Human Fc Receptor Research Articles

Enhancement of trastuzumab-mediated cellular cytotoxicity by interleukin-21

2567 Background: Interleukin-21, a cytokine produced by activated T cells, has potent effects on growth, survival and activation status of T, B and natural killer (NK) cells. In vitro studies show increased NK cytolytic function following combined human recombinant (rIL-21) stimulation and Fc receptor engagement. We studied the effect of rIL-21 on antibody-dependent cellular cytotoxicity (ADCC) against trastuzumab-coated breast cancer targets. Methods: ADCC assays were performed on peripheral blood mononuclear cell preparations (MNCs) from cynomolgus monkeys and on enriched human NK cells, following in vitro treatment with rIL-21. Cynomolgus monkeys were treated with rIL-21 as part of an in vivo model of ADCC (Hughes, et al., ASCO submitted), and the ex vivo ADCC activity of MNCs from these animals was tested. Whole blood and MNCs were characterized by flow cytometry. Results: Human NK cells incubated with rIL-21 had 2 to 4-fold increased ADCC against breast cancer targets including Her2/neu-low lines MCF7 and HCC38 (p<.02). Cynomolgus MNCs cultured with rIL-21 showed variable ADCC activation. In animals treated with rIL-21, an immediate decline in ADCC activity correlated with the extravasation of NK cells from the peripheral blood. This was followed by a substantial increase in ADCC against BT-474 cells at later timepoints. Normalization for NK cell numbers showed increased lytic efficiency of NK cells, especially after two cycles of treatment. The percentage of perforin-positive MNCs increased following treatment, as did other biomarkers of immune activation including FcγRI and FcγRIII expression. Conclusion: Treatment of cynomolgus monkeys with rIL-21 increased the activity of MNCs in trastuzumab-mediated ex vivo ADCC assays. This lytic activation is consistent with the enhanced effector function observed after in vitro incubation of NK cells or MNCs with rIL-21. Thus, adjuvant treatment with rIL-21 may enhance monoclonal antibody therapy by improving ADCC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ZymoGenetics ZymoGenetics

An extended family of Fc receptor relatives.

A surprising number of Fc receptor (FcR) relatives have been recognized recently with the potential capacity to modulate innate and adaptive immune responses. The six human FcR homologs (FcRH1-6), which belong to a phylogenetically conserved gene family, have variable numbers of extracellular immunoglobulin domains of five different subtypes. FcRH immunoregulatory potential is implicated by the presence of consensus tyrosine-based activation or inhibition motifs in their cytoplasmic tails. All but one of these new receptors, FcRH6, are expressed on B cells at different stages in differentiation. Their ligands, function, and prospective roles as diagnostic B cell markers and therapeutic targets are topics of intense interest.

Prolonged and increased expression of soluble Fc receptors, IgG and a TCR-Ig fusion protein by transiently transfected adherent 293E cells

In studies of the relation between structure and function of proteins of the immune system, there is a continuous need for screening of a large number of protein variants. To optimise the yield following transient gene expression in small or medium culture volumes, several parameters were investigated. First, secretion levels of a soluble form of human Fcγ receptor IIA (FcγRIIA) were measured after transfection of 293, 293E, 293T as well as COS-7 cell lines. The transgene was under cytomegalovirus (CMV) promoter control on the expression vector pcDNA3, which also contains an SV40 origin of replication (SV40 ori). All 293 cell lines secreted more protein than COS-7 cells. Introduction of the Epstein Barr virus (EBV) origin of replication (oriP) greatly increased the protein expression from the 293E cells, both the amount of protein produced per day and the duration of production. At best, 293E cells secreted fully functional protein for 3–4 weeks provided supernatant was harvested every 2–3 days followed by medium replacement. This method was then used for expression of soluble forms of human FcγRI, FcγRIIB, the human neonatal Fc receptor (FcRn), a T cell receptor (TCR)-immunoglobulin (Ig) fusion protein, and human IgG3. With an initial culture volume of 5 ml, the yield was approximately 200 μg for FcγRIIA, 1.5 μg for FcγRI, 5 μg for FcRn, 20 μg for FcγRIIB, 40 μg for the TCR-Ig fusion protein and 850 μg for IgG3. Culture expansion during the 3 weeks of culture further increased the yield. Protein yield was also improved by scaling up the initial volume. This approach can provide sufficient amounts of protein for screening experiments, and in the case of antibody, milligrams of recombinant protein for extensive structural analysis can be obtained from one single transient transfection. The approach should be of interest to laboratories that do not have access to a bioreactor but still have a requirement for reasonable amounts of protein to be produced in an easy and cost-effective manner.

