Low-affinity Fcγ Receptors Research Articles

Abstract 3190: Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement

Abstract Immunoglobulin G (IgG)-based mAbs have become a dominant class of cancer immunotherapy due to their excellent drug-like properties, long half-life, and generally safe profiles. Typical IgG-based therapeutic mAbs have strong binding to target antigens owing to the valency effect of two antigen-binding fragments (Fabs). However, the single crystallizable fragment (Fc) lacks the valency effect to engage immune effector elements. As Fcγ receptors (FcγR) such as FcγRIIa and FcγRIIIa and C1q complement component have low affinity for Fc, IgG-based mAbs require high antibody opsonization density to exhibit avidity for these low affinity Fcγ receptors and C1q. This may also account for the suboptimal efficacy of IgG-based mAbs against tumors with less abundant antigen. To enhance effector functions of IgG1 by overcoming such limitation, we devised a novel platform, Twin Fc-ICE, in which a single Fab region is attached by two Fc regions which allow for high avidity binding to Fcγ receptors and C1q. The two Fc regions of Twin Fc-ICE are arranged in parallel, not in tandem, to adopt natural topologies of multiple Fc regions presented by IgG1-opsonized targets. Supporting the avidity effect of Twin Fc-ICE for FcγR binding, we showed that CTN001, a Twin Fc-ICE with trastuzumab Fab, have markedly increased binding to FcγRs including FcγRI, FcγRIIa and FcγRIIIa compared with trastuzumab. The avid FcγR binding was further demonstrated by flow cytometry analysis of human blood which revealed significantly increased binding of CTN001 relative to trastuzumab for immune cells expressing activating FcγRs such as natural killer (NK) cells, monocytes, and dendritic cells (DCs). The monovalent antigen binding of Twin Fc-ICE requires Fab with high affinity. However, provided that high affinity is achieved, the monovalent Fab adds to the two Fc regions to maximize the Fc load on cancer cells expressing low levels of antigen. Indeed, flow cytometry analysis revealed that treatment of CTN001 alone results in higher Fc load on HER2-expressing cancer cells than combination of two anti-HER2 IgG1 mAbs, trastuzumab and pertuzumab. In consistent with increased Fc load on target cells and avid FcγR binding, CTN001 induces enhanced effector functions such as CDC, ADCC and ADCP against HER2-expressing cancer cells compared with trastuzumab, which will be presented in a separate poster. CTN001 exhibits comparable thermal stability with trastuzumab and can be readily purified to >95% purity with typical purification platforms. The serum half-life of CTN001 at 10 mg/kg intravenous dose in rats is found non-inferior to that of trastuzumab (~12 days versus ~7 days). Taken together, Twin Fc-ICE provides an optimized platform with drug-like properties, which leverages avidity effects of Fc to potentiate therapeutic efficacy of antibody. Citation Format: Eun Shik Choi, Hyunkyu Park, Jiseon Bae, Ki-June Lee, Eunmi Kim, Sujin Yoon, Deokjae Lee, Gi-Hyeok Yang. Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3190.

Importance of circulating monocyte subpopulations and Fcγ receptors in immune thrombocytopenia.

Monocytes have been linked to the pathogenesis of immune thrombocytopenia (ITP) because of their role in autoantibody-mediated platelet phagocytosis. However, monocytes constitute unique populations with major differences in expression for surface Fcγ receptors (FcγRs). Thus, we evaluated monocytes in whole blood samples from patients with newly diagnosed and chronic ITP. Monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 (lipopolysaccharide receptor) and of CD16 (low-affinity Fcγ receptor III) into classical (CLM), intermediate (INTM) and nonclassical (non-CLM) monocytes. We also examined the expression of FcγRI/CD64 and FcγRIII/CD16 by monocyte subpopulations. Newly diagnosed patients showed a decrease in non-CLM, expressed as a relative percentage of total monocytes compared with controls and chronic ITP patients. Both non-CLM and INTM of newly diagnosed patients closely correlated with platelet count. These monocyte subpopulations showed significantly enhanced CD64 expression in newly diagnosed patients. On the contrary, patients with chronic ITP presented higher non-CLM in percentage than controls and concomitant lower CLM and total monocytes, in percentage and number. The expression of CD64 was increased by all monocyte subpopulations, CLM, INTM, and non-CLM in chronic patients. In conclusion, differences in monocyte subpopulations, together with enhanced expression of FcγRI/CD64 are evident in patients with ITP.

