Antibody-dependent Cytolysis Research Articles

Modulation of innate and adaptive cellular immunity relevant to HIV-1 vaccine design by seminal plasma.

Mucosal exposure to HIV-1 infection generally occurs in the presence of semen. Immunomodulation by seminal plasma is well described in the reproductive biology literature. Little is known, however, about the impact of seminal plasma on innate and adaptive anti-HIV-1 cellular immunity. The study investigated the effects of seminal plasma on immune responses considered important for prophylactic HIV-1 vaccine development, namely innate and adaptive cellular immunity mediated by natural killer (NK) cells and T cells, respectively. The ability of seminal plasma to modulate direct, antibody-dependent and cytokine-stimulated NK cell activation was assessed utilizing intracellular cytokine staining. Direct and antibody-dependent cellular cytotoxicity was assessed using lactate dehydrogenase release assays. The effects of seminal plasma on T-cell activation upon stimulation with staphylococcus enterotoxin B or HIV-1 Gag peptides were assessed by intracellular cytokine staining. The impact of seminal plasma on redirected cytolysis mediated by T cells was measured using lactate dehydrogenase release assays. Both direct and antibody-dependent NK cell activation were dramatically impaired by the presence of either HIV-1-uninfected or HIV-1-infected seminal plasma in a dose-dependent manner. Additionally, seminal plasma suppressed both direct and antibody-dependent NK cell-mediated cytolysis, including anti-HIV-1 antibody-dependent cytolysis of gp120-pulsed CEM.NKr-CCR5 cells. Finally, seminal plasma attenuated both HIV-1 Gag-specific and staphylococcus enterotoxin B-induced CTL activation. Semen contains potent immunosuppressors of both NK cell and CD8 T-cell-mediated anti-HIV-1 immune responses. This could impede attempts to provide vaccine-induced immunity to HIV-1.

IFITM1 targets HIV-1 latently infected cells for antibody-dependent cytolysis.

HIV-1 persistence in latent reservoirs during antiretroviral therapy (ART) is the main obstacle to virus eradication. To date, there is no marker that adequately identifies latently infected CD4+ T cells in vivo. Using a well-established ex vivo model, we generated latently infected CD4+ T cells and identified interferon-induced transmembrane protein 1 (IFITM1), a transmembrane antiviral factor, as being overexpressed in latently infected cells. By targeting IFITM1, we showed the efficient and specific killing of a latently infected cell line and CD4+ T cells from ART-suppressed patients through antibody-dependent cytolysis. We hypothesize that IFITM1 could mark natural reservoirs, identifying an immune target for killing of latently infected cells. These novel insights could be explored to develop clinical therapeutic approaches to effectively eradicate HIV-1.

Selective Targeting of T and B Cell Populations by Alemtuzumab in the Treatment of Multiple Sclerosis

Upstream targeting of both T and B cells is a rational therapeutic approach in multiple sclerosis (MS) in view of how both cell types and the interaction between them contribute to MS pathophysiology. This article will discuss this new way of thinking in MS: the targeting of both T and B cells, with a focus on the recently developed therapy, alemtuzumab (Lemtrada®, Genzyme, UK). Alemtuzumab depletes T and B lymphocytes, mainly via complement-dependent cytolysis and antibody-dependent cytolysis; depletion of B cells is not an enduring effect compared with the depletion of T cells. After dosing, CD4+ and CD8+ T cells and CD19 B cells decrease initially but increase over the following 11 months. During repopulation after alemtuzumab treatment, there is a shift in the relative proportions of T cell and B cell subsets whereby proportions of regulatory T cells and memory-phenotype T cells are increased and the proportion of naive T cells is decreased. A switch from a pro- to an anti-inflammatory phenotype and cytokine profile caused by alemtuzumab may underpin the long-lasting suppression of MS activity that has been observed in clinical trials. Alemtuzumab treatment is also associated with a consistently good safety and tolerability profile. Further, alemtuzumab appears to promote neurorehabilitation by improving measures of physical functioning, disability, measures of quality of life, and brain volume loss. Alemtuzumab therefore has the potential to reduce disease burden and improve substantially the prognosis for patients with MS.