Fc Receptors and Their Role in Immune Regulation and Autoimmunity

The activation threshold of cells in the immune system is often tuned by cell surface molecules. The Fc receptors expressed on various hematopoietic cells constitute critical elements for activating or downmodulating immune responses and combines humoral and cell-mediated immunity. Thus, Fc receptors are the intelligent sensors of the immune status in the individual. However, impaired regulation by Fc receptors will lead to unresponsiveness or hyperreactivity to foreign as well as self-antigens. Murine models for autoimmune disease indicate the indispensable roles of the inhibitory Fc receptor in the suppression of such disorders, whereas activating-type FcRs are crucial for the onset and exacerbation of the disease. The development of many autoimmune diseases in humans may be caused by impairment of the human Fc receptor regulatory system. This review is aimed at providing a current overview of the mechanism of Fc receptor-based immune regulation and the possible scenario of how autoimmune disease might result from their dysfunction.

Role of Fc.GAMMA. receptors in immune regulation and diseases

The activation threshold of cells in the immune system is often tuned by cell surface molecules. The Fc receptors expressed on various hematopoietic cells constitute critical elements for activating or down-modulating immune responses and combines humoral and cell-mediated immunity. Thus, Fc receptors are the intelligent sensors of the immune status in the individual. However, impaired regulation by Fc receptors will lead to unresponsiveness or hyperreactivity to foreign as well as self antigens. Murine models for autoimmune disease indicate the indispensable roles of the inhibitory Fc receptor in the suppression of such disorders, whereas activating-type FcRs are crucial for the onset and exacerbation of the disease. The development of many autoimmune diseases in humans may be caused by impairment of the human Fc receptor regulatory system. This review is aimed at providing a current overview of the mechanism of Fc receptor-based immune regulation and the possible scenario of how immunological disorders might result from their dysfunction.

Identification and characterization of macaque CD89 (immunoglobulin A Fc receptor).

The interaction of the immunoglobulin A (IgA) molecule with its specific cellular receptor is necessary to trigger a variety of effector functions able to clear IgA-opsonized antigens. The human IgA-specific Fc receptor, FcalphaRI or CD89, is expressed on cells of the myeloid lineage. Recently, CD89 homologues have been identified in rats and cattle. Because non-human primates represent well established models for a variety of human diseases and for the testing of immunotherapeutic strategies, we cloned and sequenced cDNAs corresponding to the CD89 gene from rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Macaque sequences of full-length CD89 consist of five exons of length identical to the corresponding human CD89 exons. The rhesus and cynomolgus macaque derived amino acid sequences are highly homologous to each other (99.3% identity) and exhibit 86.5% and 86.1% identity to the human counterpart, respectively. Transfection of HeLa cells with plasmids containing the cloned macaque cDNAs resulted in the expression of surface molecules recognized by an anti-human CD89 antibody. Five splice variants were identified in rhesus macaques. Three of the five variants are similar to described human CD89 splice variants, whereas two variants have not been described in humans. Three splice variants were identified in cynomolgus macaques. Of the three variants, one is present also in humans and rhesus macaques, whereas the other two are shared with rhesus macaques but not humans. Similarly to the human CD89, macaque CD89 is expressed on myeloid cells from peripheral blood. The characterization of macaque CD89 represents an essential step in establishing a non-human primate model for the testing of immunotherapeutic approaches based on the manipulation of the IgA/CD89 interaction.