Exploring the Role of T VMCells in Age-Related CD8 +T Cell Dysfunction through ATAC-seq and RNA-seq Analysis

Abstract Virtual memory T (T VM) cells are a unique subset of CD8 +T cells that have both classical memory and innate-like immune function. Unlike other CD8 +T cell subsets, T VMcells are highly dependent on cytokines for their homeostasis and are selectively depleted in mice lacking IL-15. In aged mice and humans, T VMcells acquire T cell receptor-associated defects, which may contribute to the decline in overall CD8 +T cell function seen with aging. T VMcells make up a large proportion of the naive CD8 +T cell pool in aged individuals, making them an ideal target for age-specific therapeutic interventions. In this study, we performed ATAC-seq and RNA-seq analysis on young and aged mouse CD8 +T cell subsets and identified several key signatures associated with age-related dysfunction specifically in T VMcells. These signatures reveal a bifurcation in the aging process whereby memory-phenotype CD8 +T cells are affected differently by the aging environment. The aged-specific T VMsignature is distinct from that of conventional memory CD8 +T cells and results in the upregulation of immunomodulatory molecules such as Fcgr2b, which encodes the low-affinity inhibitory Fcγ receptor. Expression of FcγRIIB on CD8 +T cells has been shown to play a role in suppressing anti-tumor immunity and mitigating graft-versus-host disease. We show that a subset of aged T VMcells upregulate FcγRIIB independently of antigen exposure and are prone to apoptosis in vitro. Blockade of FcγRIIB has been shown to augment CD8 +T cell responses in vivo, making it a potential therapeutic target for restoring function in aged T VMcells. These findings provide insight into the mechanisms of age-related immune dysfunction and potential strategies for targeting T VMcells in the elderly. Supported by grants from NHMRC/ARC

FcγR Genetic Variation and HIV-1 Vaccine Efficacy: Context And Considerations.

Receptors for the crystallisable fragment (Fc) of immunoglobulin (Ig) G, Fcγ receptors (FcγRs), link the humoral and cellular arms of the immune response, providing a diverse armamentarium of antimicrobial effector functions. Findings from HIV-1 vaccine efficacy trials highlight the need for further study of Fc-FcR interactions in understanding what may constitute vaccine-induced protective immunity. These include host genetic correlates identified within the low affinity Fcγ-receptor locus in three HIV-1 efficacy trials – VAX004, RV144, and HVTN 505. This perspective summarizes our present knowledge of FcγR genetics in the context of findings from HIV-1 efficacy trials, and draws on genetic variation described in other contexts, such as mother-to-child HIV-1 transmission and HIV-1 disease progression, to explore the potential contribution of FcγR variability in modulating different HIV-1 vaccine efficacy outcomes. Appreciating the complexity and the importance of the collective contribution of variation within the FCGR gene locus is important for understanding the role of FcγRs in protection against HIV-1 acquisition.

