Activating and inhibitory Fcγ receptors in immunotherapy: being the actor or being the target

Membrane Fcγ receptors (FcγRs) can act either as potent activators of effector cell functions or as inhibitors of receptor-mediated cell activation following engagement by IgG antibodies bound to their target molecules. The remarkable ability of activating FcγRs to trigger antibody-dependent cellular cytotoxicity, cytokine release and phagocytosis/endocytosis followed by antigen presentation has stimulated the development of a number of therapeutic monoclonal antibodies whose Fc regions have been engineered to optimize their effector functions, mostly their killing activities. Conversely, the demonstration that inhibitory FcγRs can block or downmodulate effector functions has led to the concept that targeting these receptors is of interest in a number of pathologies. The use of bispecific antibodies leading to the crosslinking of FcγRIIB with activating receptors could induce immunomodulation in autoimmune or allergic diseases. Alternatively, the use of cytotoxic/antagonist anti-FcγRIIB antibodies could kill FcγRIIB-positive tumor cells or prevent the downmodulation of activating receptors. Thus, antibodies engineered to preferentially target activating or inhibitory FcγRs are currently being designed for therapeutic use.     

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.     

Complement activation is not required for IgG-mediated suppression of the antibody response

Feedback suppression of the antibody response by IgG is known to be dependent on intact Fc regions. However, it is not clear which of the Fc-mediated effector functions is required. In the present report we have studied whether ability or inability of the IgG antibodies to activate the complement system was of consequence for their immunosuppressive effect. First, a monoclonal IgG1-anti-2,4,6-trinitrophenyl (TNP) antibody, unable to activate complement via the classical or alternate pathway, was shown to be able to inhibit more than 90% of the in vivo sheep erythrocyte-specific antibody response in mice when TNP coupled to sheep erythrocytes was used as antigen. Second, we investigated the immunosuppressive ability of a non-complement-activating mutant IgG2a-anti-TNP monoclonal antibody. The mutant differs from the wild type by a single amino acid substitution in the CH2 domain leading to inability to fix complement factor C1q. However, the mutant has the same affinity for antigen and the same Fc receptor-binding capacity as the wild type antibody. It is demonstrated that the mutant was as efficient as the wild type antibody in inhibiting an in vitro antibody response to TNP-coupled sheep erythrocytes. These findings confirm the non-determinant specificity and Fc dependence of IgG-mediated suppression, and show that the Fc-mediated effector mechanism is independent of complement activation. The results instead suggest binding to Fc receptors as a necessary step in feedback immunosuppression and favor inactivation of B cells by cross-linking of Fc and antigen receptors on their surface rather than elimination of antigen by complement-dependent phagocytosis as the effector mechanism.     

Cytoplasmic Calcium Fluxes Induced in Cytotoxic Effector Cells by Engagement of Fcγ Receptors I, II, and III

Changes in the intracellular calcium ion concentration ([Ca2+]i) of monocytes, granulocytes, and NK cells have been studied following either (1) independent cross-linking of Fc gamma receptors (Fc gamma R) I, II, or III, with F(ab gamma')2 fragments of monoclonal antibodies; or (2) linking of a selected Fc gamma R to a tumour cell target with bispecific F(ab' gamma)2 antibodies. Upon cross-linking each Fc gamma R with antibody an increase in the [Ca2+]i was observed, although all receptors apart from Fc gamma RIII on NK cells required additional cross-linking with an anti-mouse Fab' gamma. These results indicate that each type of receptor can transduce signals to the cell independently. Bispecific antibodies (anti-Fc gamma R x anti-target) linking cytotoxic Fc gamma R-bearing effector cells to tumour target cells also mediated increases in [Ca2+]i for all Fc gamma R tested except for Fc gamma RIII on granulocytes. The failure to transduce a signal via this receptor may be related to the GPI link, which is in contrast to the transmembrane link of Fc gamma RIII on NK cells. Significant lysis of tumour cell targets occurred when bispecific antibodies recruited NK cells or monocytes, but not granulocytes, via Fc gamma R. Chelation of intracellular Ca2+ in the effector cells reduced the observed lysis, suggesting a role for Ca2+ in the pathways leading to cytotoxicity.     

