Peck Szee Tan

FcγRI-Deficient Mice Show Multiple Alterations to Inflammatory and Immune Responses

The inactivation of the mouse high-affinity IgG Fc receptor FcgammaRI resulted in a wide range of defects in antibody Fc-dependent functions. These studies showed the primary importance of FcgammaRI in endocytosis of monomeric IgG, kinetics, and extent of phagocytosis of immune complexes, in macrophage-based ADCC, and in immune complex-dependent antigen presentation to primed T cells. In the absence of FcgammaRI, antibody responses were elevated, implying the removal of a control point by the deletion of FcgammaRI. In addition, FcR-gamma chain-deficient mice were found to express partially functional FcgammaRI. Thus, FcgammaRI is an early participant in Fc-dependent cell activation and in the development of immune responses.

Structural Basis for FcγRIIa Recognition of Human IgG and Formation of Inflammatory Signaling Complexes

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.

Unique Monoclonal Antibodies Define Expression of FcγRI on Macrophages and Mast Cell Lines and Demonstrate Heterogeneity Among Subcutaneous and Other Dendritic Cells

The mouse FcγRI is one of the most fundamentally important FcRs. It participates in different stages of immunity, being a low affinity receptor for T-independent IgG3 and yet a high affinity receptor for IgG2a, the product of a Th1 immune response. However, analysis of this receptor has been difficult due largely to the failure to generate specific Abs to this FcR. We have made use of the polymorphic differences between BALB/c and NOD/Lt mice to generate mAb specific for the FcγRI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected FcγRI encoded by the more common Fcgr1a and Fcgr1b alleles, and although they identified different epitopes, none inhibited the binding of IgG to FcγRI. When bound to FcγRI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of FcγRI. Resting and IFN-γ-induced macrophages expressed FcγRI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed FcγRI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of FcγRI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8α, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, FcγRI on APC may act as a link between innate and adaptive immunities by binding and mediating the uptake of T-independent immune complexes for presentation, thereby assisting in the development of T-dependent immune responses.

Gain‐of‐function mutations in FcγRI of NOD mice: implications for the evolution of the Ig superfamily

It has been postulated that, during evolution of the Ig superfamily, modifications of the function of individual receptors might occur by acquisition of exons and their subsequent modification, though evidence of this is lacking. Here we have analysed the interaction of mouse IgG subclasses with high‐affinity FcγRI (CD64) which contains three Ig‐like domains and is important in innate and adaptive immunity. This analysis has identified a mechanism by which the postulated modification of newly acquired exons provides gains in function. Thus, the most widely distributed FcγRI allele in mice (e.g. BALB/c), bound only a single IgG subclass, IgG2a, with high affinity. However, non‐obese diabetic (NOD) mice expressed a unique allele that exhibits broader specificity and, in addition to binding IgG2a, FcγRI‐NOD bound monomeric IgG3 and bound IgG2b with high affinity, an IgG subclass not bound by FcγRI of other mouse strains, either as monomer or multivalent immune complexes. Analysis of mutants of FcγRI wherein segments of the interdomain junctions were exchanged between FcγRI‐BALB and FcγRI‐NOD identified these regions as having major influence in ‘gain‐of‐function’ by the NOD form of FcγRI. Nucleotide sequence analysis of intron/exon boundaries encoding the interdomain junctions of the FcγRI alleles showed these to have arisen by mutation to alter existing or create new mRNA splice donor/acceptor sites, resulting in generation of modified junctions.

Targeting CLEC9A delivers antigen to human CD141+ DC for CD4+ and CD8+T cell recognition

DC-based vaccines that initiate T cell responses are well tolerated and have demonstrated efficacy for tumor immunotherapy, with the potential to be combined with other therapies. Targeting vaccine antigens (Ag) directly to the DCs in vivo is more effective than cell-based therapies in mouse models and is therefore a promising strategy to translate to humans. The human CD141+ DCs are considered the most clinically relevant for initiating CD8+ T cell responses critical for killing tumors or infected cells, and they specifically express the C-type lectin-like receptor CLEC9A that facilitates presentation of Ag by these DCs. We have therefore developed a human chimeric Ab that specifically targets CLEC9A on CD141+ DCs in vitro and in vivo. These human chimeric Abs are highly effective at delivering Ag to DCs for recognition by both CD4+ and CD8+ T cells. Given the importance of these cellular responses for antitumor or antiviral immunity, and the superior specificity of anti-CLEC9A Abs for this DC subset, this approach warrants further development for vaccines.

Functional Expression of IgG-Fc Receptors in Human Airway Smooth Muscle Cells

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

The high-affinity receptor for IgG, FcγRI, of humans and non-human primates

Non‐human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low‐affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low‐affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high‐affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

Enhancing vaccine antibody responses by targeting Clec9A on dendritic cells

Targeting model antigens (Ags) to Clec9A on DC has been shown to induce, not only cytotoxic T cells, but also high levels of Ab. In fact, Ab responses against immunogenic Ag were effectively generated even in the absence of DC-activating adjuvants. Here we tested if targeting weakly immunogenic putative subunit vaccine Ags to Clec9A could enhance Ab responses to a level likely to be protective. The proposed “universal” influenza Ag, M2e and the enterovirus 71 Ag, SP70 were linked to anti-Clec9A Abs and injected into mice. Targeting these Ags to Clec9A greatly increased Ab titres. For optimal responses, a DC-activating adjuvant was required. For optimal responses, a boost injection was also needed, but the high Ab titres against the targeting construct blocked Clec9A-targeted boosting. Heterologous prime-boost strategies avoiding cross-reactivity between the priming and boosting targeting constructs overcame this limitation. In addition, targeting small amounts of Ag to Clec9A served as an efficient priming for a conventional boost with higher levels of untargeted Ag. Using this Clec9A-targeted priming, conventional boosting strategy, M2e immunisation protected mice from infection with lethal doses of influenza H1N1 virus.Vaccine technology: targeting the vaccine to the immune systemDuration and intensity of vaccine response can be boosted using antibodies to target pathogen fragments to specific immune system cells. Dendritic cells exist to take fragments of infectious diseases and present them to the immune system, sparking host defenses. Now, researchers led by Monash University’s Mireille Lahoud, and Ken Shortman of the Walter and Eliza Hall Institute, have successfully used antibodies to target fragments of influenza and hand, foot and mouth disease directly to dendritic cell molecules, specifically chosen to elicit a prolonged immune response. Mice inoculated with the targeted vaccine were protected from lethal influenza exposure, whereas the hand, foot and mouth disease vaccine elicited promising, but less marked results. With further development, this technology could provide a vital boost to vaccines that offer poor immunity on their own.

The Rare Anaphylaxis-Associated FcγRIIa3 Exhibits Distinct Characteristics From the Canonical FcγRIIa1

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.