Specificity of Fcgamma receptors in human malignant tissue and normal lymphoreticular tissue

The specificity of the Fcgamma receptors in normal spleen and liver and in malignant tissues was studied using hemadsorption to cryostat sections. Indicator cells (EA) were sheep erythrocytes (E) sensitized with rabbit IgG antibody (A). The binding of EA to sections of normal and malignant tissues was inhibited by pooled IgG of human, rabbit, and guinea pig origin and by human IgG1, and IgG3, and IgG4 myeloma proteins. Heat-aggregated IgG inhibited the binding to sections of liver and some malignant tissues more effectively than monomeric IgG. The Fc fragments of IgG were also inhibitory, but not the F(ab')2, Fab', and Facb fragments. The inhibition obtained increased with decreasing amounts of A used for sensitization of E. The inhibitory activity of IgG was abolished after partial reduction and alkylation. No inhibition was obtained with IgG2, IgM, IgA, or albumin. E sensitized with Facb or F(ab')2 fragments of A did not bind to normal or malignant tissues. The specificity of the Fc receptors in normal spleen and liver and in malignant tissues is apparently very similar.     

Relation between bronchial reactivity to antigen in vivo and serum IgE and IgG2a antibody levels in rats

Sprague Dawley rats were immunized with graded doses of ovalbumin (OA) together with alum. The capacity of the animals to produce a bronchial anaphylactic response to intravenous antigen challenge was related to serum OA-IgE and OA-IgG2a antibody levels estimated by radioimmunoassay. A significant correlation between bronchial anaphylactic response capacity and OA-IgE antibody levels was found under a few, but not all, of several carefully chosen experimental conditions. No correlation was demonstrable between the in vivo reactivity of the animals and their serum levels of OA-specific IgG2a antibodies.     

Tetanus and antitetanus vaccination: a study of the urban population of Londrina (PR), Brazil

The first component of guinea pig complement (C1)2) is bound in a cooperative manner to antigen-antibody complexes containing rabbit IgM antibodies, as was previously shown to be the case for IgG antibodies. The shape of the binding curves is consistent with an allosteric mechanism involving clusters of 10 interacting C1 binding sites. Similar results were obtained with IgM antibodies against an artificial hapten and against a natural constituent of the erythrocyte membrane.     

Characterization of the human immunoglobulin G Fc-binding activity in Prevotella intermedia

Many pathogenic bacteria possess cell surface receptors which can bind immunoglobulins via the Fc portion. The aim of this study was to characterize the human immunoglobulin G (IgG) Fc-binding activity of Prevotella intermedia, a suspected etiologic agent of adult chronic periodontitis. The Fc-binding activity of P. intermedia on whole cells and on extracellular vesicles was demonstrated. Incubation of P. intermedia cells in the presence of Zwittergent 3-14 allowed complete solubilization of the Fc receptor from the cell surface. This cell envelope extract was thus used to characterize the Fc-binding activity. A microtiter plate assay using alkaline phosphatase-labeled Fc fragments showed that preincubation of the cell envelope extract with human IgG, human IgG Fc fragments, or human serum completely inhibited the Fc-binding activity. Partial inhibition was obtained with human IgG F(ab')2 fragments, whereas no inhibition occurred following preincubation with human IgA, carbohydrates, and selected proteins. Preincubation of the cell envelope extract with IgG from a variety of animals demonstrated that rabbit, mouse, rat, goat, and sheep IgG did not inhibit Fc-binding activity, whereas cow, pig, and dog IgG partially inhibited Fc-binding activity. A strong inhibition comparable to that obtained with human IgG was noted with monkey IgG. The Fc receptor of P. intermedia is thus different from the six types previously reported in other nonoral bacteria. Polyacrylamide gel electrophoresis and Western blotting (immunoblotting) analysis of the cell envelope extract revealed a major band with a molecular mass of approximately 65 kDa which reacted with peroxidase-labeled human IgG Fe fragments. Transmission electron microscopy showed a uniform distribution of the Fc receptor on the bacterial surface, as revealed by gold labeling. The Fc-binding activity demonstrated in this study may act as an additional virulence factor for P. intermedia by reducing IgG reactions with the bacterial cell.     

