Antibody-Dependent Cell Cytotoxicity (ADCC)

The effector cell(s) in human antibody-dependent cell cytotoxicity (ADCC), with antibody-coated chickens erythrocytes as targets, was studied by comparison of cell suspensions from various lymphoid organs and by means of various cell fractionation methods. Effector cells (K) were found mostly in peripheral blood, spleen, and bone marrow but not in tonsils, lymph nodes, and thymus. Effector cells bear Fc receptors and can form EA rosettes with the antibody-coated target cells. About 1,5% peripheral blood lymphocytes can form 'high-avidity' EA rosettes with targets coated at low antiserum concentration. Most of the effector cells belong to this small subset, as shown by experiments of selective depletion. Removal of most monocytes, T cells, or B cells from, or addition of T-cell-specific antiserum to, the effector cell suspensions did not affect ADCC. Effector cells in this model of ADCC therefore lack the conventional B- or T-cell markers but at least some of them are likely to bear C3 receptors.     

Human peripheral Null lymphocytes I. Isolation,immunological and functional characterization

A Null lymphocyte-enriched population was isolated from human peripheral blood of healthy donors using a combination of Ficoll density gradient centrifugation followed by elimination of mononuclear phagocytes, passage through Ig-anti-Ig columns and sedimentation of E rosettes. After each separation step lymphocyte fractions were examined for morphology, cell surface markers, mitogen responsiveness and effector functions in antibody-dependent (ADCC) and spontaneous cellular cytotoxicity (SCMC) reactions against an allogeneic melanoma cell line. The final Null lymphocyte preparation was recovered at a rate of 1 to 3 % from the population passed through Ig-anti-Ig columns (fraction FFF-C). The marker analysis revealed over 99 %of surface Ig-negative lymphoid cells; 50 to 60 % of these cells were ‘real Null’ cells lacking immunological cell surface markers, 7 % formed EA, 13 % EAC and 24 %E rosettes. Regarding the mitogen responses, passage through Ig-anti-Ig columns drastically reduced concanavalin A (Con A), pokeweed mitogen (PWM)and tuberculin (PPD) responses, whereas the phytohemagglutinin (PHA) response was reduced in absolute counts but not in the stimulation index. Compared to the T cell-enriched lymphocyte fraction, the Null cells showed significantly diminished proliferative responses to PHA and Con A and slightly increased reactivity to PWM and PPD. Although depleted of high affinity Fc receptor lymphocytes, the Null cell fractions exhibited good ADCC and SCMC activities being about 4 to 6 times higher than in the T cell fraction.     

Natural killer cells in guinea pig spleen bear Fc receptors

The phenomenon of natural cytotoxicity or spontaneous cell-mediated cytotoxicity (SCMC) was investigated in guinea pigs and compared with two other in vitro cytotoxicity reactions: mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC). The same xenogeneic target cells were employed in all three cytotoxicity assays. Organ distribution studies revealed that SCMC effector cell activity was present in spleen and peripheral blood but not in thymocytes. Bone marrow cells possessed low levels of SCMC effector cell activity. The organ distribution of effector cell activities for MICC and ADCC paralleled that for SCMC. Studies of the cell surface characteristics of the SCMC effector cell revealed that spleen cells nonadherent to antigen-antibody but not to antigen-F(ab')₂ antibody-coated surfaces possessed markedly reduced SCMC effector cell activity. In addition, spleen cells depleted of Fc receptor-bearing cells by EA rosetting also possessed diminished SCMC effector cell activity, while cell populations enriched in Fc receptorbearing cells by EA resetting possessed enhanced SCMC effector cell activity. These fractionation techniques had similar effects on MICC and ADCC effector cell activity. Depletion of adherent spleen cells including macrophages by nylon wool column passage resulted in a population of cells with enhanced SCMC, MICC, and ADCC effector cell activity. Thus, in guinea pig spleen the effector cells mediating SCMC were shown to belong to a population of nonadherent Fc receptor-bearing lymphocytes possessing several cytotoxic capabilities including the potential of mediating MICC and ADCC.     

