Publications by authors named "Sietse Q Nagelkerke"

21 Publications

  • Page 1 of 1

C-Reactive Protein Enhances IgG-Mediated Cellular Destruction Through IgG-Fc Receptors .

Front Immunol 2021 15;12:594773. Epub 2021 Mar 15.

Sanquin Research and Landsteiner Laboratory, Department of Experimental Immunohematology, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, Netherlands.

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.
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http://dx.doi.org/10.3389/fimmu.2021.594773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006934PMC
March 2021

Genetic biomarkers for intravenous immunoglobulin response in chronic inflammatory demyelinating polyradiculoneuropathy.

Eur J Neurol 2021 May 6;28(5):1677-1683. Epub 2021 Feb 6.

Department of Immunology, Erasmus MC, University Medical Centre, Rotterdam, the Netherlands.

Background And Purpose: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinical and electrophysiological heterogeneous immune-mediated polyneuropathy. Intravenous immunoglobulin (IVIg), corticosteroids, and plasma exchange are proven effective treatments for CIDP. The clinical response to IVIg is variable between patients and currently unexplained. Finding biomarkers related to treatment response can help to understand the diversity of CIDP and personalise treatment choice.

Methods: We investigated whether genetic variation between patients may explain some of these differences in treatment response. Based on previous publications, we selected six candidate genes that might affect immune and axonal functions, IVIg metabolism, and treatment response in CIDP. Genetic variants were assessed in 172 CIDP patients treated with at least one course of IVIg (2 g/kg). A response to IVIg was defined by ≥1 grade improvement on the modified Rankin Scale. Blood samples were tested for variations in CNTN2, PRF1, FCGRT, FCGR2B, GJB1, and SH2D2A genes.

Results: In univariate analysis, patients with the FCGR2B promoter variant 2B.4/2B.1 responded more often to IVIg than patients with the 2B.1/2B.1 variant (odds ratio [OR] = 6.9, 95% confidence interval [CI] = 1.6-30; p = 0.003). Patients with the p.(Ala91Val) variant of PRF1 were less often IVIg responsive (OR = 0.34, 95% CI = 0.13-0.91; p = 0.038). In multivariate analysis, both PRF1 and FCGR2B showed discriminative ability to predict the chance of IVIg response (area under the curve = 0.67).

Conclusions: Variations in PRF1 and the promoter region of FCGR2B are associated with the response to IVIg in CIDP. These findings, which require validation, are a first step towards the understanding of the heterogeneity in the treatment response in CIDP.
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http://dx.doi.org/10.1111/ene.14742DOI Listing
May 2021

Treatment-associated hemolysis in Kawasaki disease: association with blood-group antibody titers in IVIG products.

Blood Adv 2020 07;4(14):3416-3426

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center (UMC), University of Amsterdam, Amsterdam, The Netherlands.

Hemolytic anemia resulting from IV Immunoglobulin (IVIG) treatment can be a serious complication, especially for those with underlying conditions with a high level of inflammation and after administration of high IVIG dosages, such as Kawasaki disease (KD), a multisystem vasculitis affecting young children. This hemolysis is caused by antibodies against blood groups A and B, but the precise mechanism for hemolysis is not known. We performed a single center, partly retrospective, partly prospective study of a cohort of 581 patients who received IVIG for treatment of KD from 2006 to 2013. Factors associated with hemolysis were identified through univariable and multivariable logistic regression. Six IVIG preparations were assayed for their hemolytic effect with serological and cellular assays to clarify the mechanism of red cell destruction. During the study period, a sudden increase in the incidence of hemolysis was observed, which coincided with the introduction of new IVIG preparations in North America that contained relatively high titers of anti-A and anti-B. These blood-group-specific antibodies were of the immunoglobulin G2 (IgG2) subclass and resulted in phagocytosis by monocyte-derived macrophages in an FcγRIIa-dependent manner. Phagocytosis was increased in the presence of proinflammatory mediators that mimicked the inflammatory state of KD. An increased frequency of severe hemolysis following IVIG administration was caused by ABO blood-group-specific IgG2 antibodies leading to FcγRIIa-dependent clearance of erythrocytes. This increase in adverse events necessitates a reconsideration of the criteria for maximum titer (1:64) of anti-A and anti-B in IVIG preparations.
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http://dx.doi.org/10.1182/bloodadvances.2020002253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391134PMC
July 2020

