FcγRIII (CD16)‐mediated ADCC by NK cells is regulated by monocytes and FcγRII (CD32)

Monocytes are known to engage in reciprocal crosstalk with NK cells but their influence on NK‐cell‐associated antibody‐dependent cellular cytotoxicity (ADCC) is not well understood. We demonstrate that in humans FcγRIII (CD16)‐dependent ADCC by NK cells is considerably enhanced by monocytes, and that this effect is regulated by FcγRII (CD32) crosslinking in healthy individuals. It is known that during HIV‐1 infection, NK cells are known to express low levels of CD16 and exhibit reduced ADCC. We show that immune regulation of CD16‐mediated NK‐cell cytotoxicity by monocytes through CD32 engagement is substantially disturbed in chronic progressive HIV‐1 infection. Expression of activating isoform of CD32 represented a compensatory mechanism for reduced expression of CD16 on NK cells during HIV‐1 infection. As a result, the regulation of NK‐cell‐associated ADCC by monocytes is skewed and eventually constitutes a novel factor that contributes to HIV‐1‐associated immune deficiency, dysregulation and pathogenesis. Our data therefore provide evidence, for the first time, that in humans monocytes act as a rheostat for FcγRIII‐mediated NK‐cell functions maintaining a well‐balanced immune response.     

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.     

FcγRII and multi-system autoimmune disease

The FcR are a crucial link in the immune response between humoral and cellular immunity and cell-based effector systems, mediating a wide variety of physiological and biochemical responses. The FcR for IgG (FcγR) and in particular the most widely expressed of these, FcγRII, are important in regulating adaptive immunity. Disruption of their function is a key factor in the development of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), which are characterized by chronic, multi-organ inflammation. Studies of the FcγRII include structure/function relationships, investigation of the associations between FcR polymorphisms and human disease and animal studies using knockout or transgenic mouse models. These investigations showed that the various forms of FcγRII interact with immune complexes to either initiate or inhibit inflammation. In conjunction with environmental antigens and genotype, the FcγRII activating and inhibitory receptors determine the nature and magnitude of response to antigens. In this review, the structure and function of the FcγRIIs and their role in immune complex-mediated auto-immunity are discussed.     

Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications

Receptors for the Fc domain of IgG (FcγR) provide a critical link between specific humoral responses and the cellular branch of the immune system. When hFcγR interact with immunoglobulin, a variety of biological responses are triggered. These include phagocytosis, endocytosis, antibody-dependent cellular cytotoxicity (ADCC), release of inflammatory mediators, and enhancement of antigen presentation. In the last few years our understanding of the Fcγ receptor structure has increased dramatically, due to the availability of monoclonal antibodies (mAb) and cDNA probes. FcγR are members of the immunoglobulin superfamily and three main classes, hFcγRI, hFcγRII, and hFcγRIII are recognized in man¹⁻³ generating at least 12 different isoforms. A further level of complexity is introduced by various genetic polymorphisms and, importantly, recent evidence points at the relevance of this FcγR heterogeneity.     

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.     

Neutrophils as immune effector cells in antibody therapy in cancer

Tumor-targeting monoclonal antibodies are available for a number of cancer cell types (over)expressing the corresponding tumor antigens. Such antibodies can limit tumor progression by different mechanisms, including direct growth inhibition and immune-mediated mechanisms, in particular complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be mediated by various types of immune cells, including neutrophils, the most abundant leukocyte in circulation. Neutrophils express a number of Fc receptors, including Fcγ- and Fcα-receptors, and can therefore kill tumor cells opsonized with either IgG or IgA antibodies. In recent years, important insights have been obtained with respect to the mechanism(s) by which neutrophils engage and kill antibody-opsonized cancer cells and these findings are reviewed here. In addition, we consider a number of additional ways in which neutrophils may affect cancer progression, in particular by regulating adaptive anti-cancer immunity.     

Defective antibody-dependent tumour cell lysis by neutrophils from cancer patients.

Neutrophil antibody-dependent cellular cytotoxicity (ADCC) against Raji target cells was studied in 32 patients with lung or gastro-intestinal carcinoma and 25 healthy controls. Seventeen of the patients (53%) had defective ADCC. Moreover, neutrophils obtained from patients with defective ADCC were found to bind IgG-coated target cells normally. Thus, the defect responsible for the impaired lysis appears to be distal to the Fc receptor (FcR)-mediated target cell binding by neutrophils.     