Natural Cytotoxicity of Human Fcγ-Receptor-positive T Lymphocytes after Surface Modulation with Immune Complexes

In humans T cells with surface receptors for the Fc fragment of IgG (Fc gamma receptors) (TG cells) are effector cells in antibody-dependent cellular cytotoxicity (ADCC) and in natural cytotoxicity. While Fc gamma receptors are required to mediate ADCC, their role in natural cytotoxicity is unknown. To investigate this question, Fc gamma receptors on effector cells were modulated by interaction with IgG immune complexes. As a consequence of this modulation, TG cells lost most of their ADCC activity but retained a significant part of their natural killer activity. Thus, these experiments demonstrate that the cytotoxic mechanisms exerted by the same cell population can be dissociated experimentally. Furthermore, they suggest that the net natural cytotoxicity of normal human lymphocytes in certain effector cell-target cell combinations is the result of distinct types of reaction.     

Relationship between expression of Fc gamma receptors or Ia antigens and cytolytic activities of alloactivated human T cells

The relationship between expression of Ia antigens or Fc gamma receptors (Fc gamma R) and different cytolytic activities of mixed leukocyte culture (MLC)-activated T-cell populations was studied. Lymphocytes mediating specific lysis of target cells bearing the stimulating alloantigens (CTL) could clearly be distinguished from cells mediating antibody-dependent cellular cytotoxicity (ADCC) in that CTL were Fc gamma R- while cells mediating ADCC were Fc gamma R+. In contrast, MLC-generated natural killer (NK) cells were distributed in both the Fc gamma R- and Fc gamma R+ cell fractions. The presence or absence of Ia antigens did not correlate with any of the cytolytic activities studied. In addition, Fc gamma R- and not Fc gamma R+ cells responded in the secondary MLC. Thus, although Fc gamma R+ cells are generated in large numbers in MLC, they do not appear to play a direct role in specific cytotoxicity nor do they proliferate in response to secondary stimulation. However, their ability, following MLC generation, to mediate NK activity and ADCC, both of which may contribute to in vitro and in vivo lysis of allogeneic cells, might explain their appearance following allogenic stimulation.     

Mumps Virus-Induced Enhancement of the in Vitro Cytotoxicity of Cord Blood Lymphocytes

Purified lymphocytes from the umbilical cord of healthy donors (CBL) displayed lower natural cytotoxicity (NK) and antibody-dependent cellular cytotoxicity (ADCC) than peripheral blood (PBL) from adult donors. In contrast, CBL treated with small amounts of UV-inactivated or live mumps virions expressed the same level of enhanced cytotoxicity (virus-dependent cytotoxicity (VDCC)) against non-infected target cells as PBL. For individual CBL donors there was no correlation between the level of NK and VDCC, indicating involvement of partly distinct effector cell populations. The heterogeneity of the effector cells active in VDCC was confirmed by cell fractionation experiments. The major CBL effector cells in NK and ADCC were found in 'non-T' lymphocyte fractions and/or in fractions containing cells with high-avidity receptors for IgG. In contrast, CBL fractions consisting of about 100% lymphocytes bearing T-cell markers and depleted of Fc gamma R+ cells were strongly cytotoxic in VDCC when T24 cells (human bladder carcinoma) were the targets. With two other target cell types of similar susceptibility to VDCC, the cytotoxic activity of T-cell-containing fractions was less pronounced, indicating that the target cells play an active role in effector cell selection. The surface marker profiles of the VDCC effector cells were the same for CBL and adult PBL. Incubation of CBL with UV-inactivated virions usually gave no significant stimulation of DNA synthesis above that seen in virus-free controls. Taken together, our results suggest that neither specific recognition of viral antigen by T cells nor mitogenic effects of viral material are involved in VDCC generation.     

Antibody-Dependent Cell-Mediated Cytotoxicity in the Mouse

The surface characteristics of the mouse spleen cells mediating antibody-dependent cytotoxicity (ADCC) against antigen-coated chicken erythrocytes have been studied by several different column fractionation methods The major effector cells in this system were shown to be surface-adherent and could be depleted from spleen cells by passage through glass-bead ovalbumin/anti-OA immune complex columns (Fc receptor-binding), glass-bead immunoglobulin/anti-mouse Ig columns (Fc receptor and surface immunoglobulin-binding), and glass-bead mouse Ig/rabbit (Fab')₂-anti-mouse Ig or Sephadex G-200/rabbit anti-mouse Ig columns (surface immunoglobulin binding) The concentration of EDTA in the medium used to fractionate the cells played a significant role in determining whether surface immunoglobulin could be demonstrated on the ADCC effector cells. From these results, the conclusion was drawn that ADCC on the part of mouse spleen cells could be mediated by surface-adherent, Fc receptor-positive cells bearing surface immunoglobulin of unknown origin. The possibility that ADCC can be mediated by a heterogenous population of cells in the mouse spleen is discussed.     