The association and functional relevance of genetic variation in low-to-medium-affinity Fc-gamma receptors with clinical platelet transfusion refractoriness.

J Thromb Haemost 2020 08 25;18(8):2047-2053. Epub 2020 Jun 25.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Background: Inadequate responses to platelet transfusions (i.e., platelet transfusion refractoriness [PLT refractoriness]) are a serious problem. Multiple factors contribute to low yields upon platelet transfusion, among which are platelet-reactive allo-antibodies. Platelet-reactive allo-antibodies occur in up to 30% of patients receiving multiple transfusions, and presumably lead to rapid destruction of the transfused platelets via receptors for IgG, the Fc-gamma receptors (FcγRs). Genetic variation in FcγRs is associated with susceptibility to immune thrombocytopenia, in which autoantibodies against platelets cause thrombocytopenia.

Objectives: We hypothesized that genetic variation in FcγRs may also influence PLT refractoriness in allo-immunized patients and could help in identifying the patients at risk.

Patients/methods: Patients with severe PLT refractoriness for whom diagnostic testing for allo-immunization was requested in the period of 2005 to 2013 were retrospectively included. A case-control study was performed comparing patients in whom platelet-reactive antibodies were detected (n = 181) with ethnically matched healthy controls (n = 180) to determine differences in all known functional copy number variations and single nucleotide polymorphisms in FcγRs.

Results And Conclusions: None of the tested FcγR genetic variations seemed associated with the development of severe PLT refractoriness. In contrast to observations in immune thrombocytopenia, genetic variation in FcγRs does not seem to influence the chance to develop PLT refractoriness. Our results do not support determination of FcγR genetic background as a means to identify patients most at risk for PLT refractoriness.
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http://dx.doi.org/10.1111/jth.14892DOI Listing
August 2020

Functional Attributes of Antibodies, Effector Cells, and Target Cells Affecting NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity.

J Immunol 2019 12 20;203(12):3126-3135. Epub 2019 Nov 20.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands;

Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell-cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell-mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell-mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa-Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.
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http://dx.doi.org/10.4049/jimmunol.1900985DOI Listing
December 2019

Genetic Variation in Low-To-Medium-Affinity Fcγ Receptors: Functional Consequences, Disease Associations, and Opportunities for Personalized Medicine.

Front Immunol 2019 3;10:2237. Epub 2019 Oct 3.

Sanquin Research and Landsteiner Laboratory, Department of Blood Cell Research, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

Fc-gamma receptors (FcγR) are the cellular receptors for Immunoglobulin G (IgG). Upon binding of complexed IgG, FcγRs can trigger various cellular immune effector functions, thereby linking the adaptive and innate immune systems. In humans, six classic FcγRs are known: one high-affinity receptor (FcγRI) and five low-to-medium-affinity FcγRs (FcγRIIA, -B and -C, FcγRIIIA and -B). In this review we describe the five genes encoding the low-to-medium -affinity FcγRs (, and , including well-characterized functionally relevant single nucleotide polymorphisms (SNPs), haplotypes as well as copy number variants (CNVs), which occur in distinct copy number regions across the locus. The evolution of the locus is also discussed. Importantly, we recommend a consistent nomenclature of genetic variants in the locus. Next, we focus on the relevance of genetic variation in the locus in auto-immune and auto-inflammatory diseases, highlighting pathophysiological insights that are informed by genetic association studies. Finally, we illustrate how specific FcγR variants relate to variation in treatment responses and prognosis amongst autoimmune diseases, cancer and transplant immunology, suggesting novel opportunities for personalized medicine.
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http://dx.doi.org/10.3389/fimmu.2019.02237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786274PMC
October 2020

Transient and chronic childhood immune thrombocytopenia are distinctly affected by Fc-γ receptor polymorphisms.