Human Neonatal Fc Receptor Mediates Transport of IgG into Luminal Secretions for Delivery of Antigens to Mucosal Dendritic Cells

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. The mechanism by which IgG reaches luminal secretions and the function of IgG in these locations are unknown. Here, we find that the human neonatal Fc receptor (FcRn) is the vehicle that transports IgG across the intestinal epithelial barrier into the lumen where the IgG can bind cognate antigen. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4 + T cells in regional organized lymphoid structures. These results explain how IgG is secreted onto mucosal surfaces and scavenges luminal antigens for recognition by the immune system.

The role of FcγRIIA and IIIA polymorphisms in autoimmune diseases

Our knowledge about the role of human Fc receptors for IgG (FcγR) has increased considerably within the last several years. These receptors vary in their affinity for IgG, their preferences for IgG subclasses, the cell type-specific expression patterns, and the intracellular signals that they elicit. Additional FcγR heterogeneity is introduced by the presence of well characterized genetic polymorphisms. Allelic variants of FcγR genes may influence phagocyte biologic activity, providing a basis for inherited predisposition to disease. Recent evidence suggests that certain FcγR alleles are genetic risk factors for systemic autoimmune diseases and the development of major manifestations of these diseases. The FcγRIIA-R/H131 polymorphism is an important determinant of predisposition to systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). FcγRIIA-R131, the low-binding IgG2 allele, seems to confer risk for APS under a recessive model, whereas its effect on SLE susceptibility probably has a dose–response character. The population-attributable fraction of lupus cases due to the R131 allele is 13% and for APS cases is at least 10%, in subjects of European descent. The FcγRIIIA-V/F158 polymorphism has a significant impact on renal involvement in lupus patients. The proportion of nephritis cases that could be attributed to the low-binding IgG1 and IgG3 F158 allele is approximately 10–14%. These genetic associations have been well documented in meta-analyses including a large number of studies. Besides the epidemiologic and pathophysiologic interest, this knowledge may be of use in the future in designing novel therapeutic interventions.

FcgammaRIIa expression with FcgammaRI results in C-reactive protein- and IgG-mediated phagocytosis.

C-reactive protein (CRP) is a pattern-recognition molecule, which can bind to phosphorylcholine and certain phosphorylated carbohydrates found on the surface of a number of microorganisms. CRP has been shown recently to bind human Fc receptor for immunoglobulin G (IgG; FcgammaR)I and mediate phagocytosis and signaling through the gamma-chain. To date, binding of monomeric CRP to FcgammaRII has been contentious. We demonstrate that erythrocytes opsonized with CRP bind FcgammaRIIa-transfected COS-7 cells. In addition, we demonstrate that FcgammaRI can use FcgammaRIIa R131 and H131 to phagocytose erythrocytes coated with IgG or purified or recombinant CRP in the absence of the gamma-chain. COS-7 cells expressing FcgammaRIIa or FcgammaRI alone did not phagocytose opsonized erythrocytes. Such phagocytosis required the cytoplasmic domain of FcgammaRIIa, as mutation of tyrosine at position 205 and truncation of the cytoplasmic domain from the end of the transmembrane region (position 206), resulting in the loss of the immunoreceptor tyrosine activatory motif, abrogated phagocytosis. FcgammaRIIa R131 was more efficient than FcgammaRIIa H131 at mediating CRP-dependent phagocytosis.

Carcinoembryonic antigen-immunoglobulin Fc fusion protein (CEA-Fc) for identification and activation of anti-CEA immunoglobulin-T-cell receptor-modified T cells, representative of a new class of Ig fusion proteins.