Comparison of the Effector Cells in Human Spontaneous Cellular Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity: Differential Sensitivity of Effector Cells to in Vivo and in Vitro Corticosteroids

The effector cells mediating antibody dependent cellular cytotoxicity (ADCC) and spontaneous cellular cytotoxicity (SCC) in humans have been reported to possess similar characteristics. Multiple cell separation techniques were employed in an attempt to physically separate and distinguish the effector cells in these two types of cellular cytotoxicity. Subpopulations of mononuclear cells obtained by a variety of fractionation procedures which either enriched or depleted monocytes, lymphocytes bearing a receptor for sheep erythrocytes (SRBC), a receptor for complement (CRL) or an Fc receptor for IgG always had similar effects on both ADCC and SCC. Aggregated gamma globulin blockade of Fc receptors produced similar dose-dependent depressions of ADCC and SCC. Despite our inability to physically separate the effector cells of ADCC and SCC, administration of in vivo dexamethasone caused a relative increase in ADCC but a profound decrease in SCC. Furthermore, in vitro dexamethasone in pharmacologic and suprapharmacologic concentrations caused no change in ADCC but significantly decreased SCC. This study demonstrates that although the effector cells cannot be physically separated, ADCC and SCC are differentially sensitive to corticosteroids and are hence functionally distinct either on the basis of different subsets of effector cells with similar surface markers or different mechanisms of cytotoxicity by the same effector cell.     

High reproducible ADCC analysis revealed a competitive relation between ADCC and CDC and differences between FcγRllla polymorphism

The anti-CD20 chimeric monoclonal antibody rituximab mediates cytotoxicity in malignant B cells via multiple mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To optimize treatment of non-Hodgkin's lymphoma, a fuller understanding of these mechanisms and their relative contributions to clinical efficacy is required. Here, we report the characteristics of the mutual impact between ADCC and CDC, the two major effector functions through the Fc receptors. To compare ADCC induced under various conditions, we developed a highly reproducible method of estimating ADCC activity using immortalized effector cells. The set of the effector cells that we established was able to calculate net ADCC with high reproducibility by comparing the cytotoxicity of effector cells expressing exogeneous FcγRIIIa to those of mock effector cells. In addition, the different property of effector cells of two FcγRIIIa single-nucleotide polymorphisms (SNP) could be also evaluated in exactly identical background. ADCC assessment in the presence of human serum directly provided the evidence of the competitive interaction of ADCC and CDC. The inhibition of ADCC of effector cells having low affinity SNP of FcγRIIIa by active complement was more potent than those having high-affinity SNP at the rituximab-concentration comparable to the serum level obtained in patients. These findings could have a profound impact on optimization of the regimen of therapeutic antibodies and on the development of antibodies that will enhance effector function.     

Effect of Histamine Receptor Blocking on Human Antibody-dependent Cell-mediated Cytotoxicity

The effect of H1 and H2 receptor-blocking agents on antibody-dependent cell-mediated cytotoxicity (ADCC) was studied. The H1 receptor-blocker clemastinum and the H2 receptor blocker cimetidine dose-dependently inhibited the antibody-dependent cytotoxic activity of normal human peripheral blood mononuclear cells on chicken erythrocytes. The inhibition cannot be explained either by a direct toxic effect on effector cells or by blocking of Fc receptors. The possible involvement of histamine receptor-bearing effector cells in human ADCC is suggested.     

Ability of human cord blood lymphocytes to mediate antibody-dependent cellular cytotoxicity against influenza virus-infected cells

Cord blood lymphocytes, monocytes, and neutrophils from newborns were shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against influenza virus-infected cells. Antibody mediating ADCC was detectable in cord plasma, indicating that all components necessary for ADCC against influenza virus-infected cells are present in newborns. Among adult lymphocytes, two effector cell populations of influenza ADCC are recognized: non-T and T gamma cells. Each of these cell types expresses an antigen recognized by monoclonal HNK-1 antibody. The proportion of HNK-1 antigen-positive lymphocytes in cord blood was markedly lower than in adult blood; furthermore, ADCC was mediated by cord blood lymphocytes which were HNK-1 negative. By lymphocyte fractionation, the effector lymphocytes in cord blood were, as in adults, non-T and T gamma cells, suggesting that HNK-1 antigen is not expressed on these cell lineages in newborns.     

Reconstitution of natural cell-mediated cytotoxicity with specific antibodies.