Blood Adv 2019 07;3(13):2003-2012

Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands.

In childhood immune thrombocytopenia (ITP), anti-platelet autoantibodies mediate platelet clearance through Fc-γ receptor (FcγR)-bearing phagocytes. In 75% to 90% of patients, the disease has a transient, self-limiting character. Here we characterized how polymorphisms of FcγR genes affect disease susceptibility, response to intravenous immunoglobulin (IVIg) treatment, and long-term recovery from childhood ITP. Genotyping of the locus was performed in 180 children with newly diagnosed ITP, 22 children with chronic ITP, and 180 healthy control children by multiplex ligation-dependent probe amplification. Children with newly diagnosed ITP were randomly assigned to a single administration of IVIg or observation, and followed for 1 year (Treatment With or Without IVIg for Kids With ITP [TIKI] trial). We defined transient ITP as a complete recovery (≥100 × 10/L) 3 months after diagnosis, including both self-limiting disease/IVIg responders and chronic ITP as absence of a complete recovery at 12 months. ITP susceptibility, as well as spontaneous recovery and response to IVIg, was associated with the genetic variants *ORF and *27W and the promoter variant 2B.4. These variants were overrepresented in patients with transient (N = 131), but not chronic (N = 43), disease. The presence of *ORF predisposed to transient ITP with an odds ratio of 4.7 (95% confidence interval, 1.9-14.3). Chronic ITP was associated with a deletion of (copy number region 1) with an odds ratio of 6.2 (95% confidence interval, 1.8-24.7). Taken together, susceptibility to transient and chronic ITP is distinctly affected by polymorphic variants of genes. Our data suggest that genotyping of the locus may be useful for prognosis and guidance of treatment decisions in newly diagnosed childhood ITP.
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http://dx.doi.org/10.1182/bloodadvances.2019000068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616256PMC
July 2019

Extensive Ethnic Variation and Linkage Disequilibrium at the Locus: Different Genetic Associations Revealed in Kawasaki Disease.

Front Immunol 2019 21;10:185. Epub 2019 Mar 21.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the -ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the -ORF haplotype showed strong LD with, among others, rs201218628 (-Q27W, = 0.63). LD between these two variants was weaker ( = 0.17) in Africans, whereas the -ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The -ORF haplotype and rs1801274 (-H131R) were in weak LD ( = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the -ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the locus with knowledge of LD and ethnic variation.
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http://dx.doi.org/10.3389/fimmu.2019.00185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437109PMC
January 2020

FcγRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils.

Front Immunol 2018 30;9:3124. Epub 2019 Jan 30.

Sanquin Research, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab') blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of causing different levels of surface FcγRIIIb expression. Individuals with one copy of showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.
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http://dx.doi.org/10.3389/fimmu.2018.03124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363688PMC
October 2019

Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-γ receptors.

Blood Adv 2018 04;2(8):941-953

Department of Blood Cell Research, Sanquin Research, Amsterdam, The Netherlands.