CD32a receptor in health and disease

Low-affinity Fcγ-receptors that recognize the Fc portion of immunoglobulin (Ig) molecules, usually being in antigen-bound state, thus representing a link between innate and adaptive immunity. They play a significant role in inflammatory and infectious diseases. Among them, a separate FcγRII family (CD32) is discerned, which is characterized by transmission of intracellular signal independently of the common γ-chain, they have one α-chain containing two extracellular immunoglobulin-like domains. FcγRII receptors are present in almost all cells of the innate immune system: monocytes and macrophages, neutrophils, eosinophils, dendritic cells, as well as on B-lymphocytes and platelets. They perform two main functions: target recognition, facilitation of phagocytosis and destruction of antibody-opsonized cells by monocytes/ macrophages (including pathogenic cells). In parallel, the phagocytes are activated via the cytokine synthesis stimulation. The FcγRIIA (CD32a) and FcγRIIC (CD32c) activating receptors, like as FcγRIIB (CD32b) inhibiting receptors are present among the members of the FcγRII family. The low-affinity FcγRII receptors bind to IgG, with immune complexes being their natural ligands. High levels of immune complexes are usually found in both chronic viral infections and autoimmune diseases. There are shown polymorphic variants of the CD32a gene, which can affect the receptor function, and, thereby, causing susceptibility for different infections, influence the development of autoimmune diseases and primary immunodeficiencies. Activation of the CD32a receptor induces the production of pro-inflammatory cytokines, including TNFα and interferons, that are involved into inflammation in systemic lupus erythematosus, Kawasaki disease, Graves’ disease and rheumatoid arthritis. It has been shown that antibacterial activity of platelets is carried out via the CD32a receptor. The study of CD32a expression in people The CD32a receptor is considered a biomarker of cells that are a reservoir of HIV infection. At the present time, however, many questions remain regarding the mechanisms of CD32a expression of on HIV-infected cells and the role of CD32a in the formation of an HIV reservoir and/or development of appropriate resistance. In addition to HIV infection, the significance of FcγR receptors is shown in other infectious diseases, for example, with influenza and dengue virus infections. Better understanding of the CD32a structure and function will help to assess its role in immunopathogenesis of different conditions. This review focuses on the role of CD32a in development of the normal immune response in normal state and various diseases.

Platelet Activation by Antiphospholipid Antibodies Depends on Epitope Specificity and is Prevented by mTOR Inhibitors.

Antiphospholipid antibodies (aPL) have been reported to activate platelets. This is considered to be one of the pathogenic properties of aPL. Even though aPL heterogeneity is quite well established, little is known, if the ability to activate platelets is common to all aPL or depends on antigen specificity. To further study this issue, we analyzed the ability of three human monoclonal aPL with distinctly different antigenic specificities to activate platelets in vitro. The results obtained with human monoclonal aPL were validated with immunoglobulin G (IgG) fractions obtained from patients with antiphospholipid syndrome (APS). A co-factor-independent human monoclonal anticardiolipin aPL had no discernible effect on human platelets. Two monoclonal aPL reactive against β2 glycoprotein I (β2GPI) induced platelet aggregation, integrin αIIbβ3 activation and P-selectin surface expression. These data could be confirmed with patient IgG fractions which could only induce aggregation, if they had anti-β2GPI activity. Anti-β2GPI aPL-induced platelet activation depended on interaction of aPL with the low affinity Fcγ-receptor IIa on the platelet surface. It was completely abolished by pretreatment of platelet-rich plasma with the mechanistic target of rapamycin (mTOR) inhibitors rapamycin or everolimus. This extends previous data showing that mTOR is involved in signaling of anti-β2GPI in monocytes and endothelial cells. In conclusion, anti-β2GPI aPL activate platelets while co-factor-independent anticardiolipin aPL have no effect. mTOR is involved in this signaling process which has implications beyond APS, because so far the role of mTOR signaling in platelets is incompletely explored and requires further study.

Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein–Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A–mediated agglutination of human erythrocytes.

Effects of an FcγRIIA polymorphism on leukocyte gene expression and cytokine responses to anti-CD3 and anti-CD28 antibodies.

The low affinity Fcγ receptor, FcγRIIA, harbors a common missense mutation, rs1801274 (G>A, Arg131His) that modifies binding affinity to human IgG2 and mouse IgG1 antibodies and is associated with increased risk of autoimmune disease. Despite the important role of the Arg131His variant, little is understood about heterozygous genotype effects on global gene expression and cytokine production during an FcγR-dependent response. To address this gap in knowledge, we treated human whole-blood samples from 130 individuals with mouse IgG1 anti-CD3 and anti-CD28 antibodies and characterized the genome-wide gene expression profiles and cytokine production among individuals stratified by rs1801274 genotype. Our analysis revealed that the levels of four cytokines (IFNγ, IL-12, IL-2, TNFα) and global gene expression patterns differed between all three genotype classes. Surprisingly, the heterozygotes showed suboptimal T cell activation compared to cells from individuals homozygous for the higher-affinity FcγRIIA allele (GG; Arg/Arg). The results of this study demonstrate that IgG response varies among all rs1801274 genotype classes and results in profound differences in both cytokine responses and gene expression patterns in blood leukocytes. Because even heterozygotes showed dampened global responses, our data may provide insight into the heterogeneity of outcomes in cytokine release assays and immunotherapy efficacy.