The apparent nonselective reactions of natural cell-mediated cytotoxicity (NCMC) are selective when tested by inhibition of cytotoxicity with competitor cells indicating a recognition of specificities by the effector cell. N cells that mediate this NCMC in humans have most of the characteristics of K cells that mediate antibody-dependent, cell-mediated cytotoxicity (ADCC) and possess Fc receptors. IgG antibodies attached loosely to N cells through their Fc region, form part of the class of lymphocytes with surface immunoglobulin. We hypothesized that ADCC and NCMS involved similar mechanisms but with the specificity of NCMC directed by the natural IgG antibodies already attached to N cells. Removal of the antibodies with trypsin and reconstitution with specific anti-HLA antibodies produced specific effector cells supporting the role of antibodies on N cells as directors of specificity in NCMC.     

Antibody-dependent cellular cytotoxicity (ADCC) against Epstein-Barr virus-determined antigens II. Induction of ADCC by FC-FC receptor bridging.

Four different methods to test antibody-dependent cellular cytotoxicity (ADCC) against Epstein-Barr virus (EBV) determined antigens were analysed. It was found that optimal ADCC was obtained if viral antigens were present during the cytotoxicity reaction and that killing probably was mediated by EBV-related immune-complexes forming Fc-Fc receptor bridges between Fc receptor-bearing effector cells and Fc receptor-bearing target cells. Only lymphoid target cells with a high expression of Fc receptors were found to be susceptible in this particular system.     

Monoclonal antibodies against leucoagglutinin-reactive human T lymphocyte surface components. Two antibodies which inhibit cell-mediated cytotoxicity at a post-binding stage

Two out of 20 monoclonal antibodies (IgM, kappa), mAb 3192 and mAb K3G, raised against leucoagglutinin-reactive components on human T cells, effectively blocked lymphocyte-mediated cytotoxicity in vitro. No antigenic polypeptide reactive with these antibodies has been identified thus far. However, they have previously been shown to react specifically with certain neutral glycolipids obtained from spleen. Both mAb inhibited the cytotoxicity of natural killer (NK) cells against K562 cells, antibody-dependent cellular cytotoxicity (ADCC) towards antibody-coated bovine erythrocytes and cytotoxic T lymphocyte activity against allogeneic target cells. In both NK and ADCC, preincubation of the lymphocytes with different antibody concentrations resulted in a dose-dependent reduction of cytotoxicity. In contrast, preincubation of the target cells had no effect indicating that the mAb inhibited cytotoxicity at the effector cell level. When studied at the single-cell level, the mAb did not alter the number of lymphocytes forming conjugates with K562 but significantly reduced the frequency of conjugates containing dead target cells. Addition of the mAb to preformed conjugates resulted in a dose-dependent reduction in the proportion of conjugates containing dead target cells. Furthermore, mAb 3192 did not reduce the number of lymphocytes forming rosettes with bovine erythrocytes, indicating that inhibition of ADCC was not due to blocking of the effector cell-target cell interaction mediated by the Fc receptor of the effector cells. Taken together, these results suggest that the mAb inhibited cytotoxicity by interfering with a post-binding step common for the different cytotoxicity systems.     

The Fc Receptors of Normal and Cancer Patients Monocytes

The ability of human monocytes from normal donors and gastric-cancer patients to form rosettes with ?0? Rh⁺(D) human erythrocytes coated with hyperimmune IgG anti-D antibody (EA_hu) and to kill the same target in antibody-dependent cellular cytotoxicity (ADCC) were assessed. Trypsin pretreatment of normal monocytes decreased their ability to form rosettes with EA_hu complexes, but their ADCC activity was unaffected. The Fc receptor (FcR) expression and ADCC activity of monocytes of cancer patients were elevated, and trypsin-treatment led to their further increase. The elevated values were related to the presence of the tumour. These results may suggest that human monocytes possess trypsin-sensitive and trypsin-resistant Fc receptors. The trypsin-resistant FcR seems to be involved in ADCC phenomenon and to be preferentially expressed on monocytes of some cancer patients.     

Lymphocyte Subpopulations in Man: Characterization of Human Killer Cells against Allogeneic Targets Sensitized with HLA Antibodies

Human killer cells mediating antibody-dependent cytotoxicity against allogeneic lymphoblasts presensitized with HLA antibodies have been studied by rosette fractionation experiments. Enriched and/or depleted cell suspensions have been tested in dose-response studies. Two different populations can act as killer cells. The major cytotoxic capacity is retained among T cells with high-avidity Fc receptors, whereas a minor cytotoxic capacity was found among non-T cells with high-avidity Fc receptors. These two populations have different dose-response curves, indicating different effector mechanisms.     