Survival of very young children with medulloblastoma (primitive neuroectodermal tumor of the posterior fossa) treated with craniospinal irradiation

We have previously shown that the ligand-binding activity of type II Fc receptor for IgG (FcgammaRIIB) on guinea pig peripheral blood polymorphonuclear leukocytes is very low and dramatically increases after treatment of the cells with proteolytic enzymes. In the present study, we analyzed the mechanism of this augmentation. We found that the protease treatment failed to enhance the binding of monomeric IgG to FcgammaRIIB, increased the binding of small immune complexes (IC) prepared under antigen-excess conditions only modestly, but markedly enhanced the binding of large IC prepared under antibody-excess conditions. These results suggest that proteolysis increases the ligand-binding avidity but not the intrinsic affinity of FcgammaRIIB. Confocal laser scanning microscopy revealed that the mobility of FcgammaRIIB on the cell surface was increased after protease treatment. In addition, transfection experiments indicated that the effect of proteolysis on IC binding to CHO cells expressing guinea pig FcgammaRIIB was strongly dependent on the receptor density. Finally, we demonstrated that the transmembrane and cytoplasmic domains of FcgammaRIIB were not involved in the proteolysis-induced augmentation of IC binding. Together our results suggest that the mobility of FcgammaRIIB, which may be restricted due to the association of the ectodomain of the receptor with unknown membrane proteins, is enhanced by proteolysis, allowing the receptors to bind multivalent ligands more readily and hence with higher avidity.     

Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.     

Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris

The influence of thapsigargin, a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+ ATPase, on the positive inotropic effects of digoxin before and after pretreatment with rimalkalim [(3S,4R)-3-hydroxy-2,2-dimethyl-4-(oxopyrrolidinyl)-6-phenyl-su lfonylchroman hemihydrate (formerly HOE 234)], a known activator of ATP-sensitive K+ channels, was studied in the guinea pig heart. The isolated papillary muscles from the guinea pig heart were used to study these effects. The following parameters were measured: force of contraction (Fc), rate of rise (+dF/dt) and rate of fall (-dF/dt) of Fc, time to peak contraction (ttp) and time to 10% of the total amplitude of force (tt10). After pretreatment with rimalkalim (1 microM), digoxin caused a significant increase in the amplitude of Fc and significant shortening of ttp and tt10 (p < 0.05 compared with the values obtained with digoxin alone). Thapsigargin (1 microM), a selective inhibitor of sarcoplasmic reticulum Ca2+ ATPase, added to rimalkalim, prevented the enhancement of the amplitude of Fc induced by digoxin after pretreatment with rimalkalim but had no significant influence on the effects of digoxin itself. The results demonstrate significant influence of activation of KATP channels on digoxin-induced positive inotropic effects in the guinea pig heart. Attenuation of this effects of rimalkalim by addition of thapsigargin suggests that activation of SR Ca2+ ATPase can be included in this interaction.     

In vivo immune evasion mediated by the herpes simplex virus type 1 immunoglobulin G Fc receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcgammaR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcgammaR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcgammaRs. NS-gE339 expresses gE and gI, is FcgammaR-, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcgammaR does not bind murine IgG; therefore, the absence of an FcgammaR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcgammaR- mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcgammaR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcgammaR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Characteristics of corneal xenograft rejection in a discordant species combination