Nonclassical FCGR2C haplotype is associated with protection from red blood cell alloimmunization in sickle cell disease

AbstractRed blood cell (RBC) transfusions are of vital importance in patients with sickle cell disease (SCD). However, a major complication of transfusion therapy is alloimmunization. The low-affinity Fcγ receptors, expressed on immune cells, are important regulators of antibody responses. Genetic variation in FCGR genes has been associated with various auto- and alloimmune diseases. The aim of this study was to evaluate the association between genetic variation of FCGR and RBC alloimmunization in SCD. In this case-control study, DNA samples from 2 cohorts of transfused SCD patients were combined (France and The Netherlands). Cases had a positive history of alloimmunization, having received ≥1 RBC unit. Controls had a negative history of alloimmunization, having received ≥20 RBC units. Single nucleotide polymorphisms and copy number variation of the FCGR2/3 gene cluster were studied in a FCGR-specific multiplex ligation-dependent probe amplification assay. Frequencies were compared using logistic regression. Two hundred seventy-two patients were included (130 controls, 142 cases). The nonclassical open reading frame in the FCGR2C gene (FCGR2C.nc-ORF) was strongly associated with a decreased alloimmunization risk (odds ratio [OR] 0.26, 95% confidence [CI] 0.11-0.64). This association persisted when only including controls with exposure to ≥100 units (OR 0.30, CI 0.11-0.85) and appeared even stronger when excluding cases with Rh or K antibodies only (OR 0.19, CI 0.06-0.59). In conclusion, SCD patients with the FCGR2C.nc-ORF polymorphism have over a 3-fold lower risk for RBC alloimmunization in comparison with patients without this mutation. This protective effect was strongest for exposure to antigens other than the immunogenic Rh or K antigens.

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR.

Characterization of conformational deformation-coupled interaction between immunoglobulin G1 Fc glycoprotein and a low-affinity Fcγ receptor by deuteration-assisted small-angle neutron scattering

A recently developed integrative approach combining varied types of experimental data has been successfully applied to three-dimensional modelling of larger biomacromolecular complexes. Deuteration-assisted small-angle neutron scattering (SANS) plays a unique role in this approach by making it possible to observe selected components in the complex. It enables integrative modelling of biomolecular complexes based on building-block structures typically provided by X-ray crystallography. In this integrative approach, it is important to be aware of the flexible properties of the individual building blocks. Here we examine the ability of SANS to detect a subtle conformational change of a multidomain protein using the Fc portion of human immunoglobulin G (IgG) interacting with a soluble form of the low-affinity Fcγ receptor IIIb (sFcγRIIIb) as a model system. The IgG-Fc glycoprotein was subjected to SANS in the absence and presence of 75%-deuterated sFcγRIIIb, which was matched out in D2O solution. This inverse contrast-matching technique enabled selective observation of SANS from IgG-Fc, thereby detecting its subtle structural deformation induced by the receptor binding. The SANS data were successfully interpreted by considering previously reported crystallographic data and an equilibrium between free and sFcγRIIIb-bound forms. Our SANS data thus demonstrate the applicability of SANS in the integrative approach dealing with biomacromolecular complexes composed of weakly associated building blocks with conformational plasticity.

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

SummaryIn order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen‐presenting cells. These self‐adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII‐PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII‐PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII‐PIGS also induced a significant cellular immune response, IFN‐γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof‐of‐principle study shows that the potent antibody Fc‐mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad‐ranging immune response.

Low copy numbers of FCGR3A and FCGR3B associated with Chinese patients with SLE and AASV.