Effect of morphine and beta-endorphin on human Fc receptor-dependent and natural killer cell functions.

Interactions between opiates and the human immune system have important clinical implications. To further evaluate these interactions, we studied in vitro and in vivo effects of morphine sulfate (morphine) and beta-endorphin (Bend) on antibody-dependent cell cytotoxicity (ADCC), natural killer cell cytotoxicity (NKCC), and effector cell expression of antibody Fc receptors. Morphine and Bend had no potent in vivo or in vitro effect on FcR expression nor did they have a significant in vitro effect on ADCC by monocytes or polymorphonuclear cells. Bend enhancement of NKCC in vitro was inhibited by coincubation of effector cells with morphine. After taking 90 to 150 mg of oral morphine, study volunteers demonstrated a significant decrease in ADCC by peripheral blood mononuclear cells. The same individuals demonstrated a consistent increase in NKCC and no change in the expression of Fc receptors. Effector cells from these individuals responded normally to in vitro incubation with interferon-gamma (IFN-gamma).     

Characterization of Effector Cells Mediating IgG and IgM Antibody-Dependent Cellular Cytotoxicity

Spleen effector cells for IgG- and IgM-induced antibody-dependent cellular cytotoxicity (ADCC) were characterized with respect to density and cell surface markers by using sheep erythrocytes (SRBC) coated with hybridoma-derived monoclonal anti-SRBC IgG or anti-SRBC IgM antibodies as targets. While basically the same effector cells are cytolytic for IgG and IgM antibody-coated SRBC, they differ with respect to their relative killing capacity for IgG- versus IgM-coated target cells. On the basis of physical and biochemical properties three populations with cytolytic capacity could be separated: (I) A light fraction of large cells had high cytolytic potential for both IgG- and IgM-coated SRBC. The cells were negative for the Fc receptor for IgG (Fc gamma-R-) and the C3-receptor (C3-R-), they carried the receptor for Helix pomatia A agglutinin (HP-A+), and reactivity was strongly reduced after treatment with anti-Thy-1 and complement (C). (II) High activity was also observed with a medium-dense fraction, preferably lysing IgG antibody-coated cells. The cells were Fc gamma-R+, partly C3-R+, mostly HP-A-, and only a minor portion of the cells were Thy-1+. (III) A dense fraction, displaying on a per cell basis low cytolytic potential, was more active in IgM than IgG ADCC. The cells were Fc gamma-R+, HP-A+ and Thy-1+. All three effector cell populations were non-adherent, non-phagocytic, and surface immunoglobulin-negative (s-Ig-).     

Characterization of the human peripheral effector cells mediating antibody dependent cellular cytotoxicity against allogenic cells.

The effector cell populations in human peripheral blood responsible for antibody-dependent cellular cytotoxicity against allogenic cells coated with HLA polyspecific antibodies were investigated using several separation techniques including preparative electrophoresis. Electrophoresis produced a marked effector cells enrichment in a range of 2--5 fractions which exhibited an intermediary electrophoretic mobility. Monocytic cells do not contribute an effector mechanism but minor subsets of polymorphonuclear cells and nylon wool non-adherent non-phagocytic lymphocytes displayed ADCC. Both effector cell populations were found to exhibit a similar electrical charge of cell surface centered around -1.05 micrometer . sec-1 V-1 cm. These observations provided a precise biophysical basis for the identification of effector cells in ADCC.     

Murine epidermal gamma/delta T cells express Fc gamma receptor II encoded by the Fc gamma R alpha gene.

Short-term bulk cultures and some long-term clones and lines of murine T cell receptor (TcR) gamma/delta-bearing epidermal T cells (dEC) were found to express an Fc gamma receptor II (Fc gamma RII), as revealed by reactivity with the monoclonal antibody 2.4G2. Northern blot analysis showed that the Fc gamma RII expressed on dEC is encoded solely by the Fc gamma R alpha gene. While all the various cultured dEC cell populations analyzed exhibit lectin-dependent cellular cytotoxicity, only those which expressed Fc gamma R alpha were also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). These results in combination with the previous demonstration of Fc gamma R alpha on mouse natural killer cells support an essential role for Fc gamma R alpha in ADCC and extend an analogy with surface CD16 (Fc gamma RIII) expression and ADCC in human natural killer cells and peripheral TcR gamma/delta T cells.