About InVivoMAb anti-human CD32 (FcγRIIA)
The IV.3 monoclonal antibody reacts with human CD32 also known as FcγRII and FCRII, a 40 kDa polymorphic transmembrane glycoprotein and an Ig superfamily member. CD32 is expressed on monocytes/macrophages, granulocytes, platelets and B cells. CD32 enables interaction between Fc γ RII-expressing cells and opsonized antigen or IgG-containing immune complexes. This allows CD32 to function in the activation or inhibition of immune responses including degranulation, phagocytosis, ADCC, cytokine release, and B cell proliferation. The IV.3 antibody has been shown to block the biological effects of CD32 in vitro. Additionally, IV.3 f(ab')2 fragments have been used to block CD32 in vivo in transgenic mice expressing human CD32.
InVivoMAb anti-human CD32 (FcγRIIA) Specifications
|Recommended Isotype Control(s)|
|Recommended Dilution Buffer|
|Immunogen||Human K562 leukemia cell line|
|Sterility||0.2 μM filtered|
|Production||Purified from tissue culture supernatant in an animal free facility|
|Molecular Weight||150 kDa|
|Storage||The antibody solution should be stored at the stock concentration at 4°C. Do not freeze.|
InVivoMAb anti-human CD32 (FcγRIIA) (Clone: IV.3)
Walsh, T. G., et al. (2015). “SDF-1alpha is a novel autocrine activator of platelets operating through its receptor CXCR4.” Cell Signal 27(1): 37-46. PubMed
Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1alpha upon platelet activation, but the ability of platelet-derived SDF-1alpha to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1alpha and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1alpha-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production. Time-dependent release of SDF-1alpha from collagen-activated platelets supports a functional role for SDF-1alpha in this regard. Using an in vitro whole blood perfusion assay, collagen-induced thrombus formation was substantially reduced with CXCR4 inhibition. In washed platelets, recombinant SDF-1alpha in the range of 20-100 ng/mL(-1) could significantly enhance platelet aggregation responses to a threshold concentration of collagen. These enhancements were completely dependent on CXCR4, but not CXCR7, which triggered TxA2 production and dense granule secretion. Rises in cAMP were significantly blunted by SDF-1alpha, which could also enhance collagen-mediated Ca2+ mobilisation, both of which were mediated by CXCR4. This potentiating effect of SDF-1alpha primarily required TxA2 signalling acting upstream of dense granule secretion, whereas blockade of ADP signalling could only partially attenuate SDF-1alpha-induced platelet activation. Therefore, this study supports a potentially novel autocrine/paracrine role for platelet-derived SDF-1alpha during thrombosis and haemostasis, through a predominantly TxA2-dependent and ADP-independent pathway.
Ben Mkaddem, S., et al. (2014). “Shifting FcgammaRIIA-ITAM from activation to inhibitory configuration ameliorates arthritis.” J Clin Invest 124(9): 3945-3959. PubMed
Rheumatoid arthritis-associated (RA-associated) inflammation is mediated through the interaction between RA IgG immune complexes and IgG Fc receptors on immune cells. Polymorphisms within the gene encoding the human IgG Fc receptor IIA (hFcgammaRIIA) are associated with an increased risk of developing RA. Within the hFcgammaRIIA intracytoplasmic domain, there are 2 conserved tyrosine residues arranged in a noncanonical immunoreceptor tyrosine-based activation motif (ITAM). Here, we reveal that inhibitory engagement of the hFcgammaRIIA ITAM either with anti-hFcgammaRII F(ab’)2 fragments or intravenous hIgG (IVIg) ameliorates RA-associated inflammation, and this effect was characteristic of previously described inhibitory ITAM (ITAMi) signaling for hFcalphaRI and hFcgammaRIIIA, but only involves a single tyrosine. In hFcgammaRIIA-expressing mice, arthritis induction was inhibited following hFcgammaRIIA engagement. Moreover, hFcgammaRIIA ITAMi-signaling reduced ROS and inflammatory cytokine production through inhibition of guanine nucleotide exchange factor VAV-1 and IL-1 receptor-associated kinase 1 (IRAK-1), respectively. ITAMi signaling was mediated by tyrosine 304 (Y304) within the hFcgammaRIIA ITAM, which was required for recruitment of tyrosine kinase SYK and tyrosine phosphatase SHP-1. Anti-hFcgammaRII F(ab’)2 treatment of inflammatory synovial cells from RA patients inhibited ROS production through induction of ITAMi signaling. These data suggest that shifting constitutive hFcgammaRIIA-mediated activation to ITAMi signaling could ameliorate RA-associated inflammation.
Flinsenberg, T. W., et al. (2014). “A novel FcgammaRIIa Q27W gene variant is associated with common variable immune deficiency through defective FcgammaRIIa downstream signaling.” Clin Immunol 155(1): 108-117. PubMed
We identified a novel Q27W FcgammaRIIa variant that was found more frequently in common variable immunodeficiency (CVID) or CVID-like children. We analyzed the possible functional consequence of the Q27W FcgammaRIIa mutation in human cells. We used peripheral blood mononuclear cells from Q27W FcgammaRIIa patients and healthy controls, and cultured cells that overexpress the Q27W and common FcgammaRIIa variants. The Q27W FcgammaRIIa mutation does not disrupt FcgammaRIIa surface expression in peripheral blood mononuclear cells. Mononuclear cells express multiple FcgammaR, precluding careful analysis of Q27W FcgammaRIIa functional deviation. For functional analysis of FcgammaRIIa function, we therefore overexpressed the Q27W FcgammaRIIa and common FcgammaRIIa variant in IIA1.6 cells that are normally deficient in FcgammaR. We show that FcgammaRIIa triggering-induced signaling is obstructed, as measured by both decrease in calcium flux and defective MAPK phosphorylation. In conclusion, we here describe a novel Q27W FcgammaRIIa variant that causes delayed downstream signaling. This variant may contribute to CVID.