Low-affinity Fcγ receptors (FcγR) act as key mediators of the pathogenic effects of autoantibodies. In this study, we aimed to determine whether copy number variations (CNVs) in FCGR3A and FCGR3B were associated with systemic lupus nephritis (SLE) and ANCA-associated systemic vasculitis (AASV) in Chinese individuals. A total of 1118 individuals were enrolled, including 415 SLE patients, 139 AASV patients, and 564 healthy controls. FCGR3A and FCGR3B copy numbers (CNs) were determined by both a paralogue ratio test and TaqMan quantitative PCR assay. In the susceptibility associations, a low FCGR3B CN was significantly associated with SLE ( p = 5.01 × 10-3; odds ratio (OR) 1.71; 95% confidence interval (CI) 1.17-2.48) and AASV ( p = 0.04; OR = 1.72; 95% CI 1.02-2.88). A low FCGR3A CN was also significantly associated with SLE ( p = 6.02 × 10-3; OR 2.72; 95% CI 1.30-5.71) and AASV ( p = 0.042; OR 2.64; 95% CI 1.00-6.93). Further subphenotype analysis revealed that low CNs of FCGR3A and FCGR3B were significantly associated with clinical manifestations in SLE and AASV patients. Therefore, in this case-control study, we identified low CNs of FCGR2A and FCGR3B to be common risk factors for SLE and AASV.

Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis.

Chemical protein biotinylation and streptavidin or anti-biotin-based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine-tuning of critical parameters is important to reduce biotinylation in a protein binding site.

Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-016-9515-8) contains supplementary material, which is available to authorized users.

Allelic and copy-number variations of FcγRs affect granulocyte function and susceptibility for autoimmune blistering diseases

Low-affinity Fcγ receptors (FcγR) bridge innate and adaptive immune responses. In many autoimmune diseases, these receptors act as key mediators of the pathogenic effects of autoantibodies. Genes encoding FcγR exhibit frequent variations in sequence and gene copy number that influence their functional properties. FcγR variations also affect the susceptibility to systemic autoimmunity, e.g. systemic lupus erythematosus and rheumatoid arthritis. This raises the question whether FcγR variations are also associated with organ-specific autoimmunity, particularly autoantibody-mediated diseases, such as subepidermal autoimmune blistering diseases (AIBD). A multitude of evidence suggests a pathogenic role of neutrophil granulocyte interaction with autoantibodies via FcγR. In a two-stage study, we analyzed whether the FcγR genotype affects neutrophil function and mRNA expression, and consequently, bullous pemphigoid (BP) disease risk. We compared this to findings in pemphigus vulgaris/foliaceus (PV/PF), two Fc-independent AIBDs. Our results indicate that both allele and copy number variation of FcγR genes affect FcγR mRNA expression and reactive oxygen species (ROS) release by granulocytes. Susceptibility of BP was associated with FcγR genotypes that led to a decreased ROS release by neutrophils, indicating an unexpected protective role for these cells. BP and PV/PF differed substantially regarding the FcγR genotype association patterns, pointing towards different disease etiologies.

Single-nucleotide polymorphisms and copy number variations of the FCGR2A and FCGR3A genes in healthy Japanese subjects

FcγRII and FcγRIII are low-affinity Fcγ receptors that are encoded by the FCGR2A and FCGR3A genes, respectively. These genes contain functional single-nucleotide polymorphisms (SNPs), which alter the binding affinities of these receptors for the γ chain of the Fc fragment of immunoglobulin G. The known SNPs in FCGR2A and FCGR3A are rs1801274 (A>G; H131R) and rs396991 (T>G; F158V), respectively. It is also known that there are copy number variations (CNVs) in the genetic locus (1q23) where FCGR2A and FCGR3A are located. However, the frequencies of these SNPs and CNVs have not been determined in the Japanese population. The aim of this study was to investigate SNPs and CNVs in FCGR2A and FCGR3A among 113 healthy individuals. The SNPs and CNVs in FCGR2A and FCGR3A were determined using the TaqMan® SNP Genotyping and the TaqMan® Copy Number assays. Our results revealed that the incidence of FCGR2A (rs1801274) genotypes were as follows: A/A, 69.9%; A/G, 29.2%; and G/G, 0.9%. The incidence of the FCGR3A (rs396991) genotypes were as follows: T/T, 56.7%; T/G, 38.9%; and G/G, 4.4%). No CNVs were detected for FCGR2A. To the best of our knowledge, this finding has not been previously reported in the Japanese population. By contrast, CNVs were observed in FCGR3A (3 subjects were found to harbour a gene deletion and 5 subjects had 3 copies of the gene). Using simple commercially available assays we were able to confirm previous findings regarding FCGR2A and FCGR3A alleles and CNVs. These assays may provide a basis for the investigation of the role of these genes in the efficacy of antibody-based drugs, such as trastuzumab and rituximab, in Japanese subjects.

