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.     

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen‐specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcγ receptor (FcγR)‐mediated antigen uptake and enhance antigen presentation resulting in augmented T‐cell proliferation. We examined the impact of antigen presentation and T‐cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene‐deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcγR‐deficient mice. Lung DC of WT, but not FcγR‐deficient mice, induced increased antigen‐specific CD4⁺ T‐cell proliferation when pulsed with anti‐OVA IgG immune complexes. Intranasal application of anti‐OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen‐specific T‐cell proliferation in vivo. Finally, antigen‐specific IgG in the serum of sensitized mice led to a significant increase of antigen‐specific CD4⁺ T‐cell proliferation induced by WT, but not FcγR‐deficient, lung DC. We conclude that FcγR‐mediated enhanced antigen presentation and T‐cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.     

Low‐affinity FcγR interactions can decide the fate of novel human IgG‐sensitised red blood cells and platelets

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1‐sensitised counterparts. However, non‐destructive splenic retention of G1Δnab‐coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab‐sensitised RBCs did not cause FcγRI‐mediated monocyte activation, FcγRIIIa‐mediated antibody‐dependent cell‐mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low‐affinity binding to this receptor class. Additional contacts via P‐selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII‐dependent activation by G1Δnab. These results emphasise the physiological relevance of low‐affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab‐coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab‐sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions.     

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody‐dependent cellular cytotoxicity towards cancer cells

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti‐cancer antibodies act through different mechanisms, including antibody‐dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47‐SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab‐coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa‐131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa‐131R. Furthermore, ADCC was consistently enhanced by targeting CD47‐SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47‐SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47‐SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.     

Functional advantage of educated KIR2DL1+ natural killer cells for anti‐HIV‐1 antibody‐dependent activation

Evidence from the RV144 HIV‐1 vaccine trial implicates anti‐HIV‐1 antibody‐dependent cellular cytotoxicity (ADCC) in vaccine‐conferred protection from infection. Among effector cells that mediate ADCC are natural killer (NK) cells. The ability of NK cells to be activated in an antibody‐dependent manner is reliant upon several factors. In general, NK cell‐mediated antibody‐dependent activation is most robust in terminally differentiated CD57⁺ NK cells, as well as NK cells educated through ontological interactions between inhibitory killer immunoglobulin‐like receptors (KIR) and their major histocompatibility complex class I [MHC‐I or human leucocyte antigen (HLA‐I)] ligands. With regard to anti‐HIV‐1 antibody‐dependent NK cell activation, previous research has demonstrated that the epidemiologically relevant KIR3DL1/HLA‐Bw4 receptor/ligand combination confers enhanced activation potential. In the present study we assessed the ability of the KIR2DL1/HLA–C2 receptor/ligand combination to confer enhanced activation upon direct stimulation with HLA‐I‐devoid target cells or antibody‐dependent stimulation with HIV‐1 gp140‐pulsed CEM.NKr‐CCR5 target cells in the presence of an anti‐HIV‐1 antibody source. Among donors carrying the HLA‐C2 ligand for KIR2DL1, higher interferon (IFN)‐γ production was observed within KIR2DL1⁺ NK cells than in KIR2DL1^– NK cells upon both direct and antibody‐dependent stimulation. No differences in KIR2DL1⁺ and KIR2DL1^– NK cell activation were observed in HLA‐C1 homozygous donors. Additionally, higher activation in KIR2DL1⁺ than KIR2DL1^– NK cells from HLA–C2 carrying donors was observed within less differentiated CD57^– NK cells, demonstrating that the observed differences were due to education and not an overabundance of KIR2DL1⁺ NK cells within differentiated CD57⁺ NK cells. These observations are relevant for understanding the regulation of anti‐HIV‐1 antibody‐dependent NK cell responses.     

NK cell function and antibodies mediating ADCC in HIV-1-infected viremic and controller patients

Natural killer (NK) cells have been suggested to play a protective role in HIV disease progression. One potent effector mechanism of NK cells is antibody-dependent cellular cytotoxicity (ADCC) mediated by antiviral antibodies binding to the FcγRIIIa receptor (CD16) on NK cells. We investigated NK cell-mediated ADCC function and the presence of ADCC antibodies in plasma from 20 HIV-1-infected patients and 10 healthy donors. The HIV-positive patients were divided into two groups: six who controlled viremia for at least 8 y without treatment (controllers), and 14 who were persistently viremic and not currently on treatment. Plasma from both patient groups induced NK cell IFN-γ expression and degranulation in response to HIV-1 envelope (Env) gp140-protein-coated cells. Patient antibodies mediating ADCC were largely directed towards the Env V3 loop, as identified by a gp140 protein lacking the V3 loop. Interestingly, in two controllers ADCC-mediating antibodies were more broadly directed to other parts of Env. A high viral load in patients correlated with decreased ADCC-mediated cytolysis of gp140-protein-coated target cells. NK cells from both infected patients and healthy donors degranulated efficiently in the presence of antibody-coated HIV-1-infected Jurkat cells. In conclusion, the character of ADCC-mediating antibodies differed in some controllers compared to viremic patients. NK cell ADCC activity is not compromised in HIV-infected patients.     