A cell permeant peptide containing the cytoplasmic tail sequence of Fc receptor type IIA reduces calcium signaling and phagolysosome formation in neutrophils

Receptors for the Fc domain of IgG mediate target recognition, signal transduction, and effector functions including antibody-dependent cytolysis, phagocytosis, and phagolysosome formation. To better understand FcR-mediated functions and to identify potential therapeutic strategies, we employed cell-penetrating (“Trojan”) peptides to deliver “wild-type” (LTL) or modified (AAA) FcγRIIA tail sequences to the neutrophil’s cytoplasm. The Trojan-LTL peptide appeared to label the endoplasmic reticulum whereas the Trojan-AAA peptide distributed throughout the cytoplasm. The Trojan-LTL peptide, but not the Trojan-AAA peptide, decreased Ca 2+ signaling at the phagosome and reduced phagolysosome formation. These studies suggest that FcγRIIA’s tail can act as a peptide decoy thereby blunting FcγRIIA-mediated processes, which, in turn, suggests a possible route in managing inflammatory tissue damage.

Monoclonal LYM-1 antibody-dependent cytolysis by human neutrophils exposed to GM-CSF: auto-regulation of target cell attack by cathepsin G.

Murine monoclonal antibody (mAb) Lym-1 is an immunoglobulin G2a specific for certain human leukocyte antigen-DR variants expressed on the surface of malignant B cells. It has been proposed for serotherapy in patients with B lymphomas. We have previously shown that mAb Lym-1 synergizes with granulocyte macrophage-colony stimulating factor to promote Raji B-lymphoid cell lysis by human neutrophils via the intervention of neutrophil Fc receptors type II and D-mannose-inhibitable interactions between CD11b-CD18 integrins and CD66b glycoproteins. Here, we provide evidence that the process is oxygen-independent by inference related to the release of primary granules and is regulated by cathepsin G activity. The lysis was indeed reproduced by replacing normal neutrophils with cells from three patients suffering from chronic granulomatous disease, i.e., neutrophils genetically incapable of generating oxidants. Moreover, the lysis was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin and by Z-glycyl-leucyl-phenyl-chloromethyl ketone (Z-Gly-Leu-Phe-CMK), which blocks cathepsin G. Conversely, the lysis was unaffected by N-methoxysuccinyl-alanyl-alanyl-prolyl-alanyl-CMK (MeOSuc-Ala-Ala-Pro-Ala-CMK; elastase inhibitor) and MeOSuc-Ala-Ala-Pro-valine (Val)-CMK, which inhibits elastase and proteinase 3. The ability of neutrophils, engaged in cytolysis, to release cathepsin G was proved by detecting this enzymatic activity spectrophotometrically and immunocytochemically. Moreover, inhibition of cathepsin G activity by concentrations of Z-Gly-Leu-Phe-CMK, incapable of affecting elastase activity, was found to reduce the release of elastase and myeloperoxidase from neutrophils under conditions similar to those used for cytolytic assays. These findings suggest that neutrophils auto-regulate their lytic efficiency by controlling the exocytosis of primary granules via their cathepsin G activity.

Monoclonal Lym-1 Antibody-Dependent Cytolysis by Neutrophils Exposed to Granulocyte-Macrophage Colony-Stimulating Factor: Intervention of FcγRII (CD32), CD11b-CD18 Integrins, and CD66b Glycoproteins