Correlation between effector lymphocytes in natural and antibody-mediated cytotoxicity

Human sera enhanced spontaneous cell-mediated cytotoxicity (SCMC), while anti-IgG (Fab') 2 treatment decreased this cytotoxic activity of human lymphocytes for an in vitro growing cell line (K--562). Trypsin treatment of the effector cells considerably decreased the cytotoxic potential. However, a significant cytotoxic activity could always be found in serum-free medium. While these findings suggest the involvement of antibodies in the SCMC, they also reflect the existence of serum-indpendent (sui generis) SCMC activity of lymphocytes. Removal of SCMC of Fc receptor bearing effector cells was performed by target cell adherence (rosetting). Separation of the target cell-bound lymphocytes was done by centrifugation on special Ficoll gradient. The depletion of SCMC effector cells resulted in a 62% reduction of SCMC and in a 39% reduction of ADCC. On the other hand, removal of Fc bearing effector cells showed a similar reduction in both ADCC (66%) and SCMC (78%). Our results suggest that SCMC represents a complex activity, arising partly from the interactions of certain serum-derived or lymphocytes surface-bound antibodies and partly from a spontaneous cytotoxic function of the effector cells. It is possible that the effector cells involved in both SCMC and ADCC derive from the same lymphocyte population and the differences are due mainly to the lower number of SCMC effector cells.     

Wheat-germ agglutinin initiates monocytoid cell killing of non-antibody-coated erythrocytes

Cells bearing alien surface antigens can be specifically destroyed by antibodies directed to the antigens and certain Fc receptor-bearing lymphoid or monocytoid cells through a killing mechanism known as antibody-dependent cellular cytotoxicity (ADCC). The present study was undertaken to see if lectins could substitute for specific antibody in ADCC and initiate lysis of non-antibody-coated erythrocytes. Human peripheral blood leucocytes and mouse PU-5 monocytoid cell lines were used as effector cells. Target-cell destruction was measured by the specific release of 51Cr. Our data indicated that Wheat-germ agglutinin (WGA), but not concanavalin A or soybean agglutinin, could activate killing of non-antibody-coated erythrocytes by human peripheral blood leucocytes and PU-5 cells. The membrane structure on the effector cell that was triggered by WGA directly related to N-acetyl-glucosamine and may be adjacent to, if not part of, the Fc receptor.     

The functional properties of Fc gamma RI, II and III on myeloid cells: a comparative study of killing of erythrocytes and tumor cells mediated through the different Fc receptors

Three distinct Fc receptors for IgG, Fc gamma RI, Fc gamma RII and Fc gamma RIII are known to be associated with human myeloid cells. Using mAb specific for these receptors, and the hydridoma cells lines that produce these mAb, we have examined the ability of each of these receptors on different myeloid cells and cell lines to mediate killing of tumor and red cell targets. Hybridoma cells (HC) expressing anti-Fc gamma RI, Fc gamma RII or Fc gamma RIII upon their surface were used as model self-directed tumor targets. Chicken erythrocytes (CE) were used as another type of target cell and in this case effector cell cytotoxicity was mediated by heteroantibodies (HA) composed of Fab fragments of anti-Fc gamma R mAb covalently linked to Fab fragments of rabbit anti-CE antibodies. Monocytes, lymphocytes, polymorphonuclear cells (PMNs) and the myeloid cell lines U937, HL-60 and THP-1 were used as effector cells either in their native state or after activation with rIFN-gamma. Direct comparison of cytotoxicity by the same effector cell population against both tumor and erythroid targets has permitted definitive evaluation of the ability of the different Fc gamma R to promote cytolysis under two different conditions. Monocytes were able to utilize Fc gamma RI, Fc gamma RII and Fc gamma RIII in killing both CE and HC targets, and incubation with rIFN-gamma augmented their ability to kill CE, particularly through Fc gamma RI. Fc gamma RII and Fc gamma RIII mediated killing of CE by untreated neutrophils. rIFN-gamma induced PMNs to express Fc gamma RI and to mediate killing of CE through this receptor. Moreover, HC targets were not lyzed by untreated neutrophils, but rIFN-gamma activated neutrophils killed HC bearing surface anti-Fc gamma RI and anti-Fc gamma RII, but not anti-Fc gamma RIII. Myeloid cell lines HL-60 and U937 were unable to perform cytotoxicity without prior culture with rIFN-gamma, following which they killed CE through Fc gamma RI and Fc gamma RII, but were still incapable of HC lysis. THP-1, another myeloid cell line, was cytotoxic to CE through Fc gamma RI and Fc gamma RII without activation. Following rIFN-gamma treatment, cytotoxicity through these two Fc gamma R increased and was also mediated by Fc gamma RIII but these cells were still unable to kill HC.(ABSTRACT TRUNCATED AT 400 WORDS)     

The proliferative capacity of human T lymphocyte subpopulations in a continuous culture system.