Polyanions Inhibit Murine Macrophage Fc Receptor Mediated ADCC and Binding

We examined the effect of various polyanions on mouse complement hemolytic activity, Fc receptor (FcR) subclass mediated antibody-dependent cellular cytotoxicity (ADCC) as well as binding by mouse peritoneal macrophages (MΦ) of sheep erythrocyte targets. All the polyanions tested (dextran sulfate, carrageenan, polyvinyl sulfate, pentosan polysulfate and polyanethol sulfonic acid) inhibited the hemolytic activity of mouse serum complement to varying degrees. Polyanions inhibited ADCC mediated by either IgG2a or IgG2b in a reversible manner. FcR subclass mediated binding studies at 4°C indicated that the various polyanions compete for FcR binding of sheep erythrocytes opsonized with murine IgG2a, IgG2b and polyclonal IgG. Polyanethol sulfonic acid was uniformly the most potent inhibitor of mouse CH50 and FcR dependent ADCC and binding functions, but: did not affect C3b receptor mediated binding.     

Effect of rIFN-gamma on antibody-mediated cytotoxicity via human monocyte IgG Fc receptor II (CD32).

Human monocytes and macrophages express an isoform of IgG Fc receptor II (Fc gamma RII), Fc gamma RIIa. Two allotypic variants of this receptor could be distinguished with respect to their ability to bind murine (m)IgG1 complexes either strongly or weakly, defined as high-responder (HR) and low-responder (LR), respectively. We investigated the effect of recombinant (r)IFN-gamma on the ability of freshly isolated monocytes, and those cultured for 40 h and 9 days, to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Using human erythrocytes (E) sensitized with mIgG1 as target cells, Fc gamma RII was studied selectively. Cells which had been cultured for 40 h exhibit a significantly decreased Fc gamma RII expression, and Fc gamma RII-mediated ADCC activity as compared with freshly isolated monocytes. Co-culture with rIFN-gamma (40 h) reversed this decrease. Short-term rIFN-gamma-cultured cells, and fresh cells express similar numbers of Fc gamma RII, and exhibit comparable Fc gamma RII-mediated ADCC activity. Phagocytic activity was not affected. Prolonged culture of monocytes for 9 days, co-cultured with rIFN-gamma either from day 0 or from day 7, did not affect expression or functional activity of Fc gamma RII. Furthermore, the effects were observed in both HR and LR individuals. Our results show that rIFN-gamma has strong effects on Fc gamma RII-mediated responses specifically during the early stages of monocyte maturation, most likely by affecting receptor expression levels.     

Polyanions Inhibit Murine Macrophage Fc Receptor Mediated ADCC and Binding

We examined the effect of various polyanions on mouse complement hemolytic activity, Fc receptor (FcR) subclass mediated antibody-dependent cellular cytotoxicity (ADCC) as well as binding by mouse peritoneal macrophages (MΦ) of sheep erythrocyte targets. All the polyanions tested (dextran sulfate, carrageenan, polyvinyl sulfate, pentosan polysulfate and polyanethol sulfonic acid) inhibited the hemolytic activity of mouse serum complement to varying degrees. Polyanions inhibited ADCC mediated by either IgG2a or IgG2b in a reversible manner. FcR subclass mediated binding studies at 4°C indicated that the various polyanions compete for FcR binding of sheep erythrocytes opsonized with murine IgG2a, IgG2b and polyclonal IgG. Polyanethol sulfonic acid was uniformly the most potent inhibitor of mouse CH50 and FcR dependent ADCC and binding functions, but: did not affect C3b receptor mediated binding.     

Anti‐HIV‐1 antibody‐dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG

Antibodies with antibody‐dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV‐1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV‐specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV‐1 envelope gp140 and elicited high titers of anti‐gp140‐binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ‐receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose‐dependent manner. Antibody‐dependent killing was observed in the presence of anti‐HIV‐1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab’)₂ fragments. ADCC activity was primarily mediated by CD14⁺ monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte‐mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti‐HIV colostrum IgG have robust HIV‐1‐specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV‐1 infection. This could assist the development of novel Ab‐mediated approaches for prevention of HIV‐1 transmission.     