Chimeric immunoglobulin-T-cell receptor (IgTCR)-modified T cells ("designer T cells") kill tumor cells based on antibody-redirected recognition of tumor-associated antigen. Anti-carcinoembryonic antigen (CEA) designer T cells have been prepared and applied in adoptive cellular immunotherapy regimens for CEA-positive cancers. A CEA-immunoglobulin Fc (CEA-Fc) fusion protein was created from the A3B3 region of CEA and the Fc portion of human IgG for the purposes of activation and detection of anti-CEA designer T cells. CEA-Fc was expressed at high yield in CHO cells and purified to homogeneity in a single step on a protein A affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that CEA-Fc formed disulfide-linked dimers with a molecular weight of about 170 kDa and a monomer size of 85kDa. The A3B3 CEA component of the CEA-Fc bound to anti-CEA monoclonal antibody MN-14, as well as to the single-chain Fv (sFv) derived from this antibody that was expressed in the IgTCR on the surface of designer T cells. The Fc portion of CEA-Fc was recognized by anti-human IgG Fc antibody and bound by human monocyte Fc receptors. CEA-Fc activated the anti-CEA designer T cells as plate-bound or monocyte-bound form but not as soluble form, as measured by CD69 expression and T-cell proliferation. Our results indicate that the CEA-Fc fusion protein can be used to detect the expression of the anti-CEA IgTCR chimeric receptors on the modified T cells, as well as to serve as an antigen to activate the anti-CEA IgTCR modified T cells. CEA-Fc is the prototype for a new class of antigen-Fc molecules that may significantly augment the analytic and therapeutic goals of adoptive designer T-cell immunotherapies.

Cloning and characterization of an immunoglobulin A Fc receptor from cattle.

Here, we describe the cloning, sequencing and characterization of an immunoglobulin A (IgA) Fc receptor from cattle (bFcalphaR). By screening a translated EST database with the protein sequence of the human IgA Fc receptor (CD89) we identified a putative bovine homologue. Subsequent polymerase chain reaction (PCR) amplification confirmed that the identified full-length cDNA was expressed in bovine cells. COS-1 cells transfected with a plasmid containing the cloned cDNA bound to beads coated with either bovine or human IgA, but not to beads coated with bovine IgG2 or human IgG. The bFcalphaR cDNA is 873 nucleotides long and is predicted to encode a 269 amino-acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail devoid of known signalling motifs. Genetically, bFcalphaR is more closely related to CD89, bFcgamma2R, NKp46, and the KIR and LILR gene families than to other FcRs. Moreover, the bFcalphaR gene maps to the bovine leucocyte receptor complex on chromosome 18. Identification of the bFcalphaR will aid in the understanding of IgA-FcalphaR interactions, and may facilitate the isolation of FcalphaR from other species.

Purification, crystallization and X-ray diffraction analysis of the extracellular part of the human Fc receptor for IgA, FcalphaRI (CD89).

FcalphaRI is the predominant receptor for IgA in the serum. Nevertheless, the interaction between the molecules that finally leads to an immune response is poorly understood. To investigate the structural requirements for IgA binding, the extracellular region of FcalphaRI was cloned and overexpressed in Escherichia coli. The resulting inclusion-body protein was refolded and purified. Despite its deglycosylated state, this recombinant FcalphaRI retained its ability to bind human IgA. The protein crystallized spontaneously as microcrystalline needles. Recrystallization yielded crystals belonging to a primitive monoclinic space group. A complete 2.8 A resolution X-ray diffraction data set was collected using synchrotron radiation.

IgG Fc receptor polymorphisms in human disease: Implications for intravenous immunoglobulin therapy

Polymorphisms of human Fc receptors (FcRs) have been described that are associated with the development or progression of autoimmune diseases. The FcR polymorphisms affect the affinity with which FcRs interact with immunoglobulin molecules. Intravenous immunoglobulin is administered as therapy for many autoimmune diseases and might exert its effects by interacting with FcRs. Thus, FcR polymorphisms might influence the efficacy of intravenous immunoglobulin therapy for patients with certain autoimmune diseases. In this article we review FcR polymorphisms in relation to autoimmune diseases for which intravenous immunoglobulin is used therapeutically. (J Allergy Clin Immunol 2003;111:697-703.)