PURPOSE: To characterize the fate of Lewis rat corneas transplanted to Hartley guinea pigs. METHODS: Full-thickness Lewis rat corneal buttons were grafted orthotopically to Hartley guinea pigs (xenografts), ACI rats (allografts), or Lewis rats (isografts). Two panels of recipients were presensitized with xenogeneic skin grafts or allogeneic skin grafts. Serum samples were collected pre- and post-transplant and analyzed by flow cytometry and indirect immunofluorescence. RESULTS: Unlike vascularized xenografts that reject within 30 min, corneal xenografts had a mean survival time of 8 days. Presensitization with guinea pig skin grafts increased recipient IgM and IgG xenoantibody levels, as measured by flow cytometry on guinea pig hematopoietic cells, and significantly accelerated corneal xenograft rejection with a mean survival time of 5 days. Presensitization with allogeneic ACI skin grafts had no effect on xenoantibody levels or xenogeneic corneal graft survival. Guinea pig corneas stained by indirect immunofluorescence with normal rat serum exhibited low (1+) but significant binding of IgG and IgM, primarily on epithelium and stroma. Serum from Lewis rats that rejected a corneal xenograft had elevated IgG and IgM xenoantibodies that reacted strongly (4+) with guinea pig cornea and heart. CONCLUSIONS: In the discordant guinea pig-to-rat species combination, donor corneas express xenoantigens; rejection of corneal xenografts stimulates IgM and IgG xenoantibody production; sensitization to xenoantigens can accelerate corneal xenograft rejection; and discordant corneal xenografts, unlike vascularized organs, are not hyperacutely rejected.     

Fc gamma RI blockade and modulation for immunotherapy

Splenectomy and corticosteroids are the treatment of choice for patients with immune thrombocytopenic purpura (ITP). However, for the 10%-15% of patients who do not respond to conventional therapy, high-dose i.v. IgG can induce life-saving transient responses. The benefits of i.v. IgG have been attributed to Fc receptor blockade; however, the involvement of the individual Fc receptors for IgG (Fc gamma R) in ITP remain to be more completely defined. Recently a mAb, designated mAb H22, which recognizes an epitope on Fc gamma RI (CD64) outside the ligand-binding domain, was humanized. Because mAb H22 is a human IgG1 and Fc gamma RI has a high affinity for human IgG1 antibodies, we predicted that mAb H22 would bind to the Fc gamma RI ligand-binding site through its Fc domain and to its external Fc gamma RI epitope through both Fab domains. These studies demonstrate that mAb H22 blocked Fc gamma RI-mediated phagocytosis of opsonized red blood cells more effectively than an irrelevant IgG. Moreover, cross-linking Fc gamma RI with mAb H22 down-modulated Fc gamma RI expression on monocytes, an effect seen within 2 h.     

Vaccination with DNA encoding an immunodominant myelin basic protein peptide targeted to Fc of immunoglobulin G suppresses experimental autoimmune encephalomyelitis

We explore here if vaccination with DNA encoding an autoantigenic peptide can suppress autoimmune disease. For this purpose we used experimental autoimmune encephalomyelitis (EAE), which is an autoaggressive disease in the central nervous system and an animal model for multiple sclerosis. Lewis rats were vaccinated with DNA encoding an encephalitogenic T cell epitope, guinea pig myelin basic protein peptide 68-85 (MBP68-85), before induction of EAE with MBP68-85 in complete Freund's adjuvant. Compared to vaccination with a control DNA construct, the vaccination suppressed clinical and histopathological signs of EAE, and reduced the interferon gamma production after challenge with MBP68-85. Targeting of the gene product to Fc of IgG was essential for this effect. There were no signs of a Th2 cytokine bias. Our data suggest that DNA vaccines encoding autoantigenic peptides may be useful tools in controlling autoimmune disease.     