Activity of Two Types of Fc Receptors, FcγRI and FcγRII, in Human Monocyte Cytotoxicity to Sensitized Erythrocytes

We investigated the cytotoxicity of human monocytes mediated by two types of receptors for the Fc portion of IgG, Fc gamma RI and Fc gamma RII. Erythrocytes sensitized with human IgG (EA-human IgG) were used to assay Fc gamma RI function, and erythrocytes sensitized with mouse IgG1 (EA-mouse IgG1) were used to assay Fc gamma RII. Both types of Fc gamma R were observed to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), which was further characterized by using different monoclonal anti-Fc gamma R antibodies (MoAb) and monomeric IgG. Lysis of EA-human IgG was inhibited by both monomeric human IgG and mouse IgG2a in a dose-dependent way, and also by anti-Fc gamma RI MoAb 10.1. Cytolysis of EA-mouse IgG1 was inhibited by monomeric mouse IgG1 and by two anti-Fc gamma RII MoAb, IV.3 and CIKM5. Antibodies of the mouse IgG2b isotype affected neither type of ADCC. The effectiveness of cytotoxicity mediated by either of the Fc gamma R was studied by means of targets sensitized with a calibrated number of IgG molecules. At least 20 times more IgG molecules per target cell were necessary to obtain half-maximal cytotoxicity mediated by Fc gamma RII than for Fc gamma RI-mediated cytolysis. Furthermore, the previously described polymorphism of Fc gamma RII was also reflected in Fc gamma RII-dependent cytotoxicity. These studies demonstrate that Fc gamma RII can mediate ADCC, although a higher degree of target cell sensitization is required than for Fc gamma RI-mediated ADCC.     

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.     

Multiple Loss of Effector Cell Functions in FcRγ-Deficient Mice

Immunoglobulin Fc receptor (FcR) γ subunit is a component of low affinity receptor for IgG, FCγRIII, as well as high affinity receptor for IgE, FcεRI. This subunit is required for efficient surface expression of these FcRs on various cells in immune system. The FcRγ-deficient mice, generated by gene targeting in embryonic stem cells, exhibit multiple defects in FcR-mediated effector cell responses, including absence of phagocytic activity against opsonized red blood cells by activated macrophages, loss of antibody-dependent cell-mediated cytotoxicity manifested by IL-2-induced splenic NK cells, and unresponsiveness of mast cells to crosslinking of IgE on these cells. These results demonstrate an indispensable role of FcRγ for functional expression of FcRs, and clearly indicate the importance of FCγRIII as well as FcεRI for these effector functions. Since FcRγ-deficient mice is unable to mount the type II and type III hypersensitivity reactions, it is suggested that FcRs play pivotal roles in initiating these reaction cascades. The mutant mice should prove to be useful in evaluating FcRs in various humoral and cellular immune responses, and in developing new strategies for treatment of immunodeficiency as well as autoimmune disorders.     

High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

PurposeHost genetic variants in activating natural killer (NK) cell receptors may contribute to differences in severity of COVID-19. NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) responses play, however, a controversial role in SARS-CoV-2 infections. It is unclear whether proinflammatory and cytotoxic SARS-CoV-2-specific ADCC responses limit disease severity or rather contribute to the immunopathogenesis of severe COVID-19.MethodsUsing a genetic association approach and subsequent in vitro antibody-dependent NK cell activation experiments, we investigated whether genetic variants in the FcγRIIIa-encoding FCGR3A gene, resulting in expression of either a low-affinity or high-affinity variant, and individual SARS-CoV-2-specific ADCC response contribute to COVID-19 severity.ResultsIn our study, we showed that the high-affinity variant of the FcγRIIIa receptor, 158-V/V, is significantly over-represented in hospitalized and deceased patients with COVID-19, whereas the low-affinity FcγRIIIa-158-F/F variant occurs more frequently in patients with mild COVID-19 (P < .0001). Furthermore, functional SARS-CoV-2 antibody-specific NK cell-mediated ADCC assays revealed that significantly higher proinflammatory ADCC responses occur in hospitalized patients with COVID-19, and are especially observed in NK cells expressing the FcγRIIIa-158-V/V variant (P < .0001).ConclusionOur study provides evidence that pronounced SARS-CoV-2-specific NK cell-mediated ADCC responses are influenced by NK cell FcγRIIIa genetic variants and are a hallmark of severe COVID-19.     