Murine monoclonal antibody (MoAb) Lym-1 is an IgG2a able to bind HLA-DR variants on malignant B cells and suitable for serotherapeutic approaches in B-lymphoma patients. We have previously shown that Lym-1 can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to trigger neutrophil cytolysis towards Raji cells used as a model of B-lymphoma targets. Here we provide evidence for the intervention of certain neutrophil receptors or surface molecules in this model of cell-mediated lysis. The lysis was completely inhibited by the anti-FcgammaRII MoAb IV.3 and unaffected by the anti-FcgammaRIII MoAb 3G8. This suggests that neutrophil cytolysis involves FcgammaRII without cooperation of this receptor with FcgammaRIII. Moreover, the lysis was inhibited by an anti-CD18 MoAb (MEM48) and by a MoAb specific for carcinoembryonic antigen (CEA)-like and glycophosphatidyl inositol (GPI)-linked glycoproteins (CD66b). Using an immunofluorescence staining procedure, cross-linking of CD66b induced the redistribution of CD11b on neutrophils with distinct areas of CD11b clustering via a process susceptible of inhibition by D-mannose. This is consistent with the ability of CD11b-CD18 and CD66b to undergo lectin-like physical interactions on the neutrophil surface. Such a type of interaction is presumably instrumental for neutrophil cytolytic activity in that the lysis was inhibited by D-mannose and enhanced by the MoAb VIM-12, which mimics the cooperation between CD11b and GPI-anchored molecules by specifically interacting with CD11b lectin-like sites. Therefore, the present results prove the absolute requirement for FcgammaRII in neutrophil GM-CSF/Lym-1-mediated cytolysis and, on the other hand, define the crucial role of CD66b and CD11b/CD18 in the expression of the cell lytic potential.

There is no relationship between the activities of mono- and polyclonal antirhesus immunoglobulinsin vitro andin vivo

Monoclonal anti-D antibodies induce lysis of rhesus-positive erythrocytes in the antibody-dependent cytolysis test with blood mononuclear cells, the activity of the best antibodies being 10–15 times higher than of polyclonal IgG. The activities of anti-D antibodies in the cytolysis test with monocytes and lymphocytes varied: only one monoclonal antibody was highly active in both tests, whereas polyclonal IgG caused no lysis of erythrocytes in the cytolysis test with monocytes but was active in the same test with lymphocytes. Monoclonal antibodies intravenously administered to rhesus-negative recipients after transfusion of rhesus-positive erythrocytes stimulated their clearance, but its rate was slower than with polyclonal antibodies. Erythrocytes sensitizedin vitro with monoclonal antibodies were completely removed from circulation within 3 h. It is not clear the results of which of the studied tests correlate with the capacity of anti-D immunoglobulins to block rhesus sensitization.

Antibody-dependent cytotoxic activity on human cancer cells expressing UK 114 tumor membrane antigen

The monoclonal antibody (P-3 mAb) and the rabbit immune sera (RIS) recognizing a 14 kDa perchloric acid soluble protein extracted from goat liver (UK 114), locate this antigen on the cell membrane of several human cancer cell lines. UK 114-positive cells (e.g. HT 29 and KATO VI cells) undergo antibody-dependent cytolysis bl vitro. In nu/nu mice bearing xenografted HT 29 cells, tumor growth was markedly impaired by peri-tumoral injection of anti-UK 114 antibodies. These experiments suggest that human tumors expressing UK 114 over the cell membrane may undergo antibody-mediated cytolysis and growth control.

Protection of mice against an influenza virus infection by oral vaccination with viral nucleoprotein incorporated into immunostimulating complexes.