Rat IgG subclasses mediating binding and phagocytosis of target cells by homologous macrophages

Attachment and ingestion of 51Cr-labelled TNP-SRBC sensitized by rat IgG1, IgG2a or IgG2b-type antibodies by homologous, elicited peritoneal macrophages were studied. IgG1 was found to be the most efficient isotype in mediating these functions. The antibody doses required for a significant attachment were found to differ with the isotype of Ab, while doses needed for a significant phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) varied between 400-700 Ab/SRBC with all the isotypes studied. Both binding and phagocytosis were also influenced by the degree of hapten conjugation when target cells were sensitized by IgG1. Inhibition of these functions by soluble immune complexes and monomeric immunoglobulins suggests the involvement of two Fc gamma R in binding of the three isotypes. Based on the present work and on previous results we conclude that IgG2a interacts with a receptor binding complexed IgG only (Fc gamma RII), IgG2b binds to a different receptor which appears to bind monomeric ligand as well (Fc gamma RI), while IgG1 seems to interact with both types of receptor. We propose that phagocytosis can be mediated by both Fc gamma RI and Fc gamma RII.     

Structural bases of Fc gamma R functions

This review describes structures which determine the biological activities triggered by Fc gamma R and account for the cell-mediated functions of IgG antibodies in physiology and pathology. The binding specificity and affinity of Fc gamma R depend primarily on IgG-binding structures, in their immunoglobulin-like extracellular domains. Binding is however also influenced by subunits that associate to multichain Fc gamma R. Effector and regulatory intracytoplasmic sequences that are unique to molecules of the Fc gamma RIIB family determine the internalization properties of these receptors. Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) are intracytoplasmic effector sequences shared by Fc gamma R and other receptors involved in the recognition of antigen, which trigger cell activation and internalization. Immunoreceptor Tyrosine-based Inhibition Motifs (ITIMs) are intracytoplasmic sequences, shared by Fc gamma RIIB and a growing number of negative coreceptors which negatively regulate cell activation via ITAM-bearing receptors. Altogether, these structures enable IgG antibodies to exert a variety of finely tuned biological effects during the immune response.     

Maturation of postsynaptic acetylcholine receptors in cochlear outer hair cells

Sound transduction in the inner ear is controlled by olivocochlear efferents terminating predominantly at outer hair cells (OHC). Development of efferent fibers and thereby of postsynaptic OHC receptors was studied immunohistochemically in 13 cochleae from fetal guinea pigs. The gestational ages of the animals ranged from gestational day (GD) 35 to GD 56. To visualize nicotinic acetylcholine receptors (nAChR), sera were used from myasthenia gravis patients with confirmed nAChR antibodies. At GD 53 no staining was observed, whereas at GD 58 a striking nAChR-immunoreactivity was found. In cochleae from adult animals postsynaptic receptors were visualized at the bases of all three rows of OHCs. The region of the inner hair cells (IHC) was not stained. The present results indicate that nAChRs in guinea pig cochleae develop between GD 53 and GD 58. Maturation of the postsynaptic nAChRs coincides with development of OHC motile properties.     

HLA-specific antibodies in highly sensitized patients can cause a positive crossmatch against pig lymphocytes

BACKGROUND: The presence of IgG HLA-specific antibodies in the serum of patients awaiting transplantation indicates T- and B-cell priming and would result in acute rejection of a poorly matched human allograft. Recent advances in xenotransplantation, with the amelioration of hyperacute rejection using transgenic pig kidneys, may benefit such patients. However, accelerated cellular rejection might result from the primed T-cell recognition of antigenic epitopes shared between pig and human MHC molecules. METHODS: We have compared the reactivity of IgG antibodies from 8 nonsensitized (NS) and 13 highly sensitized (HS) patients with human and pig lymphocytes by flow cytometry. Xenoreactive natural antibodies (XNA) were absorbed with pig red blood cells, and HLA class I-specific antibodies were further absorbed with pooled human platelets. RESULTS: Before XNA absorption, 20 of the 21 patients had a positive IgG crossmatch with pig lymphocytes, and there was no difference between NS and HS patients. In contrast, after XNA absorption, none of the 8 NS patients were positive, compared with 9 of the 13 HS patients (mean of the median channel fluorescence values of 7.7 and 86.5, respectively; P=<0.001). For XNA-absorbed HS patient sera, 20 of 30 (67%) pig lymphocyte crossmatch combinations were positive, with a mean median channel fluorescence value of 125 (range 31 to 294) compared with 9.5 (range 7 to 13) for the 10 crossmatch-negative combinations. Platelet absorption resulted in a concomitant reduction in antibody binding to pig lymphocytes in three of six HS patient sera, indicating that HLA class I-specific antibodies are responsible, at least in part, for the positive crossmatch. CONCLUSION: These results suggest that some IgG HLA-specific antibodies can bind to pig lymphocytes, analogous to a positive crossmatch with allogeneic donors.     