Independent evolution of Fc‐ and Fab‐mediated HIV‐1‐specific antiviral antibody activity following acute infection

Fc‐related antibody activities, such as antibody‐dependent cellular cytotoxicity (ADCC), or more broadly, antibody‐mediated cellular viral inhibition (ADCVI), play a role in curbing early SIV viral replication, are enriched in human long‐term infected nonprogressors, and could potentially contribute to protection from infection. However, little is known about the mechanism by which such humoral immune responses are naturally induced following infection. Here, we focused on the early evolution of the functional antibody response, largely driven by the Fc portion of the antibody, in the context of the evolving binding and neutralizing antibody response, which is driven mainly by the antibody‐binding fragment (Fab). We show that ADCVI/ADCC‐inducing responses in humans are rapidly generated following acute HIV‐1 infection, peak at approximately 6 months postinfection, but decay rapidly in the setting of persistent immune activation, as Fab‐related activities persistently increase. Moreover, the loss of Fc activity occurred in synchrony with a loss of HIV‐specific IgG3 responses. Our data strongly suggest that Fc‐ and Fab‐related antibody functions are modulated in a distinct manner following acute HIV infection. Vaccination strategies intended to optimally induce both sets of antiviral antibody activities may, therefore, require a fine tuning of the inflammatory response.     

Human Fcγ receptors compete for TGN1412 binding that determines the antibody's effector function

The first‐in‐human clinical trial of the CD28‐specific monoclonal antibody (mAb) TGN1412 resulted in a life‐threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγ receptor (FcγR) interactions, these interactions contributed to TGN1412‐induced T‐cell activation. Using cell lines (TFs) expressing human FcγRI, ‐IIa, ‐IIb, or ‐III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412‐decorated T cells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest T cell proliferation, while high‐affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T‐cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T‐cell proliferation. In line with this, FcγRI^?FcγRII⁺ but not FcγRI⁺FcγRII⁺ monocytes mediate TGN1412‐induced T‐cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR‐mediated effector function, which has major implications for the design of therapeutic mAbs.     

Non‐neutralizing epitopes induce robust hepatitis C virus (HCV)‐specific antibody‐dependent CD56+ natural killer cell responses in chronic HCV‐infected patients

Natural killer (NK) cell‐mediated antibody‐dependent cellular cytotoxicity (NK‐ADCC) is of considerable interest in viral infection. However, little is known about NK‐ADCC responses in chronic hepatitis C virus (HCV) infection. In this study, impaired non‐specific antibody‐dependent CD56⁺ NK cell responses were observed in chronic HCV infection, as shown by decreased degranulation (extracellular CD107a expression) and interferon (IFN)‐γ production in response to antibody‐bound P815 cells. A peptide pool composed of epitopes recognized by anti‐HCV‐E1/E2 antibodies could induce pronounced HCV‐specific antibody‐dependent NK cell responses in sera from approximately half the chronic HCV carriers. Additionally, HCV‐specific epitopes with the capacity to induce robust NK‐ADCC activity were identified. Five linear NK‐ADCC epitopes (aa211‐aa217, aa384‐aa391, aa464‐aa475, aa544‐aa551 and aa648‐aa659 of the HCV envelope) were identified and do not overlap with putative linear neutralizing epitopes. This study revealed the dysfunctional characteristics of antibody‐dependent CD56⁺ NK cell responses in chronic HCV carriers. The key non‐neutralizing NK‐ADCC epitopes identified in this study may act as new targets for immunological intervention.     

In vivo depletion of CD4+FOXP3+ Treg cells by the PC61 anti‐CD25 monoclonal antibody is mediated by FcγRIII+ phagocytes

Depletion of CD4⁺CD25⁺FoxP3⁺ Treg using PC61 mAb (anti‐murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated ～70% of CD4⁺FoxP3⁺ cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcγ receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcγ receptor (FcγR)III^?/? mice were resistant to Treg depletion. FcγRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab‐dependent‐cellular‐cytotoxicity and Ab‐dependent‐cellular‐phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab‐dependent‐cellular‐phagocytosis of CD25⁺ cells by WT or FcγRIIB^?/?, but not FcγRIII^?/?, macrophages. Altogether these data demonstrate the critical role of FcγRIII⁺ phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.     

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.     