Definition of an Fc receptor-related gene (FcRX) expressed in human and mouse B cells.

The recent identification of five human Fc receptor (FcR) homologs, hFcRH1-5, has extended the known FcR family and identified an unanticipated richness of the chromosome 1q region in genes encoding potential Ig-binding proteins. In a database search for additional relatives of this family we identified expressed sequence tag representatives of a new FcR-related molecule (hFcRX) and its mouse ortholog (mFcRX). The FcRX cDNAs were cloned from human lymph node and mouse spleen cDNA libraries. hFcRX is located centromeric of FcgammaRII and FcgammaRIII at 1q23, and its mouse ortholog resides in a syntenic region of chromosome 1. The genes encode proteins with 67% interspecies identity that lack both N-linked glycosylation sites and transmembrane regions. Two of the four FcRX domains are Ig-like, and share characteristics similar to FcgammaRI domains 2 and 3, having 28% overall extracellular identity with hFcgammaRI and 27% identity with mFcgammaRI respectively. FcRX transcripts are found primarily in secondary lymphoid tissues, where they are expressed by B lineage cells. FcRX thus may function as a secreted or intracellular protein in normal and neoplastic B cells.

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors.

A virally encoded, high-affinity Fc receptor (FcR) is found on herpes simplex virus type 1 (HSV-1) particles and infected cells where its binding of non-immune IgG protects cells from host-mediated lysis. Whilst mutation or aglycosylation of the IgG CH2 domain reduced binding to human FcR, the interaction with HSV-1 FcR was not affected. However, the HSV-1 FcR, unlike human FcR, discriminates between human IgG1 allotypes, being sensitive to changes at positions 214 (CH1) and 356/358 (CH3), away from its proposed binding site at the CH2-CH3 interface. The biological consequences are not known but this is the first evidence of a major functional difference between IgG1 allotypes.

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors

IgA Fc receptor (FcalphaR) cross-linking recruits tyrosine kinases, phosphoinositide kinases and serine/threonine kinases to glycolipid rafts.

The human IgA Fc receptor (FcalphaR, CD89) triggers several important physiological functions, including phagocytosis, NADPH oxidase activation and antigen presentation. Efforts are underway to delineate FcalphaR signal-transduction pathways that control these functions. In a previous study, we demonstrated that cross-linking of FcalphaR increased its partitioning into membrane glycolipid rafts and was accompanied by gamma-chain-dependent recruitment and phosphorylation of the tyrosine kinases Lck/Yes-related novel protein tyrosine kinase (Lyn) and Bruton's tyrosine kinase (Btk). Here we have performed a more extensive characterization of signalling effectors recruited to rafts on FcalphaR cross-linking. We demonstrate that in addition to tyrosine kinases Lyn and Btk, FcalphaR cross-linking also recruits B-lymphocyte kinase (Blk) and spleen tyrosine kinase (Syk) to rafts. We show recruitment of phosphoinositide kinases, including 3-phosphoinositide 3-kinase and phospholipase Cgamma2, and serine/threonine kinases such as protein kinase C (PKC) alpha, PKCepsilon, and protein kinase B (PKB) alpha. This suggests that lipid rafts serve as sites for FcalphaR-triggered recruitment of multiple classes of signalling effectors. We further demonstrate that tyrosine kinases and PKCalpha have a sustained association with rafts, whereas phosphoinositide 3-kinase and its downstream effectors have a transient association with rafts. This is consistent with temporally regulated divergence of FcalphaR signalling pathways in rafts. Furthermore, we suggest the spatial separation of signalling effectors by transport of phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, PKBalpha and PKCepsilon to endocytic compartments containing internalized FcalphaR.