Monoclonal antibody to the type II Fc receptor for human IgG blocks potentiation of monocyte and neutrophil IgG-induced respiratory burst activation by aggregated C-reactive protein

The acute phase protein, CRP, when heat-aggregated (Agg-CRP), binds to human monocytes and neutrophils and potentiates the respiratory burst stimulated by heat-aggregated IgG (Agg-IgG). Earlier data from our laboratory and others have indicated that CRP binds to phagocytic cells at membrane sites associated with IgG Fc receptors. The present study utilized monoclonal antibodies (MAb) to determine whether the Agg-CRP potentiation of oxidative metabolism could be linked to activation through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Preincubation of monocytes with MAb 32.2, which recognizes an Fc gamma RI epitope distinct from its IgG binding site, had only a minimal (20%) inhibitory effect on Agg-IgG-induced luminol chemiluminescence (CL) and exerted no significant effect on its enhancement by Agg-CRP. MAb 10.1, which blocks IgG binding to Fc gamma RI, reduced Agg-IgG-induced monocyte CL by 40%, but did not alter the Agg-CRP-mediated enhancement. In contrast, exposure to MAb IV.3, which binds to Fc gamma RII on monocytes and neutrophils and blocks IgG binding to this receptor, resulted in a greater than 70%, inhibition of Agg-IgG-induced CL and also significantly suppressed the enhancement by Agg-CRP. MAb Leu-11b, which reacts with Fc gamma RIII on neutrophils, reduced Agg-IgG-induced CL by 70% but did not suppress the Agg-CRP potentiation. Preincubation of monocytes and neutrophils with anti-Leu-M1, anti-CR1, or anti-CR3 failed to block Agg-IgG-induced CL or its enhancement by Agg-CRP. Although the potentiating effect of Agg-CRP on Agg-IgG-elicited CL was blocked by MAb IV.3, this antibody failed to reduce binding of Agg-CRP to either monocytes or neutrophils. These results indicate that, although Agg-CRP does not bind to phagocytic cells at the IgG-binding determinant of Fc gamma RII, it alters Agg-IgG-induced cell activation through this receptor.     

Aneurysm of sinus of valsalva associated with rheumatic valvular disease

Supernatants of alloantigen-activated T cells contain a number of factors, including an immunoglobulin-binding factor (IBF) which inhibits complement-induced hemolysis of sheep erythrocytes coated with anti-Forssman IgG antibodies and a factor which suppresses IgM antibody synthesis in vitro. These two factors may be identical, since they are simultaneously retained on Sepharose beads to which IgG has been coupled and can be recovered by elution at pH 2.8. They do not bind to Sepharose beads to which IgM of F(ab')2 fragment of IgG has been coupled, demonstrating that they have a selective affinity for the Fc region of IgG. In addition, the fixation of IBF on the Fc portion of IgG reversibly inhibits subsequent binding of the first component of complement (C1), thus indicating that IBF does not irreversibly alter the C1 binding site(s) of IgG.     