Tissue-resident macrophages in the spleen play a major role in the clearance of immunoglobulin G (IgG)-opsonized blood cells, as occurs in immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Blood cells are phagocytosed via the Fc-γ receptors (FcγRs), but little is known about the FcγR expression on splenic red pulp macrophages in humans, with only a few previous studies that showed conflicting results. We developed a novel method to specifically isolate red pulp macrophages from 82 human spleens. Surface expression of various receptors and phagocytic capacity was analyzed by flow cytometry and immunofluorescence of tissue sections. Red pulp macrophages were distinct from splenic monocytes and blood monocyte-derived macrophages on various surface markers. Human red pulp macrophages predominantly expressed the low-affinity receptors FcγRIIa and FcγRIIIa. In contrast to blood monocyte-derived macrophages, red pulp macrophages did not express the inhibitory FcγRIIb. Red pulp macrophages expressed very low levels of the high-affinity receptor FcγRI. Messenger RNA transcript analysis confirmed this expression pattern. Unexpectedly and despite these differences in FcγR expression, phagocytosis of IgG-opsonized blood cells by red pulp macrophages was dependent on the same FcγRs as phagocytosis by blood monocyte-derived macrophages, especially in regarding the response to IV immunoglobulin. Concluding, we show the distinct nature of splenic red pulp macrophages in human subjects. Knowledge on the FcγR expression and usage of these cells is important for understanding and improving treatment strategies for autoimmune diseases such as ITP and AIHA.
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http://dx.doi.org/10.1182/bloodadvances.2017015008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916003PMC
April 2018

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody-dependent cellular cytotoxicity towards cancer cells.

Eur J Immunol 2018 02 2;48(2):344-354. Epub 2017 Nov 2.

Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti-cancer antibodies act through different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47-SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab-coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa-131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa-131R. Furthermore, ADCC was consistently enhanced by targeting CD47-SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47-SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47-SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.
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http://dx.doi.org/10.1002/eji.201747215DOI Listing
February 2018

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

Blood 2017 11 12;130(19):2121-2130. Epub 2017 Sep 12.

Department of Blood Cell Research, Sanquin Research, and Landsteiner Laboratory.

Red 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 genes has been associated with various auto- and alloimmune diseases. The aim of this study was to evaluate the association between genetic variation of 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 gene (2C.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 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.
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http://dx.doi.org/10.1182/blood-2017-05-784876DOI Listing
November 2017

Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcγ Receptor Profile.

J Immunol 2017 07 31;199(1):204-211. Epub 2017 May 31.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands.

Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene-matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1-4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.
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http://dx.doi.org/10.4049/jimmunol.1700116DOI Listing
July 2017

RhIg-prophylaxis is not influenced by FCGR2/3 polymorphisms involved in red blood cell clearance.

Blood 2017 02 12;129(8):1045-1048. Epub 2017 Jan 12.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1182/blood-2016-05-716365DOI Listing
February 2017

Fc-gamma receptor polymorphisms differentially influence susceptibility to systemic lupus erythematosus and lupus nephritis.

Rheumatology (Oxford) 2016 May 8;55(5):939-48. Epub 2016 Jan 8.

Department of Rheumatology, Amsterdam Rheumatology and immunology Center, VU University Medical Center.

Objective: To determine relevant Fc-gamma receptor (FcγR) polymorphisms in relation to susceptibility to SLE and LN, and to determine the functional consequences of genetic associations found.

Methods: Using multiplex ligation-dependent probe amplification, copy number regions (CNRs) and relevant known functional single nucleotide polymorphisms of FcγRII and FcγRIII were determined in a LN-enriched cohort of 266 Dutch Caucasian SLE patients and 919 healthy Caucasian controls. Expression of FcγRs on leukocytes was assessed using flow cytometry.

Results: In multivariable analysis, low copy number of CNR1 (including FCGR3B; odds ratio (OR) 2.04; 95% CI: 1.29, 3.23), FCGR2A-131RR (OR 2.00; 95% CI: 1.33, 2.99), and the 2B.4 haplotype of FCGR2B (OR 1.59; 95% CI: 1.13, 2.24), but not FCGR2C open reading frame, were significantly (all P < 0.01) and independently associated with susceptibility to SLE. The 2B.4 haplotype was negatively associated with LN and led to surface expression of FcγRIIb on neutrophils and monocytes.