Humanized mouse models of FcR clearance in immune platelet disorders

Transgenic mouse lines expressing physiologic levels of human platelet Fc receptor (FcR) for IgG, FcγRIIA, on platelets and macrophages were generated. Anti-CD9 antibody activated platelets of FcγRIIA transgenic mice and, following injection in vivo, caused rapid and severe thrombocytopenia compared with nonactivating antiplatelet antibody. Anti-CD9 injected in FcγRIIA transgenic mice crossed with FcR γ-chain knockout (γ-KO) mice caused thrombosis and shock in all mice, and death in 16 of 18 mice. Histologic examination of lung vasculature of anti-CD-treated FcγRIIA transgenic × γ-KO mice showed extensive platelet-fibrin thrombi. Taken together, these observations suggest that in FcγRIIA transgenic × γ-KO mice there is an important interplay of intravascular platelet activation and splenic clearance. Reduction of splenic clearance surgically or functionally also facilitated anti-CD-9-mediated shock in FcγRIIA transgenic mice. Thus, antibodies that activate platelets in an FcγRIIA-dependent manner lead to thrombosis, shock, and death. These mouse model findings have implications for human immune-mediated thrombocytopenic disorders. Genetic factors may be important in the interindividual variability seen clinically, and with nonactivating platelet antibody the spleen is largely responsible for the thrombocytopenia. This is likely the case in typical idiopathic thrombocytopenic purpura (ITP). For several other immune thrombocytopenic disorders, the spleen probably plays a second, protective role in removing antibody-coated platelets from the circulation.

The structure of Fc receptor/Ig complexes: considerations on stoichiometry and potential inhibitors

Recently, Fc receptors for immunoglobulins moved further into the focus of pure and applied research as a role in the induction and development of autoimmune diseases and allergies is becoming more probable. Indeed by connecting the humoral with the cellular immune response FcRs possess an important role in the immune system in which the initial, crucial step is the binding of an immunoglobulin to the cell bound receptor. Thereafter a variety of effector functions depending on the cell and the receptor is triggered. This key event could recently be visualised with the solution of the crystal structure of a human Fc receptor in complex with its native ligand the Fc fragment of IgG1. In this paper the reasons for a 1:1 stoichiometry between Fc receptor and Fc Fragment and the medical applications of potential inhibitors of complex formation are highlighted.

Neutrophil lactoferrin release induced by IgA immune complexes differed from that induced by cross-linking of fcalpha receptors (FcalphaR) with a monoclonal antibody, MIP8a.

The human IgA Fc receptor (FcalphaR, CD89) plays an important role in host defence against invading pathogens. To study the properties of the receptor, 12 MoAbs, namely, MIP7c, MIP8a, MIP9a, MIP10c, MIP11c, MIP14b, MIP15b, MIP38c, MIP59c, MIP65c, MIP68b and MIP71a, were generated. The inhibitory effects of the antibodies on FcalphaR functions were tested. Three of the antibodies, MIP7c, MIP8a and MIP59c, were able to block up to 90% of soluble FcalphaR binding to IgA-coated beads and 70-80% of neutrophil phagocytosis of IgA immune complexes (IC). MIP8a could also inhibit IgA IC-induced neutrophil lactoferrin release, while cross-linking of FcalphaR with MIP8a and anti-mouse IgG could elicit neutrophil lactoferrin release. However, IgA IC-induced lactoferrin release required both extracellular calcium and magnesium, whereas MIP8a-induced release did not require extracellular magnesium and only partially required extracellular calcium. In addition, the time course of IgA IC-induced lactoferrin release was slow. Lactoferrin was not detectable if the incubation time was less than 0.5 h. In contrast, MIP8a-induced lactoferrin release was fast. Lactoferrin could be detected within 5 min of incubation. Therefore, neutrophil lactoferrin release induced by IgA IC differed from that induced by cross-linking of FcalphaR with MIP8a.