FcγRIa–γ-chain complexes trigger antibody-dependent cell-mediated cytotoxicity (ADCC) in CD5+ B cell/macrophage IIA1.6 cells

Most receptors for immunoglobulins exist as multi-subunit complexes, with unique ligand binding α-chains, combined with accessory signalling (γ-, β-, or ζ-) chains. The myeloid class I receptor for IgG (FcγRIa) has been shown to be dependent on the FcR γ-chain for surface expression in vivo. In this study we assess the capacity of FcγRIa–γ-chain complexes expressed in IIA1.6 cells to trigger phagocytosis and ADCC. An intact immunoreceptor tyrosine-based activation motif (ITAM) signalling motif proved essential for triggering of biological function via the FcγRIa receptor complex. Both the FcR γ-chain and the FcγRIIa–ITAM proved active in directing phagocytosis of Staphylococcus aureus and ADCC of erythrocytes, triggered by the FcγRIa complex. The capacity of FcγRIa to trigger phagocytic and cytolytic activity by IIA1.6 cells, both considered ‘professional phagocyte’ functions, motivated us to re-evaluate the cell lineage and developmental stage of IIA1.6 cells. Although originally described as mouse B lymphocytes, the IIA1.6 cells proved positive for non-specific esterase activity and expressed the CD5 antigen. These combined characteristics place the IIA1.6 cells within a unique CD5⁺ B cell/macrophage lineage, optimally suited for cell biological analyses of phagocyte receptors.     

Human immunodeficiency virus infection of monocytes: relationship to Fc-gamma receptors and antibody-dependent viral enhancement.

Antibodies that augment human immunodeficiency virus (HIV) infectivity of monocytes through Fc receptor (FcR) type III for IgG have been found in the blood of sero-positive patients and immunized chimpanzees. This study investigated the effect of acute and chronic HIV infection, as well as protein kinase C activators capable of up-regulating latent HIV, on the expression of these receptors. In addition, the frequency of this antibody-dependent enhancement (ADE) phenomenon was estimated using purified IgG from HIV-1 seropositive individuals at various clinical stages of infection. The existence of an FcR-dependent pathway for ADE of HIV-1 infection in peripheral blood monocytes and promonocytic U937 cells was confirmed in sera from a small subset of patients, and the phenomenon extended to FcR types I and II. The level of ADE activity was minimal, however, and no relationship between the presence or magnitude of the ADE phenomenon and clinical stage was uncovered. Finally, perturbations which activate a latent HIV infection were shown to concomitantly up-regulate FcR on infected and uninfected cells. This suggests a positive feedback loop linking up-regulation of latent infection, enhanced expression of low affinity HIV receptors such as FcR, and viral spread.     

Functional and clinical consequences of changes to natural killer cell phenotypes driven by chronic cytomegalovirus infections

Cytomegalovirus (CMV) infections may affect natural killer (NK) cells and are implicated in age-related disorders—notably poor vascular endothelial function. Changes may be greater in renal transplant recipients (RTR) as they have a high burden of CMV and may influence antibody-dependent cellular cytotoxicity (ADCC) responses to viral antigen. We obtained blood mononuclear cells from RTR stable after transplantation (n = 27) and age- and sex-matched controls (n = 28). Natural killer (NK) cells were assessed for expression of CD107a or TNF-α, after stimulation with autologous antibodies bound to CMV glycoprotein B (measuring ADCC) or anti-CD16 (measuring NK cell activation). Alleles of FCRG3A (encoding CD16; rs396991) were determined by the Taqman assay. The vascular endothelial function was assessed using flow-mediated dilatation (FMD) of the brachial artery. Proportions of NK cells expressing CD16 ex vivo were lower in RTR. Frequencies of NK cells expressing NKG2C or LIR-1 or lacking FcRγ were highest in CMV-seropositive RTR. ADCC was affected by rs396991 genotype and CMV gB antibody levels, but not by RTR status or detection of CMV DNA in plasma. Responses of FcRγ-NK cells to anti-CD16 were lower compared to FcRγ+ NK cells. Increased percentages of LIR-1 ⁺ and FcRγ− NK cells correlated with lower FMD. In summary, CMV evokes substantial and similar ADCC responses in CMV seropositive RTR and controls. The equivalence may reflect higher titers of CMV reactive antibody in RTR, as NK responses stimulated by ligation of CD16 were lower. NK cells that were LIR-1 ⁺ and/or FcRγ− were induced by CMV and correlated inversely with vascular endothelial function.     

Association of the p56lck protein tyrosine kinase with the FCγRIIIA/CD16 complex in human natural killer cells

The multimeric FcγRIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50–70-kDa transmembrane glycoprotein Fcγ receptor (CD16), the T cell receptor (TCR)-ζ chain, and the FcεRIγ chain. Cross-linking FcγRIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the ζ, subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the FcγRIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human FcγRIIIA is complexed with p56^lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56^lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several FcγRIIIA-associated proteins are identified in immune complex kinase assays including the TCR-ζ, subunit, a p70–90 ζ-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56^lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56^lck is physically associated with FcγRIIIA(CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.