Conclusion: This study is the first to investigate the most relevant and functional single nucleotide polymorphisms and copy number variations of FcγRII and FcγRIII polymorphisms in one study population, enabling the determination of the individual contribution of each polymorphism in multivariable analysis. Three polymorphisms were shown to be independently associated with susceptibility to SLE. The novel findings of a negative association of the 2B.4 haplotype with LN, and increased expression of FcγRIIb on neutrophils and monocytes as a result of this 2B.4 haplotype warrant future research in the role of these cells and FcγRs in the pathogenesis of SLE and LN.
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http://dx.doi.org/10.1093/rheumatology/kev433DOI Listing
May 2016

Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus.

PLoS One 2015 6;10(11):e0142379. Epub 2015 Nov 6.

Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, SO16 6YD, United Kingdom.

Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142379PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636148PMC
June 2016

Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing.

Cancer Immunol Res 2015 Dec 25;3(12):1316-24. Epub 2015 Sep 25.

Immunotherapy Laboratory, Laboratory for Translational Immunology, UMC Utrecht, the Netherlands.

Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressing effector cells and therefore initiate different effector mechanisms in vivo compared with IgG. Here, we studied killing of tumor cells coexpressing EGFR and HER2 by the IgG mAbs cetuximab and trastuzumab and their IgA variants. In the presence of a heterogeneous population of effector cells (leukocytes), the combination of IgG and IgA mAbs to two different tumor targets (EGFR and HER2) led to enhanced cytotoxicity compared with each isotype alone. Combination of two IgGs or two IgAs or IgG and IgA against the same target did not enhance cytotoxicity. Increased cytotoxicity relied on the presence of both the peripheral blood mononuclear cell and the polymorphonuclear (PMN) fraction. Purified natural killer cells were only cytotoxic with IgG, whereas cytotoxicity induced by PMNs was strong with IgA and poor with IgG. Monocytes, which coexpress FcγRs and FcαRI, also displayed increased cytotoxicity by the combination of IgG and IgA in an overnight killing assay. Coinjection of cetuximab and IgA2-HER2 resulted in increased antitumor effects compared with either mAb alone in a xenograft model with A431-luc2-HER2 cells. Thus, the combination of IgG and IgA isotypes optimally mobilizes cellular effectors for cytotoxicity, representing a promising novel strategy to improve mAb therapy.
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http://dx.doi.org/10.1158/2326-6066.CIR-15-0099-TDOI Listing
December 2015

Immunomodulation by IVIg and the Role of Fc-Gamma Receptors: Classic Mechanisms of Action after all?

Front Immunol 2014 21;5:674. Epub 2015 Jan 21.

Department of Blood Cell Research, Sanquin, University of Amsterdam , Amsterdam , Netherlands ; Department of Pediatric Hematology, Immunology and Infectious Disease, Emma Children's Hospital at the Academic Medical Center, University of Amsterdam , Amsterdam , Netherlands.

Intravenous IgG (IVIg) contains polyclonal immunoglobulin G (IgG) from thousands of donors. It is administered at a low dose at regular intervals as antibody replacement therapy and at a higher dose as immunomodulatory treatment in various auto-immune or auto-inflammatory diseases. The working mechanism of immunomodulation is not well understood. Many different explanations have been given. During the last decade, we have focused on classical antibody binding via the Fc-domain of the IgG molecules to the common IgG receptors, i.e. the Fcγ receptors (FcγRs). Variation in the genes encoding human FcγRs determines function as well as expression among immune cells. As described here, NK cells and myeloid cells, including macrophages, can express different FcγR variants, depending on the individual's genotype, copy number variation (CNV), and promoter polymorphisms. B-cells seem to only express the single inhibitory receptor. Although these inhibitory FcγRIIb receptors are also expressed by monocytes, macrophages, and only rarely by NK cells or neutrophils, their presence is unlikely to explain the immunomodulatory capacity of IVIg, nor does the sialylation of IgG. Direct IVIg effects at the level of the activating FcγRs, including the more recently described FcγRIIc, deserve renewed attention to describe IVIg-related immunomodulation.
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http://dx.doi.org/10.3389/fimmu.2014.00674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301001PMC
February 2015

Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages.