Influenza A virus nucleoprotein (NP) was integrated into immunostimulating complexes (ISCOMs) after attachment of bacterial lipopolysaccharide to the antigen. Oral immunization with these NP-ISCOMs protected mice fully against an otherwise lethal challenge infection with an unrelated influenza virus subtype without the appearance of severe clinical signs or extensive pathological lesions in the lungs. Mice immunized with analogous bovine serum albumine-incorporated ISCOMs all died. After oral immunization, high titers of NP-specific antibodies, particularly IgA, could be detected in the bronchoalveolar fluid and in the blood serum. No cytotoxic lymphocytes could be demonstrated in the spleens or the lungs of vaccinated mice, and no anti-NP antibody-dependent cytolysis of infected host cells was mediated by complement or in the form of an antibody-dependent cell cytotoxicity. However, a vigorous delayed-type hypersensitivity reaction was produced after probing vaccinated animals with purified NP. No comparable protective immunity or antibody response was induced by a strictly intragastric administration of NP-ISCOMs. It appears, therefore, that the general and local immune response in the lungs was primarily stimulated through contact of NP-ISCOMs with the mucous membrane of the oro-pharyngeal cavity and that cytotoxic effects did not play a major role for the establishment of the protective immunity. Partial protection against a lethal challenge was observed in chickens immunized with NP-ISCOMs in the drinking water.

Chimeric mouse-human anti-carcinoma antibodies that mediate different anti-tumor cell biological activities

Two chimeric mouse-human antibodies, ING-1 (IgG1, kappa) and ING-2 (IgG1, lambda), have been constructed starting from anticarcinoma mouse hybridomas. These antibodies bind to different tumor-associated antigens which are present on human breast carcinoma cell lines at 10(5)-10(6) antigens/cell; ING-1 binds to a 40-kD membrane glycoprotein, while ING-2 binds to a glycoprotein of greater than 300 kD. In competitive binding experiments, both chimeric antibodies have identical binding activity to the parental mouse antibodies. The antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytolysis (CDC) activities of these antibodies were studied on carcinoma target cell lines. ING-1 mediates potent ADCC, but ING-2 had undetectable or very weak ADCC activity. ING-2 ADCC activity was significantly reduced by the addition of human serum, but ING-1 ADCC was unaffected. Neither ING-1 nor ING-2 mediated CDC of breast carcinoma cell lines, but ING-1 mediated CDC of a colon carcinoma cell line. ING-1 antibody-antigen complexes are stable on the target cell surface for at least 2 hours, while much of bound ING-2 is lost from the surface of cells due to internalization or shedding. The activities of these antibodies confirm that the target antigen plays an important role in the biological effector functions triggered by cell-surface-bound antibodies. Both of these chimeric antibodies are candidates for further study as immunoconjugates for cancer diagnosis or therapy, and the unconjugated ING-1 antibody has promise for cancer therapy due to its potent activation of ADCC.

Impaired Immunity in AIDS

The inability of CD4+ T cells of HIV-1-infected patients to mount an effective immune response is widely believed to explain the increased susceptibility of these patients to opportunistic infections. Although the full explanation for T-cell dysfunction in HIV-1 infection is not yet understood, at least two fundamentally distinct mechanisms are thought to contribute: depletion of CD4+ T cells and qualitative CD4+ T-cell dysfunction independent of T-cell depletion. Many HIV-1-infected patients manifest reduced T-cell responses to recall antigens prior to measurable CD4+ T-cell depletion, and among the proposed explanations for this phenomenon are gp120-mediated interference with T-cell activation by way of inhibition of CD4-class II major histocompatibility complex (MHC) determinant interactions, gp41-mediated inhibition of protein kinase C-dependent T-cell activation, formation of gp41 cross-reactive antibodies that react with MHC class II determinants, transforming growth factor-beta (TGF-beta)-mediated immunosuppression, and decreased functions of antigen-presenting and antigen-processing cells (macrophages and bone marrow-derived dendritic cells). Despite their detection in most HIV-1-infected patients, these qualitative T-cell defects do not herald the onset of life-threatening disease. The appearance of severe clinical manifestations of AIDS, particularly opportunistic infections, occurs primarily in patients whose CD4+ T-cell count is significantly reduced. Depletion of CD4+ T cells may be a direct consequence of HIV-1 infection that occurs as a result of syncytia formation, autoantibody-mediated cytolysis, gp120-specific antibody-dependent cytolysis, and/or gp120-specific T-cell mediated cytolysis. The thymus is severely affected in patients with late-stage disease, and although there is no proof that the failure of the thymus to regenerate new T cells contributes to T-cell depletion in patients with AIDS, the likelihood seems high that this is the case. Indeed, if prolonged suppression of HIV-1 replication can be achieved with newer anti-HIV drugs or combinations of drugs, reconstitution of a normal immune system seems likely, provided that the capacity to regenerate T cells has not been irrevocably lost as a consequence of viral infection. In summary, available evidence indicates that HIV-1 uses a complex array of mechanisms to disrupt T-cell mediated immunity, but because most of these involve a direct role for HIV-1 proteins, such mechanisms are likely to be reversible if suppression of HIV-1 replication can be achieved.