Trist, H. M., et al. (2014). “Polymorphisms and interspecies differences of the activating and inhibitory FcgammaRII of Macaca nemestrina influence the binding of human IgG subclasses.” J Immunol 192(2): 792-803. PubMed
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 FcgammaRIIa (mnFcgammaRIIa) and inhibitory FcgammaRIIb (mnFcgammaRIIb) and predicted their structures using the huIgGFc/huFcgammaRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcgammaRIIa and mnFcgammaRIIb compared with their human FcR counterparts. MnFcgammaRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcgammaRIIa (His(131)). In contrast, mnFcgammaRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcgammaRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcgammaRIIa showed that Pro(159) and Tyr(160) impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcgammaRIIb was shown to be dependent on His(131) and Met(132). Significantly, both His(131) and Met(132) are conserved across FcgammaRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcgammaRIIa wherein proline at position 131, also an important polymorphic site in huFcgammaRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcgammaRIIa His(131). 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.
Stemerding, A. M., et al. (2013). “Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FLIPr) and its homologue FLIPr-like are potent FcgammaR antagonists that inhibit IgG-mediated effector functions.” J Immunol 191(1): 353-362. PubMed
To evade opsonophagocytosis, Staphylococcus aureus secretes various immunomodulatory molecules that interfere with effective opsonization by complement and/or IgG. Immune-evasion molecules targeting the phagocyte receptors for these opsonins have not been described. In this study, we demonstrate that S. aureus escapes from FcgammaR-mediated immunity by secreting a potent FcgammaR antagonist, FLIPr, or its homolog FLIPr-like. Both proteins were previously reported to function as formyl peptide receptor inhibitors. Binding of FLIPr was mainly restricted to FcgammaRII receptors, whereas FLIPr-like bound to different FcgammaR subclasses, and both competitively blocked IgG-ligand binding. They fully inhibited FcgammaR-mediated effector functions, including opsonophagocytosis and subsequent intracellular killing of S. aureus by neutrophils and Ab-dependent cellular cytotoxicity of tumor cells by both neutrophils and NK cells. In vivo, treatment of mice with FLIPr-like prevented the development of an immune complex-mediated FcgammaR-dependent Arthus reaction. This study reveals a novel immune-escape function for S. aureus-secreted proteins that may lead to the development of new therapeutic agents in FcgammaR-mediated diseases.
van der Aar, A. M., et al. (2013). “Langerhans cells favor skin flora tolerance through limited presentation of bacterial antigens and induction of regulatory T cells.” J Invest Dermatol 133(5): 1240-1249. PubMed
The mechanisms preventing detrimental T-cell responses against commensal skin bacteria remain elusive. Using monocyte-derived and skin-derived dendritic cells (DCs), we demonstrate that epidermal Langerhans cells (LCs), the DCs in the most superficial layer of the skin, have a poor capacity to internalize bacteria because of low expression of FcgammaRIIa. Furthermore, LCs show deficiency in processing and major histocompatibility complex II (MHC-II)-restricted presentation of bacterial antigens, as a result of a decreased expression of molecules involved in these functionalities. The reduced capacity to take up, process, and present bacterial antigens cannot be restored by LC activation by ectopically expressed Toll-like receptors or by cytokines. Consequently, bacteria-primed LCs poorly restimulate antibacterial memory CD4(+) T cells and inefficiently induce bacteria-specific effector CD4(+) T cells from naive T cells; however, they initiate the development of regulatory Foxp3(+)CD4(+) T cells, which are able to suppress the proliferation of autologous bystander T cells specific for the same bacteria. In contrast, dermal DCs that reside in the deeper dermal layer of the skin efficiently present bacterial antigens and provoke robust antibacterial naive and memory CD4(+) T-cell responses. In conclusion, LCs form a unique DC subset that is adapted at multiple levels for the maintenance of tolerance to bacterial skin flora.
Veri, M. C., et al. (2007). “Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization.” Immunology 121(3): 392-404. PubMed
Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.
Brown, M. G., et al. (2006). “A dominant role for FcgammaRII in antibody-enhanced dengue virus infection of human mast cells and associated CCL5 release.” J Leukoc Biol 80(6): 1242-1250. PubMed
Dengue virus is a major mosquito-borne human pathogen with four known serotypes. The presence of antidengue virus antibodies in the serum of individuals prior to dengue virus infection is believed to be an important risk factor for severe dengue virus disease as a result of the phenomenon of antibody-dependent enhancement operating on Fc receptor (FcR)-bearing cells. In addition to blood monocytes, mast cells are susceptible to antibody-enhanced dengue virus infection, producing a number of inflammatory mediators including IL-1, IL-6, and CCL5. Using the human mast cell-like lines KU812 and HMC-1 as well as primary cultures of human cord blood-derived mast cells (CBMC), we aimed to identify the participating FcRs in antibody-enhanced mast cell dengue virus infection, as FcRs represent a potential site for therapeutic intervention. CBMC expressed significant levels of FcgammaRI, FcgammaRII, and FcgammaRIII, and mast cell-like HMC-1 and KU812 cells expressed predominantly FcgammaRII. All four serotypes of dengue virus showed antibody-enhanced binding to KU812 cells. Specific FcgammaRII blockade with mAb IV.3 was found to significantly abrogate dengue virus binding to KU812 cells and CBMC in the presence of dengue-specific antibody. Dengue virus infection and the production of CCL5 by KU812 cells were also inhibited by FcgammaRII blockade.