Blood 2014 Dec 28;124(25):3709-18. Epub 2014 Oct 28.

Department of Blood Cell Research, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

In immune thrombocytopenia and warm autoimmune hemolytic anemia, circulating immunoglobulin G (IgG)-opsonized blood cells are cleared from the circulation by macrophages. Administration of intravenous immunoglobulin (IVIg) can prevent uptake, but the exact working mechanism is not known. The prevailing theory from murine studies, which states that Fc-sialylated IgG alters the balance between activating and inhibitory Fc-gamma receptors (FcγRs) by inducing upregulation of the inhibitory FcγRIIb on effector macrophages, is currently debated. We studied phagocytosis of IgG-opsonized blood cells in a human system, assessing the effect of IVIg and blocking anti-FcγR F(ab')2 fragments on uptake by monocyte-derived macrophages (both M1 and M2 macrophages). Phagocytosis was remarkably sensitive to administration of IVIg, but unexpectedly, recombinant Fc-sialylated IgG or sialic acid-enriched IVIg were equally active as unsialylated IgG fractions in mediating this inhibition, independent of FcγRIIb expression. Instead, IVIg inhibited phagocytosis by direct blockade of FcγRs. IgG fractions enriched for IgG dimers with enhanced avidity for FcγRs showed increased inhibition compared with monomeric IgG fractions. Together, our data demonstrate that inhibition of IgG-mediated phagocytosis in human macrophages by IVIg is dependent on the capacity to directly bind FcγRs but is independent of FcγRIIb or sialylation of the Fc fragment in the human setting.
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http://dx.doi.org/10.1182/blood-2014-05-576835DOI Listing
December 2014

FcγRIIa cross-talk with TLRs, IL-1R, and IFNγR selectively modulates cytokine production in human myeloid cells.

Immunobiology 2015 Feb 25;220(2):193-9. Epub 2014 Jul 25.

Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Electronic address:

Myeloid antigen-presenting cells (APCs) tailor immune responses to the pathogen involved through the production of specific pro- and anti-inflammatory cytokines. It is becoming increasingly clear that the ultimate cytokine profile produced by myeloid APCs crucially depends on interaction between multiple pathogen recognizing receptors. In this respect, we recently identified an important role for cross-talk between Fc gamma receptor IIa (FcγRIIa) and Toll-like receptors (TLRs) in human dendritic cells (DCs), which induces anti-bacterial immunity through the selective induction of TNFα and Th17-promoting cytokines. Here, we show that FcγRIIa-TLR cross-talk is not restricted to DCs, but is a common feature of various human myeloid APC subsets including monocytes and macrophages. Interestingly, FcγRIIa-TLR cross-talk in monocytes resulted in the induction of a cytokine profile distinct from that in DCs and macrophages, indicating that FcγRIIa stimulation induces cell-type and tissue specific responses. Surprisingly, we show that the FCGR2A H131R single nucleotide polymorphism (SNP), which is known to greatly affect FcγRIIa-mediated uptake of IgG2-opsonized bacteria, did not affect FcγRIIa-dependent cytokine production, indicating that these processes are differently regulated. In addition, we demonstrate that FcγRIIa selectively synergized with TLRs, IL-1R, and IFNγR, but did not affect cytokine production induced by other receptors such as C-type lectin receptor Dectin-1. Taken together, these data demonstrate that FcγRIIa-dependent modulation of cytokine production is more widespread than previously considered, and indicate that cross-talk of FcγRIIa with various receptors and in multiple cell types contributes to the induction of pathogen and tissue-specific immunity.
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http://dx.doi.org/10.1016/j.imbio.2014.07.016DOI Listing
February 2015