Antibody-mediated basophil accumulations in cutaneous hypersensitivity reactions of guinea pigs

Cutaneous basophil hypersensitivity (CBH) was studied in guinea pigs by using simplified histologic techniques. Animals immunized with oxazolone or picryl conjugates of keyhole limpet hemocyanin (KLH) emulsified with complete (CFA) or incomplete Freund's adjunvant (IFA) were found to have hapten-specific cutaneous basophil reactions when skin tested at 1 week with oxazolone or picryl chloride contant painting or intradermal injection of oxazolone or picryl-conjugated human serum albumin, respectively. Thus, hapten-specific cutaneous basophil reactions were present in guinea pigs immunized with CFA for classical delayed hypersensitivity, and in animals immunized with IFA for Jones-Mote reactions. Hapten-specific 24-hr cutaneous basophil reactions were passively transferred with immune serum from donors sensitized with conjugates of oxazolone or picryl-KLH in CFA or IFA, and with serum from oxazolone contact-sensitized animals as well. As little as 0.5 ml sera obtained from donors 1 week after immunization could systemically transfer cutaneous basophil reactions. It is likely that hapten-specific cutaneous basophil reactions are mediated by small quantities of serum antibodies. We conclude that antibody-mediated cutaneous basophil reactions may be distinctive hypersensitivity responses that can be distinguished from classical anaphylactic, Arthus, and delayed hypersensitivities. It is suggested that CBH reactions are heterogeneous and that antibody products of B lymphocytes, and factors probably derived from T lymphocytes, play a role in basophil accumulations at cutaneous hypersensitivity reactions.     

General versus specific trait anxiety measures in the prediction of fear of snakes, heights, and darkness

The histochemical changes in succinate dehydrogenase (SDH) were investigated in pectoralis major muscle of guinea pig, rat and mouse after low level X-irradiation (72 R and 240 R) and compared with control animals. Biochemical studies were carried out on liver, kidney, muscle (pectoralis major), adrenal and spleen of these animals after low dose local X-irradiation and compared with control animals. Changes in SDH activity were studied up to 72-h post-irradiation, which shows that low dose local X-irradiation leads to increased enzymic activity. The increase in enzymic activity was remarkable in mouse tissues as compared with guinea pig and rat. Adrenals of all the three animals showed significant activation after all the doses of radiation studied. The significance of these results, with special reference to oxidative metabolism, has been discussed.     

FcalphaRI (CD89) as a novel trigger molecule for bispecific antibody therapy

Promising results from clinical trials with unconjugated antibodies stimulated renewed interest in immune effector mechanisms of monoclonal antibodies (MoAbs). We investigated the potential of IgA as antibody isotype for cell- or complement-mediated tumor cell lysis and assessed the potential of its myeloid Fc receptor, FcalphaRI (CD89), as trigger molecule for bispecific antibody (BsAb)-mediated immunotherapy. Comparing hapten-directed antibodies of human IgA2 with IgG1 or IgG3 isotypes, we found all three to mediate effective killing of sensitized tumor target cells in whole blood assays. Analysis of effector mechanisms showed IgG-mediated lysis to be predominantly complement-dependent, whereas IgA-dependent killing was primarily effector cell-mediated. A comparison of effector cell populations in antibody-dependent cell-mediated cytotoxicity (ADCC) showed neutrophils to be most important for IgA-dependent tumor cell killing, involving FcalphaRI as shown with Fc receptor blocking antibodies. Reverse ADCC experiments against target cells sensitized with Fc receptor antibodies, or assays with FcalphaRI-directed bispecific antibodies confirmed FcalphaRI as effective trigger molecule in polymorphonuclear neutrophil (PMN)-mediated lysis. During granulocyte colony-stimulating factor (G-CSF ) therapy, (FcalphaRI x HER-2/neu) bispecific antibodies induced enhanced killing of HER-2/neu positive SK-BR-3 breast cancer cells in whole blood assays. This enhanced cytotoxicity was paralleled by increased PMN counts, which lead to higher effector to target cell ratios in G-CSF-primed blood. Furthermore, bispecific antibodies, directed to FcalphaRI and Candida albicans, enhanced neutrophils' phagocytosis of fungi. In summary, these results identify IgA as an effective antibody isotype for immunotherapy, working primarily via FcalphaRI on neutrophils. They suggest FcalphaRI-directed bispecific antibodies and G-CSF to be an attractive combination for malignant or infectious diseases.