegration mechanism was independent of temperature over the range of 10-37 degrees C, the slow disintegration mechanism exhibited a direct dependence on the incubation temperature. Furthermore, we observed that supernatants obtained after the termination of lytic programing by ethylene diaminetetraacetic acid could effect the slow lysis of fresh NK-susceptible target cell lines. These results support the utilization of at least two distinct mechanisms for target cell lysis by human NK cells.     

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

The monoclonal antibody CJA3 down-regulates the susceptibility of human tumor cell lines to natural cell-mediated cytotoxicity

While developing monoclonal antibodies (MoAb) to colorectal carcinoma (CRC) cells, we noted that one MoAb, termed CJA3, down-regulated natural cell-mediated cytotoxicity (NCMC) against CRC cell lines SW480 and SW620. The MoAb CJA3 was developed by immunizing a BALB/c mouse with fresh human colorectal adenocarcinoma cells. The antigen recognized by the MoAb CJA3 was expressed on several solid tumor cell lines and on one of the six lymphoreticular cell lines tested, but was not detected on normal peripheral blood lymphocytes (PBL). SDS-PAGE analysis of the antigen immunoprecipitated by the MoAb CJA3 from the CRC cell lines SW480 and SW620 and from the melanoma cell line MALME-3M revealed a component with a m.w. of 150,000. Preincubation of CRC cell lines SW480 and SW620 with the MoAb CJA3 for 16 hr reduced their susceptibility to NCMC by about 50%. Kinetic experiments showed that prolongation of the incubation of target cells with the MoAb CJA3 resulted in a time-dependent increase in the amount of MoAb bound. Maximum binding of the MoAb CJA3 was reached after 4 hr of incubation. The increase in antigen expression chronologically paralleled the decrease in NCMC target cell sensitivity, suggesting that the membrane alterations induced by the MoAb CJA3 were important for NCMC against these two cell lines.     

Identification of three major target molecules of IgM antilymphocyte autoantibodies in systemic lupus erythematosus

Three cell lymphocyte antigens of m.w. 55,000, 70,000, and 105,000 to 110,000 were identified by Western blotting as targets of IgM autoantibodies in serum from a group of 49 patients with systemic lupus erythematosus. The 55- and 70-kDa antigens were well expressed on unstimulated peripheral T cells, whereas the 105- to 110-kDa target was demonstrable only on mitogen-activated T cells and lymphoblastoid T cell lines. Localization of these molecules to the plasma membrane was established by cytoabsorption experiments in which IgM antibody staining of blotted antigens was specifically absorbed from systemic lupus erythematosus serum during 4 degrees C incubations with intact lymphocytes, and by their detection in purified lymphocyte plasma membranes. While the identity of these target antigens vis a vis known surface determinants was not defined, their expression on peripheral T cells from multiple donors and on cell lines of both undifferentiated (HSB-2) and phenotypically mature (Jurkat; HUT 78) types excluded alloantigens, major histocompatibility complex-encoded determinants, and most T cell differentiation antigens as candidates in this regard. Expression of the IgM autoantibody targets on HSB-2 cells argues against discrete T subset specificities as well. IgM reactivity with the 55-, 70-, and 105- to 110-kDa antigens by blotting was highly correlated with antilymphocyte antibody activity in complement-dependent cytotoxicity assays (Fisher's p less than 0.001), and paralleled flow microfluorimetric and microcytotoxicity quantitation of IgM antibody activity in serial observations of individual patients studied during different phases of disease activity. Taken together, these data suggest that IgM lymphocytotoxic antibodies in systemic lupus erythematosus are directed predominantly against a limited number of non-T cell subset-specific antigens.     

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder

Summary Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8⁺/CD4^–). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.     

Human alveolar macrophage FcR-mediated cytotoxicity. Heteroantibody- versus conventional antibody-mediated target cell lysis

Human alveolar macrophage have three distinct receptors for IgG: FcRI, FcRII, and FcRIII. In order to compare the ability of these receptors to mediate target cell lysis, three different assay systems were examined. First, we studied lysis of chicken E (CE) opsonized with heteroantibodies, which are synthetic antibodies composed of Fab fragments with anti-FcR activity covalently linked to Fab fragments with anti-CE activity. We found alveolar macrophage readily lysed heteroantibody-opsonized CE via each of the three FcR classes (FcRI, 20 +/- 5%; FcRII, 27 +/- 7%; and FcRIII, 13 +/- 13%, p less than 0.05). Non-FcR-dependent lysis of anti-beta 2-microglobulin x anti-CE heteroantibody-opsonized CE was not detected. Second, lysis of hybridoma cell lines bearing anti-FcR antibodies on their cell surface was examined to assess killing of "tumor-like" target cells. Whereas peripheral blood monocytes and lymphocytes were able to lyse hybridoma cell lines bearing surface anti-FcR mAb, alveolar macrophages were not. Third, activity of alveolar macrophage FcR was examined in a conventional antibody-dependent cellular cytotoxicity assay by using O+ (R1,R2) human RBC opsonized with human anti-D and anti-CD serum as target cells. We found lysis of anti-D and anti-CD opsonized human RBC was mediated exclusively via FcRI. No activity of FcRII or FcRIII was detected in these latter assays even if performed under conditions that impair FcRI activity. Thus, all three FcR present on alveolar macrophage mediate lysis of heteroantibody-opsonized CE; in contrast, with the use of a conventional antibody-dependent cellular cytotoxicity assay, only FcRI activity was detected. We were unable to demonstrate lysis of anti-FcR-bearing hybridoma cell lines by alveolar macrophages.     

Potentiation of NK cytotoxicity by antibody-C3b/iC3b heteroconjugates

The interaction of two Burkitt lymphoma lines, Raji and Rael, with human C and NK cells was analyzed. Raji cells activate the alternative C pathway (ACP) and then bind C3 fragments. Consequently, the cells become more sensitive to lysis by CR3-bearing NK cells but not to C lysis. In contrast, Rael cells are poor ACP activators, do not bind C3 fragments, and are therefore resistant to C-dependent NK lysis. As suggested earlier, the difference between Raji and Rael could be attributed to the presence or absence of CR2, respectively, on their surface. To potentiate C- and NK-dependent lysis of target cells, we generated heteroconjugates composed of a murine antitransferrin receptor mAb and of human C C3b or iC3b. Antibody-C3b conjugates induced C3 deposition on Rael cells and elevated C3 deposition on Raji cells in human serum. Both Raji and Rael cells coated with antibody-C3b conjugates were efficiently lyzed by the cytolytic ACP in human serum. This conjugate had a small enhancing effect on target cell lysis by NK cells which could be markedly increased by combined treatment of the target cell with antibody-C3b conjugate and C5-depleted human serum. On the other hand, antibody-iC3b conjugates efficiently potentiated lysis of target cells by NK cells in the absence of serum. The iC3b-directed cytotoxicity was mediated by CR3-bearing NK effector cells. Anti-C3 but not anti-mouse Ig antibodies abrogated the activity of the antibody-iC3b conjugate. These results further demonstrate that NK cytotoxicity may be potentiated by opsonizing the target cells with C3 fragments and suggest that antibody-C3b/iC3b conjugates could be potent tools for targeting and potentiation of the lytic action of both C and NK cells against tumor cells.