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.

Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.

Biologic Properties In Vitro of a Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM-CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM-CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.

Tumors undergoing rejection induced by monoclonal antibodies of the IgG2a isotype contain increased numbers of macrophages activated for a distinctive form of antibody-dependent cytolysis.

Monoclonal antibodies of the IgG2a isotype specifically inhibit the growth of human tumors in nude mice; tumors in mice given no antibody, an antibody of the IgG2a isotype that does bind to the tumor cells, or an antibody of the IgG2b isotype grow progressively. In the present work it is demonstrated that tumors of mice given the IgG2a antibody are essentially masses of necrosis, while tumors from the control mice are sheets of healthy tumor cells. Tumors of the treated mice, in comparison to controls, contain an increased number of macrophages, and these macrophages are activated for a distinct form of cytolysis dependent on antibodies of the IgG2a isotype. The data suggest that changes in the number and function of intratumoral macrophages are a major component of tumor destruction mediated by IgG2a antibodies.

Antibody-dependent cytolysis (ADCC) of tumor cells by activated murine macrophages is a two-step process: Quantification of target binding and subsequent target lysis

To analyze the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction between tumor cells and activated murine macrophages in detail, it must be first determined if physical binding occurred between the two cell types. Over 15–20 min in vitro, antibody-coated HSB neoplastic targets became so firmly attached to the activated macrophages that they resisted removal with 4 vigorous washes. When a quantitative assay of binding was employed, attachment of tumor cells to activated macrophages was found to depend on the concentration of antibody and on the density of the macrophages. These two variables also determined the subsequent extent of cytolysis. Binding of antibody-coated targets by macrophages elicited with thioglycollate broth or activated by bacillus Calmette-Guerin (BCG) was comparable. Lysis by the activated macrophages, however, was far greater. Binding occurred at 4, 22, or 37 °C, while the subsequent lytic reaction occurred only at 37 °C. Thioglycollate broth effectively inhibited lysis but had no effect on binding. A porous filter placed between activated macrophages and targets resulted in abrogation of binding and lysis, even when antibody-coated targets were placed beneath the filters. When labeled, uncoated targets were added to cultures of macrophages in the presence of unlabeled antibody-coated targets, no lysis of the bystander (i.e., uncoated) targets was seen. The data suggest that ADCC is a multistep reaction, that vigorous physical binding of antibody-coated targets by activated macrophages is an initial and necessary step in ADCC, that such binding is not sufficient for ADCC, that such binding controls the selectivity of lysis in ADCC, and that the second step in ADCC results in target lysis.

Mechanisms of lectin and antibody-dependent polymorphonuclear leukocyte-mediated cytolysis.

The mechanisms of tumor lysis by polymorphonuclear leukocytes (PMNs) were investigated. In antibody-dependent PMN-mediated cytolysis (ADPC), sensitized tumor cells were specifically lysed via Fc receptors on PMNs. On the other hand, lectin-dependent PMN-mediated cytolysis (LDPC) caused nonspecific lysis of several murine tumors after recognition of carbohydrate moieties on the cell membrane of both PMNs and tumor cells. Both ADPC and LDPC depended on glycolysis, and cytotoxicity was mediated by reactive oxygen species; LDPC was dependent on superoxide and ADPC on the myeloperoxidase system. The participation of reactive oxygen species in PMN cytotoxicity was also demonstrated by pharmacological triggering with phorbol myristate acetate. These results indicate that reactive oxygen species have an important role In tumor killing by PMNs and that ADPC and LDPC have partly different cytolytic processes as well as different recognition steps.