FcγRIII (CD16) equips immature 6-sulfo LacNAc–expressing dendritic cells (slanDCs) with a unique capacity to handle IgG-complexed antigens

AbstractBinding and uptake of immune complexes (ICs) via low-affinity Fcγ receptors (FcγRs) on dendritic cells (DCs) is well known as a booster of immune responses. It can be helpful when stimulating immunity against pathogenic microbes but may be harmful when antibodies form complexes with autologous antigens. To date, no human DC subtype specialized in handling ICs has been identified. By incubating human blood mononuclear cells with ICs and studying their cellular binding, we identified 6-sulfo LacNAc-expressing DCs (slanDCs) as having an outstanding capacity to bind ICs compared with other myeloid DCs, plasmacytoid DCs, or monocytes. Using selective blocking of different (FcγRs), we identified CD16 (FcγRIII) as the major IC-binding structure on slanDCs. In addition, CD16 proved critical for phagocytosis of IgG-coated erythrocytes, and CD16-targeted antigen led to a more efficient proliferation of CD4+ T cells than CD32 (FcγRII)-targeted antigen. Interestingly, these CD16-mediated functions are short-lived and restricted to the immature stage of slanDCs in blood. We show that CD16 is rapidly shed from the surface of maturing slanDCs, resulting from the combined action of the metalloproteinases ADAM10 and ADAM17. In conclusion, these data provide strong evidence that slanDCs play an important role in IC-driven immune responses.

At diagnosis, diffuse large B‐cell lymphoma patients show impaired rituximab‐mediated NK‐cell cytotoxicity

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma in adults. It is generally treated by a combination of chemotherapy and CD20-specific mAbs, such as rituximab, which act, at least partially, by activating antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC involves NK cells, particularly the CD56(dim) NK-cell subset expressing CD16, the low affinity Fcγ receptor. Here, we show that CD16 expression levels are decreased in a cohort of 36 newly diagnosed DLBCL patients compared with those in 20 healthy controls (HCs). CD137, a co-stimulatory molecule expressed on activated NK cells, was also expressed at lower levels in patients compared with controls. Cells sampled from our cohort also showed severely reduced degranulation activity when challenged with rituximab-coated tumor cells, which could not be corrected by stimulation with high doses of IL-2. These results suggest that rituximab-induced NK-cell ADCC could be defective in some DLBCL patients at diagnosis. These patients should be closely monitored and attempts made to improve their NK-cell function.

The binding of soluble recombinant human Fcγ receptor I for human immunoglobulin G is conferred by its first and second extracellular domains

Human FcγRI is a high affinity receptor for the Fc portion of human immunoglobulin G (IgG), and has extracellular, transmembrane and cytoplasmic regions. The extracellular region of human FcγRI, which is the part that interacts with human IgG, is comprised of three immunoglobulin-like domains. Unlike low affinity Fcγ receptors (FcγRII and FcγRIII), FcγRI has a unique third extracellular domain (D3). This study investigated the contribution of D3 to the binding between recombinant human FcγRI (rhFcγRI) and human IgG. The three extracellular domains and the first and second extracellular domains of human FcγRI were expressed by Escherichia coli as rhFcγRI and rhFcγRI-D1D2, respectively. The binding specificity of rhFcγRI-D1D2 to human IgG subclasses was the same as that of rhFcγRI. From surface plasmon resonance analysis, the binding affinity of rhFcγRI-D1D2 for human IgG1/κ was high (the equilibrium dissociation constant: KD=8.04×10−10M), but slightly lower than that of rhFcγRI (KD=2.59×10−10M). While the association of rhFcγRI-D1D2 with human IgG1/κ was same as that of rhFcγRI, the dissociation of rhFcγRI-D1D2 was faster than that of rhFcγRI. From these results, D3 of rhFcγRI would not contribute directly to the binding specificity and association of rhFcγRI, but to the holding bound human IgG.