The continuous growth of subpopulations of human T lymphocytes was investigated using a culture system containing conditioning factors from mitogen-stimulated lymphocytes. Cultures of purified peripheral blood lymphocytes rapidly became enriched for T lymphocytes, detected as sheep erythrocyte rosette-forming cells, and could be maintained for up to a month in an actively growing state. Subpopulations of T lymphocytes bearing Fc receptors for IgM(T.M) or IgG(T.G) could not be detected in these cultures unless the cells were first washed and cultured for 3 days in medium devoid of conditioning factors. During this second culture step, many of the cells reverted from a large, blast-like state to a small lymphocyte morphology and proportion of the T lymphocytes re-expressed Fc receptors. The proportions of T.M and T.G lymphocytes so detected remained constant throughout the continuous culture period indicating that the system permitted the proliferation of all T lymphocytes. Fractionation studies supported this conclusion by demonstrating that purified T, T.M and T.G but not B lymphocyte populations proliferated when cultured in the presence of conditioned medium. The majority of cells in cultures of purified T.M lymphocytes re-expressed IgM Fc receptors following reculture in unconditioned medium. However, the re-expression of IgG Fc receptors by cultured T.G lymphocytes could not be achieved.     

Co-expression of different types of Fc receptors on murine peritoneal macrophages

Simultaneous expression of particular immunoglobulin Fc receptors (FcR) was studied on the plasma membranes of murine peritoneal macrophages. This was facilitated by the use of sheep red blood cells (SRBC) and/or synthetic microspheres coated with monoclonal antibodies of different isotypes. It was concluded that a majority of macrophages bear more than one type of FcR; macrophages bearing at least three types of FcR were present in the peritoneal cavity; macrophages bearing Fc mu R did not bind IgE, IgA or IgG; all macrophages bearing Fc alpha R also expressed Fc gamma 2bR, Fc gamma 3R and Fc epsilon R; all macrophages bearing Fc epsilon R also expressed Fc gamma 2bR and Fc alpha R. Except for Fc alpha R, essentially equivalent numbers of FcR-bearing macrophages were detected when antibody-coated SRBC or polymeric microspheres were used. Simultaneous applications of these reagents permitted the most detailed and direct investigations yet performed of multiple FcR expression on individual cells.     

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.     

Fc gamma receptors and cancer

FcγRs are a family of heterogeneous molecules that play opposite roles in immune response and control the effector functions of IgG antibodies. In many cancers, IgG antibodies are produced that recognize cancer cells, form immune complexes and therefore, activate FcγR. The therapeutic efficacy of monoclonal IgG antibodies against hematopoietic and epithelial tumors also argue for an important role of IgG antibodies in anti-tumor defenses. Since the 1980s, a series of lines of evidence in experimental models and in humans strongly suggest that FcγR are involved in the therapeutic activity of monoclonal IgG antibodies by activating the cytotoxic activity of FcγR-positive cells such as NK cells, monocytes, macrophages and neutrophils and by increasing antigen presentation by dendritic cells. Since many cell types co-express activating and inhibitory FcγR, the FcγR-dependent effector functions of IgG anti-tumor antibodies are counterbalanced by the inhibitory FcγRIIB. In addition, some tumor cells express FcγR either constitutively, such as B cell lymphomas or ectopically, such as 40% of human metastatic melanoma. The tumor FcγR isoform is preferentially FcγRIIB, which is functional at least in human metastatic melanoma. This review summarizes these data and discusses how FcγRIIB expression may influence the anti-tumor immune reaction and how beneficial or deleterious this expression could be for the efficiency of therapeutics based on monoclonal anti-tumor antibodies.     

Immunoglobulin Fc receptor molecules on Xenopus laevis splenocytes.