Intraperitoneal Immunization with Cry1Ac Protoxin from Bacillus thuringiensis Provokes Upregulation of Fc‐Gamma‐II/and Fc‐Gamma‐III Receptors Associated with IgG in the Intestinal Epithelium of Mice

In humans, intestinal epithelial FcRn is expressed throughout life and mediates the bidirectional transport of IgG, but in mice, it is markedly expressed in neonatal intestine. In adults, its expression is only faintly upregulated after intestinal IgG induction such as that elicited by i.p. immunization with Cry1Ac protoxin (pCry1Ac) Bacillus thuringiensis. This led us to suggest that additional Fcγ receptors (Fcγ‐R) may be participating in epithelial IgG uptake. So, first we determined whether CD16/32 [an epitope shared by Fcγ‐RII (CD32) and Fcγ‐RIII (CD16)] was expressed in the intestinal epithelia of mice. Using confocal microscopy and flow cytometry, we detected co‐localization of IgG and CD16/32 in epithelial cells, whose frequency was increased by immunization with pCry1Ac. Western blot and cross‐immunoprecipitation results with anti‐CD16/32 and IgG antibodies in epithelial cell extracts suggested that epithelial cells bear both Fcγ‐RII and Fcγ‐RIII and contained IgG associated with Fcγ‐RII/RIII. Using anti‐CD32 and anti‐CD16 antibodies, we confirmed by Western blot, confocal microscopy and flow cytometry that both Fcγ‐RII and Fcγ‐RIII were expressed and suggested that upregulation occurred upon immunization in intestinal epithelia. Finally, we examined the in vitro effect of anti‐CD16/32, anti‐CD16 and anti‐CD32 antibodies on IgG uptake and transport by intestinal epithelial cells and found that it was partially reduced. Although further studies are still required, our results suggest that Fcγ‐RII and Fcγ‐RIII might participate in the uptake and/or transport of IgG through the intestinal epithelia of adult mice.     

An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of FcɛRI with FcγRIIb

Background IgE binds to mast cells and basophils via its high‐affinity receptor, Fc?RI, and cross‐linking of Fc?RI‐bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross‐linking of Fc?RI with FcγRIIb, an ITIM‐containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti‐idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to Fc?RI‐bound IgE, via its Fab regions, and the negative regulatory receptor, FcγRIIb, via its Fc region. Objective To assess the ability of human 2G10 to inhibit anti‐IgE and allergen‐driven basophil degranulation through cross‐linking of Fc?RI‐bound IgE with FcγRIIb. Methods 2G10 was assessed for its ability to bind to FcγRIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti‐IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL‐4 expression intracellularly, using flow cytometery. Results Human 2G10 was able to bind to FcγRIIb on transfected cells and on purified basophils, and induce a dose‐dependent inhibition of both anti‐IgE and Der p 1‐driven degranulation of basophils. Conclusion The inhibition of basophil degranulation by the human IgG1 anti‐idiotype 2G10 highlights the therapeutic potential of IgE‐reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor FcγRIIb.     

Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans

Removal of the fucose residue from the N‐glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody‐dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2‐Å structure of the complex formed between nonfucosylated IgG1‐Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N‐glycosylation sites. The crystal structure shows that one of the two N‐glycans of sFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex. However, fucosylation of the Fc N‐glycans inhibits this interaction, because of steric hindrance, and furthermore, negatively affects the dynamics of the receptor binding site. Our results offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation.     

Differential inhibition of trastuzumab- and cetuximab-induced cytotoxicity of cancer cells by immunoglobulin G1 expressing different GM allotypes

Antibody-dependent cell-mediated cytotoxicity (ADCC), which links the innate and the adaptive arms of immunity, is a major host immunosurveillance mechanism against tumours, as well as the leading mechanism underlying the clinical efficacy of therapeutic antibodies such as cetuximab and trastuzumab, which target tumour antigens, human epidermal growth factor receptor (HER)1 and HER2, respectively. Immunoglobulin (Ig)G antibody-mediated ADCC is triggered upon ligation of Fcγ receptor (FcγR) to the Fc region of IgG molecules. It follows that genetic variation in FcγR and Fc could contribute to the differences in the magnitude of ADCC. Genetic variation in FcγR is known to contribute to the differences in the magnitude of ADCC, but the contribution of natural genetic variation in Fc, GM allotypes, in this interaction has hitherto not been investigated. Using an ADCC inhibition assay, we show that IgG1 expressing the GM 3+, 1-, 2- allotypes was equally effective in inhibiting cetuximab- and trastuzumab-mediated ADCC of respective target cells, in the presence of natural killer (NK) cells expressing either valine or phenylalanine allele of FcγRIIIa. In contrast, IgG1 expressing the allelic GM 17+, 1+, 2+ allotypes was significantly more effective in inhibiting the ADCC - mediated by both monoclonal antibodies - when NK cells expressed the valine, rather than the phenylalanine, allele of FcγRIIIa. These findings have important implications for engineering antibodies (with human γ1 constant region) against malignancies characterized by the over-expression of tumour antigens HER1 and HER2 - especially for patients who, because of their FcγRIIIa genotype, are unlikely to benefit from the currently available therapeutics.     