Induction of polymorphonuclear leukocyte-mediated cytolysis by wheat germ agglutinin and antitumor antibody.

The role of polymorphonuclear leukocytes (PMNs) as effector cells in tumor lysis was investigated in vitro. PMNs were obtained from the peritoneal cavity of C3H/He Mice injected ip with 2 ml of 12% casein sodium. These PMNs could lyse murine MM46 tumor cells in the presence of the plant lectin, wheat germ agglutinin (lectin-dependent PMN-mediated cytolysis). PMNs alone did not cause cytolysis. PMNs obtained 6 hr after casein injection were effective against tumor target cells but those obtained 3 hr after the injection were not. Other lectins, such as concanavalin A, phytohemagglutinin, pokeweed mitogen, soybean agglutinin, Ulex europaeus agglutinin, Lens culinaris hemagglutinin and Bauhinia purpurea agglutinin, did not cooperate with PMNs in tumor lysis. Antitumor antibody was another mediator that induced tumor lysis in cooperation with PMNs (antibody-dependent PMN-mediated cytolysis). These results indicate that PMNs can lyse tumor cells in the presence of appropriate mediators.

Antibody-Dependent Cytolysis of the Human Leukemia Cell Line K-562 in the Absence of Effector Cells or Complement

K-562 human leukemia cells grown in the presence of specific goat anti-K-562 gamma globulin, F(ab)'2, Fab', or Fab showed a decrease in DNA and protein syntheses and a loss of cell viability within several hours. Eventually all cells died and lysed within 2-5 days. These events occurred in the absence of added effector cells or complement. The effect on the cell was not due to proteolytic or other external lytic activity generated at the cell surface, since 51Cr-labeled bystander cells or antibody-sensitized bystander cells (chicken erythrocytes or K-562 cells) were not lysed when added to cultures of K-562 cells incubated with immune globulin. The anti-K-562 gamma globulin was not observed to patch or cap on K-562 cells, but the ability of bound immunoglobulin to fix complement decreased markedly (50% in 60 min), so that no complement-mediated cytolysis could be observed at 2 hr. However, the immunoglobulin, at least portions of it, reacted with fluorescein--labeled rabbit anti-goat gamma globulin for about 16 hr. 125I-labeled immune globulin appeared to be internalized and released into the medium as small degradation products. 125I-labeled Fab did not appear to be internalized, since it was released intact and more rapidly from cells than the intact globulin.

Antibody-dependent cytolysis of the human leukemia cell line K-562 in the absence of effector cells or complement

K-562 human leukemia cells grown in the presence of specific goat anti- K-562 gamma globulin, F(ab)'2, Fab', or Fab showed a decrease in DNA and protein syntheses and a loss of cell viability within several hours. Eventually all cells died and lysed within 2–5 days. These events occurred in the absence of added effector cells or complement. The effect on the cell was not due to proteolytic or other external lytic activity generated at the cell surface, since 51Cr-labeled bystander cells or antibody-sensitized bystander cells (chicken erythrocytes or K-562 cells) were not lysed when added to cultures of K- 562 cells incubated with immune globulin. The anti-K-562 gamma globulin was not observed to patch or cap on K-562 cells, but the ability of bound immunoglobulin to fix complement decreased markedly (50% in 60 min), so that no complement-mediated cytolysis could be observed at 2 hr. However, the immunoglobulin, at least portions of it, reacted with fluorescein--labeled rabbit anti-goat gamma globulin for about 16 hr. 125I-labeled immune globulin appeared to be internalized and released into the medium as small degradation products. 125I-labeled Fab did not appear to be internalized, since it was released intact and more rapidly from cells than the intact globulin.