The existence of membrane-associated Fc receptors for IgY (Fc nu R) or IgM (Fc mu R) was demonstrated on a large percentage of Xenopus splenocytes. The Fc receptors were detected by direct fluorescent staining in which the spleen cells were incubated with fluorescein isothiocyanate (FITC)-conjugated antigen-complexed IgY antibodies or with FITC-conjugated heat-aggregated IgM. Results showed that 28.9% (SD +/- 5.1) and 5.3% (SD +/- 2.2) of the cells bear Fcnu or Fc mu receptors, respectively. The specificity of the receptors was tested after incubation of the cells in the presence of the following fluorochrome-conjugated reagents: non-aggregated Xenopus Igs and human IgG, aggregated Xenopus albumin, Fc6 nu and Fab mu fragments and human IgG, and antigen-complex Fab2 nu fragments. Results indicated that the receptors are specific for Xenopus immunoglobulins, with the restriction that the latter must be presented in a complexed or aggregated form to the cells and that the precise binding site is located on the Fc portion of IgY or IgM. The identity of a large percentage (22.4 +/- 2.8%) of cells bearing Fc nu R was established by direct simultaneous double-fluorescent staining of surface membrane IgM and Fc nu R. All Xenopus splenic B lymphocytes bearing sIgM carry also Fc nu R, while 8.8% of splenocytes, of yet unknown lineage, bear Fc nu R alone on their surface. The presence of Fc receptors on Xenopus B lymphocytes (leaving aside their eventual presence in other leucocyte types) suggests, once again, a high degree of evolution of the immune system in these lower vertebrates.     

The effect of cytokines on the expression and function of Fc receptors for IgG on human myeloid cells

We examined expression and cytotoxic triggering capability of the three Fc receptors for IgG (Fc gamma R) on human monocytes, PMNs and myeloid cell lines after in vitro culture with various cytokines. Fc gamma R expression was evaluated using specific anti-Fc gamma R monoclonal antibodies (mAb). The cytotoxic capability of each Fc gamma R was examined after the effector cells were treated with the recombinant cytokines IFN-gamma. TNF alpha, GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, or IL-6. Hybridoma cell lines (HC) bearing antibody directed to Fc gamma RI (HC 32), Fc gamma RII (HC IV.3) or Fc gamma RIII (HC 3G8) were used as targets, as were chicken erythrocytes (CE) sensitized with heteroantibodies composed of anti-Fc gamma R mAbs (32, IV.3, 3G8) linked to anti-CE antibody. Only IFN-gamma treatment significantly increased Fc gamma R expression and then only Fc gamma RI. IFN-gamma dramatically up-regulated Fc gamma RI expression on all cells tested. However, ADCC was enhanced by treatment with a number of cytokines other than IFN-gamma. GM-CSF, TNF, and IFN-gamma treatment enhanced killing of HC 32 and HC IV.3 by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through Fc gamma RII, whereas PMN killing of HC through Fc gamma RIII could not be induced by any of the cytokines studied. Although only IFN-gamma treatment increased ADCC of CE by monocytes, GM-CSF treatment as well as IFN-gamma treatment augmented ADCC of CE by PMNs. In addition to IFN-gamma treatment, IL-6 treatment enabled U937 cells to lyse CE. Whereas IFN-gamma-treated U937 cells killed CE through both Fc gamma RI and Fc gamma RII, IL-6-treated U937 cells killed CE only through Fc gamma RI. In addition to IFN-gamma treatment, G-CSF treatment enabled HL-60 cells to lyse CE through both Fc gamma RI and Fc gamma RII. These results demonstrate that although IFN-gamma appears unique in regulating Fc gamma R expression on myeloid cells, cytokines other than IFN-gamma affect ADCC by these cells in a receptor-specific manner.     

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.     

Generation of virus-specific cytolytic activity in human peripheral lymphocytes after vaccination with vaccinia virus and measles virus

Human peripheral blood lymphocytes (PBL) harvested after vaccination with vaccinia or measles virus showed a specific activity against virusinfected target cells. This activity peaked on day 7 and was specific for the target cells infected with the virus used for the vaccination. The cytotoxic activity was not related to HLA markers. The cells involved in the cytolytic process were lymphocytes bearing Fc receptors. In addition, the cytotoxic activity was abrogated by more than 90% by rabbit Fab'2 anti-human IgG. It is therefore likely that two subpopulations of lymphocytes are involved: an antibody-secreting cell providing specific antiviral antibody and an effector cell bearing Fc receptor (K cells). Finally, these experiments suggest that antibody-dependent cell cytotoxicity may play a major role in the recovery from virus infection in man.     