A constant threat for HIV: Fc‐engineering to enhance broadly neutralizing antibody activity for immunotherapy of the acquired immunodeficiency syndrome

Passive immunotherapy with polyclonal or hyperimmune serum immunoglobulin G (IgG) preparations provides an efficient means of protecting immunocompromised patients from microbial infections. More recently, the use of passive immunotherapy to prevent or to treat established infections with the human immunodeficiency virus (HIV) has gained much attention, due to promising preclinical data obtained in monkey and humanized mouse in vivo model systems, demonstrating that the transfer of HIV‐specific antibodies can not only prevent HIV infection, but also diminish virus load during chronic infection. Furthermore, an array of broadly neutralizing HIV‐specific antibodies has become available and the importance of the IgG constant region as a critical modulator of broadly neutralizing activity has been demonstrated. The aim of this review is to summarize the most recent findings with regard to the molecular and cellular mechanisms responsible for antibody‐mediated clearance of HIV infection, and to discuss how this may help to improve HIV therapy via optimizing Fcγ‐receptor‐dependent activities of HIV‐specific antibodies.     

Anti‐FcγRIIB (CD32) Antibodies Differentially Modulate Murine FVIII‐Specific Recall Response in vitro

Fc gamma receptors (FcγRs) for IgG regulate adaptive immune responses by modulating activating and inhibitory signalling pathways within immune cells. Data from a haemophilia A mouse model demonstrate that genetic deletion or blockade of the inhibitory FcγR (CD32) suppresses the formation of antibody‐secreting cells (ASCs) in vitro. Mechanisms preventing the FVIII‐specific recall response, however, remain unclear. Here, the potential role of CD32 inhibition was studied by differentially modulating receptor activity with selected anti‐CD32 monoclonal antibodies (mAbs). Splenocytes from immunized FVIII^‐/‐ mice were restimulated with FVIII in the absence or presence of different anti‐CD32 mAbs over 6 days. At day 6, cytokine release was quantified from cell culture supernatant and the formation of FVIII‐specific ASCs assessed. Binding of FVIII‐containing immune complexes (F8‐ICs) to bone marrow‐derived dendritic cells (BMdDCs) was also investigated. The antagonistic CD32 mAb AT128 suppressed the formation of FVIII‐specific ASCs and reduced secretion of IFN‐γ and IL‐10. In contrast, the agonistic mAbs AT130‐2 and AT130‐5, and their F(ab’)₂ fragments, allowed the formation of FVIII‐specific ASCs, even though the full IgG of AT130‐2 reduced binding of F8‐ICs to CD32. Data suggest that an inhibitory signal is transmitted when F8‐ICs bind to CD32 and that this signal is required during memory B cell (MBC) activation to support formation of FVIII‐specific ASCs. If the inhibitory signal is lacking due to CD32 deletion or blockade with antagonistic anti‐CD32 mAbs, FVIII‐specific T cell stimulation and ASC formation are suppressed, whereas agonistic stimulation of CD32 restores T cell stimulation and ASC formation.     

Neutrophil Fcγ and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fcγ receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fcγ and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

Matrix metalloproteinases inhibition promotes the polyfunctionality of human natural killer cells in therapeutic antibody‐based anti‐tumour immunotherapy

Activation of human natural killer (NK) cells is associated with the cleavage of CD16 from the cell surface, a process mediated by matrix metalloproteinases (MMPs). In this report, we examined whether inhibition of MMPs would lead to improved NK cell antibody‐dependent cell‐mediated cytotoxicity (ADCC) function. Using an in‐vitro ADCC assay, we tested the anti‐tumour function of NK cells with three different therapeutic monoclonal antibodies (mAbs) in the presence of MMPs inhibitor GM6001 or its control. Loss of CD16 was observed when NK cells were co‐cultured with tumour targets in the presence of specific anti‐tumour antibodies, and was found particularly on the majority of degranulating NK responding cells. Treatment with MMPs inhibitors not only prevented CD16 down‐regulation, but improved the quality of the responding cells significantly, as shown by an increase in the percentage of polyfunctional NK cells that are capable of both producing cytokines and degranulation. Furthermore, MMPs inhibition resulted in augmented and sustained CD16‐mediated signalling, as shown by increased tyrosine phosphorylation of CD3ζ and other downstream signalling intermediates, which may account for the improved NK cell function. Collectively, our results provide a foundation for combining MMPs inhibitors and therapeutic mAbs in new clinical trials for cancer treatment.