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Ab-dependent cellular cytotoxicity, phagocytosis, and Ag presentation are key mechanisms of action of Abs arising in vaccine or naturally acquired immunity, as well of therapeutic mAbs. Cells expressing the low-affinity FcγRs (FcγRII or CD32 and FcγRIII or CD16) are activated for these functions when receptors are aggregated following the binding of IgG-opsonized targets. Despite the diversity of the Fc receptor proteins, IgG ligands, and potential responding cell types, the induction of all FcγR-mediated responses by opsonized targets requires the presentation of multiple Fc regions in close proximity to each other. We demonstrated that such "near-neighbor" Fc regions can be detected using defined recombinant soluble (rs) dimeric low-affinity ectodomains (rsFcγR) that have an absolute binding requirement for the simultaneous engagement of two IgG Fc regions. Like cell surface- expressed FcγRs, the binding of dimeric rsFcγR ectodomains to Ab immune complexes was affected by Ab subclass, presentation, opsonization density, Fc fucosylation, or mutation. The activation of an NK cell line and primary NK cells by human IgGopsonized influenza A hemagglutinin correlated with dimeric rsFcgRIIIa binding activity but not with Ab titer. Furthermore, the dimeric rsFcgR binding assay sensitively detected greater Fc receptor activity to pandemic H1N1 hemagglutinin after the swine influenza pandemic of 2009 in pooled human polyclonal IgG. Thus these dimeric rsFcγR ectodomains are validated, defined probes that should prove valuable in measuring the immune-activating capacity of IgG Abs elicited by infection or vaccination or experimentally derived IgG and its variants. [ABSTRACT FROM AUTHOR]

Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His131). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro159 and Tyr160 impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His131 and Met132. Significantly, both His131 and Met132 are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His131. These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques. [ABSTRACT FROM AUTHOR]

Ab-dependent cellular cytotoxicity (ADCC) responses are of growing interest in the HIV vaccine field but current cell-based assays are usually difficult to reproduce across laboratories. We developed an ELISA and multiplex assay to model the cross-linking of Fcg receptors (FcgR) by Abs, which is required to initiate an ADCC response. Our FcgR dimer ELISA readily detected Abs in samples from two separate cohorts of the partially efficacious Thai RV144 HIV vaccine efficacy trial. The FcgR dimer-binding Abs induced by the RV144 regimen correlated well with a functional measure of ADCC as well as IgG subclasses. The highthroughput multiplex assay allowed us to simultaneously measure FcgR dimer-binding Abs to 32 different HIV Ags, providing a measure of the breadth of FcgR-binding Abs induced by the RV144 trial. FcgR-binding Abs specific to V regions 1 and 2 were strongly associated with increased breadth of recognition of different Env proteins, suggesting anti-V regions 1 and 2 Abs may be a marker of ADCC breadth. This FcgR dimer provides an important tool for the further analysis and refinement of ADCCinducing HIV and other antiviral vaccine regimens. [ABSTRACT FROM AUTHOR]

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly. [ABSTRACT FROM AUTHOR]

Staphylococcal superantigen-like protein 10 (SSL10) is a highly conserved member of the SSL family secreted by Staphylococcus aureus that displays structural but not functional similarity to superantigens. SSL10 bound to fibrinogen and fibronectin from plasma and in addition displayed striking specificity toward the gamma-1 subclass of human Igs. SSL10 also bound strongly to primate IgG but not to any other species tested, including rabbit, pig, guinea pig, cow, sheep, or mouse. A soluble form of the 12-kDa beta-grasp C-terminal domain of SSL10 (SSL10(95-197)) retained fibrinogen and fibronectin binding but lost the ability to bind IgG1, indicating that SSL10 bound to IgG1 primarily through its N-terminal oligonucleotide binding fold domain. SSL10 blocked the binding of IgG1 to FcgammaRs on monocytes and neutrophil phagocytosis of IgG1-opsonized bacteria. Mutagenesis of human IgG1 at key sites significantly reduced SSL10 binding including Lys(322) that is important for C1q binding, a combination of Leu(234) and Leu(235) that are important for FcgammaR binding, and a combination of Lys(274) and Asp(276) that together are unique to IgG1. These mutations suggest that the most likely site bound by SSL10 is the outer face of the Cgamma2 domain in close proximity to both the FcgammaR and C1q binding sites. SSL10 is a potential virulence factor for S. aureus targeting IgG1-mediated immunity.

Clearance of IgG-opsonized erythrocytes is impaired in HIV-1-infected patients, suggesting defective FcgammaR-mediated phagocytosis in vivo. We have previously shown defective FcgammaR-mediated phagocytosis in HIV-1-infected human monocyte-derived macrophages (MDM), establishing an in vitro model for defective tissue macrophages. Inhibition was associated with decreased protein expression of FcR gamma-chain, which transduces immune receptor signals via ITAM motifs. FcgammaRI and FcgammaRIIIa signal via gamma-chain, whereas FcgammaRIIa does not. In this study, we showed that HIV-1 infection inhibited FcgammaRI-, but not FcgammaRIIa-dependent Syk activation in MDM, showing that inhibition was specific for gamma-chain-dependent signaling. HIV-1 infection did not impair gamma-chain mRNA levels measured by real-time PCR, suggesting a posttranscriptional mechanism of gamma-chain depletion. HIV-1 infection did not affect gamma-chain degradation (n = 7, p = 0.94) measured in metabolic labeling/chase experiments, whereas gamma-chain biosynthesis was inhibited (n = 12, p = 0.0068). Using an enhanced GFP-expressing HIV-1 strain, we showed that FcgammaR-mediated phagocytosis inhibition is predominantly due to a bystander effect. Experiments in which MDM were infected in the presence of the antiretroviral drug 3TC suggest that active viral replication is required for inhibition of phagocytosis in MDM. These data suggest that HIV-1 infection may affect only gamma-chain-dependent FcgammaR functions, but that this is not restricted to HIV-1-infected cells.

The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcgammaRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcgammaRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcgammaRIIa may be an essential feature of the FcgammaRIIa signaling cascade.