Cytotoxicity mediated by human Fc receptors for IgG

The Fc receptors for IgG(Fc gamma R) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of Fc gamma R-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these Fc gamma R in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monoclonal antibodies specific for Fc gamma R on human leukocytes has established the existence of three distinct Fc gamma R and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of Fc gamma R-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different Fc gamma R is discussed. The results of these studies suggest that the ability of a given Fc gamma R to trigger killing is sometimes dependent on the type of Fc gamma R, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.     

Relationship between anti-cardiolipin and anti-endothelial cell antibodies in systemic lupus erythematosus

Anti-endothelial cell antibodies (AECA) have been detected in 51 lupus sera by cell surface radioimmunoassay with a prevalence of 39.2% for IgG and 45.1% for IgM-AECA. No correlations were found between AECA and different clinical or laboratory parameters, including the presence of anti-cardiolipin antibodies and signs associated with the presence of anti-phospholipid antibodies. However, absorption with cardiolipin liposomes partially inhibited endothelial cell binding, and affinity purified anti-cardiolipin antibodies were able to react with intact human endothelial cells. The binding did not occur via Fc receptors since blocking of Fc receptors with rabbit IgG did not affect the endothelial cell reactivity. Taken together our findings support the hypothesis that antibodies directed against negatively charged phospholipids can be part of the anti-endothelial cell antibodies in certain lupus sera. The possible role of these cross-reacting antibodies in the pathogenesis of vascular thrombosis in lupus patients is discussed.     

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.     

Antibody-mediated tumor cytotoxicity of microglia

The status of microglial cells as potent effector cells in antibody-mediated tumor cytotoxicity (ADCC) could be established. Microglia (greater than or equal to 99.9% pure) derived from brain cortices of newborn mice were shown to lyse human tumor cell lines expressing different levels of epidermal growth factor (EGF) receptors in the presence of MAb 425, a monoclonal murine anti-primate EGF receptor antibody. MAb 425 mediates microglial ADCC (MiADCC) at concentrations as low as 10(-11) M. Antibody ligands binding unilaterally to either EGF receptors on target cells or Fc receptors on microglia have little effect on MiADCC. At 10(-10) M MAb 425, a 10(3)-fold excess of MAb 425 F(ab')2 fragments or irrelevant antibodies of identical isotype did not block MAb-425-induced MiADCC. Formation of effector-target cell contacts seems to be critical for MiADCC and MiADCC could not be inhibited by anti-tumor necrosis factor-alpha antibodies. In addition to its stimulatory effect on MiADCC, MAb 425 bound to EGF receptors exerted a microgliotrophic effect. Factor(s) derived from astrocytes enhance MiADCC.     

Antibody- and interferon-dependent killer cells are part of the NK cell receptor positive subpopulation of human peripheral blood cells.

Cytotoxicity by human non-adherent peripheral blood lymphocytes was analysed after effector cell activation with either interferon (IF) or by target cell specific IgG antibodies (T-IgG). Four different cell lines were used as target cells that differed in susceptibility to natural killer cell (NK) activity; a highly susceptible T cell line (Molt-4), a medium susceptible B lymphoma line (Daudi), a resistant B cell line established by Epstein-Barr virus transformation (LCL-LS) and a resistant mouse mastocytoma line (P815). Three different parameters influencing killing were investigated; lytic potential, target cell binding and efficiency of the lytic phase from which the absolute number of effector cells and their recycling capacity could be estimated. It was found that, when using human target cell lines, IF and T-IgG influenced the system in a similar way by activating the lytic phase and the effector cell recycling but not the early binding phase. With the NK resistant mouse mastocytoma cell line P815 a comparatively small target binding population was found which, however, increased markedly with T-IgG treatment. Taken together, the results indicate that the effector population responsible for antibody-induced killing belong to a subpopulation of cells that have the ability to spontaneously conjugate to the present target cells by virtue of naturally occuring undefined cell surface receptors designated NKR (NK receptor) and that the role of T-IgG in the present system is similar to that of IF. In contrast, if target cells are used that do not express binding structures for NKR receptors, T-IgG may also fulfill a receptor function through Fc receptors for IgG.