Fc γ receptors

Fibrinogen-like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM-derived DC and T-cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2-specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FccR)IIB and FccRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FccRIIB and FccRIII, or deficiency of these receptors, abrogated FGL2 binding. IntroductionFibrinogen-like protein 2 (FGL2), also known as fibroleukin, was first cloned from cytotoxic T lymphocytes and was classified as a member of the fibrinogen superfamily due to its homology (36%) with fibrinogen b and g chains [1]. Predicted as a type II transmembrane glycoprotein [2], cell membraneassociated FGL2 was shown to exhibit novel prothrombinase activity when associated with cell membranes/phospholipid vesicles [3,4], which has been implicated in the pathogenesis of Ã These authors contributed equally to this study.Correspondence: Dr. Gary Levy e-mail: glfgl2@attglobal.net DOI 10.1002/eji.200838338 Eur. J. Immunol. 2008. 38: 3114- [7]. The procoagulant activity was shown to depend on the serine 89 at the linear N terminal domain of FGL2 [4].The C terminal globular portion of FGL2 lacks the prothrombinase activity and contains a classical fibrinogen-related domain (FRED), which has been suggested to possess immunomodulatory activity based on several lines of evidence. First, other fibrinogen superfamily members including fibrinogen [8] Results Recombinant FcFGL2 protein binds to APCRecombinant FcFGL2 was generated and purified as described in the Materials and methods. The molecular size of the recombinant proteins was examined by SDS-PAGE, silver staining and Western blotting. FcFGL2 had a molecular weight of approximately 440 kDa under non-reducing conditions, which was confirmed by gel filtration (data not shown), and 110 kDa under reducing conditions (Fig. 1A). These data suggested that FcFGL2 exists as a tetramer, consistent with previous reports [13,21]. The Fc tag had a molecular size of 64 kDa under non-reducing conditions and 33 kDa under reducing conditions, suggesting that Fc is dimeric. Biotinylated FcFGL2 bound to the RAW264.7 cells (mouse macrophage cell lines), BMDC, the B-cell line A20 (which expresses only one Fcg receptor, FcgRIIB), thioglycolate-elicited peritoneal exudates cells (495% macrophages) from C57BL/6J mice, but FcFGL2 did not bind to the B-cell line A20IIA1.6, which does not express FcgRIIB (Fig. 1B) [22]. EL4 cells (a mouse T-cell line) did not bind FcFGL2 (data not shown). As expected, the Fc tag protein alone failed to bind to any of these cells and thus, Fc provided an appropriate negativ...

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“…[26]. However, FcgRIII expression has been reported on antigen-activated CD8 1 T cells [27] and anti-CD3-activated g/d T cells [28].…”

Section: Discussionmentioning

confidence: 99%

The FGL2‐FcγRIIB pathway: A novel mechanism leading to immunosuppression

et al. 2008

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“…Human immune cells express six immunoglobulin (IgG) binding FcγRs (FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb) that enable various functions, including phagocytosis, cytokine production, superoxide production, release of serotonin, and inhibition of B cell activation (1,2). An additional IgG binding receptor, the neonatal Fc receptor (FcRn), plays a critical role in IgG homeostasis by mediating antibody recycling via intracellular trafficking, thus preventing degradation in lysosomes (3).…”

mentioning

confidence: 99%

“…1B). In this study, we focused on the isolation of aglycosylated IgG that bound to FcγRI, as its role in ADCC is far less understood than that of other activating receptors (1,2). It has been suggested that because of its high affinity, FcγRI on effector cells is occupied by circulating IgG and thus is involved in ADCC only during the early stage of the immune response when antibody concentration is low (13).…”

mentioning

confidence: 99%

Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells

Jung¹,

Reddy²,

Kang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

143

118

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The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRIIa, FcγRIIIa) nor to the inhibitory receptor, FcγRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293T cells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.antibody engineering | bacterial display | bacterial expression | directed evolution | effector function

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“…Therefore the cellular response depends on the relative expression of activating and inhibitory receptors. This ratio is also influenced by the cytokine environment (Cohen-Solal et al, 2004). Moreover, the presence of allelic variants, with different affinities for selective the antibody isotypes support their role in the regulation of the inflammatory process due to the humoral immune response.…”

Section: Receptors For the Fc Of Iggmentioning

confidence: 91%