Publications by authors named "Timo K van den Berg"

123 Publications

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

m6A methylation potentiates cytosolic dsDNA recognition in a sequence-specific manner.

Open Biol 2021 Mar 10;11(3):210030. Epub 2021 Mar 10.

Sanquin Research, University of Amsterdam, Amsterdam, The Netherlands.

Nucleic acid sensing through pattern recognition receptors is critical for immune recognition of microbial infections. Microbial DNA is frequently methylated at the N position of adenines (m6A), a modification that is rare in mammalian host DNA. We show here how that m6A methylation of 5'-GATC-3' motifs augments the immunogenicity of synthetic double-stranded (ds)DNA in murine macrophages and dendritic cells. Transfection with m6A-methylated DNA increased the expression of the activation markers CD69 and CD86, and of , and mRNA. Similar to unmethylated cytosolic dsDNA, recognition of m6A DNA occurs independently of TLR and RIG-I signalling, but requires the two key mediators of cytosolic DNA sensing, STING and cGAS. Intriguingly, the response to m6A DNA is sequence-specific. m6A is immunostimulatory in some motifs, but immunosuppressive in others, a feature that is conserved between mouse and human macrophages. In conclusion, epigenetic alterations of DNA depend on the context of the sequence and are differentially perceived by innate cells, a feature that could potentially be used for the design of immune-modulating therapeutics.
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http://dx.doi.org/10.1098/rsob.210030DOI Listing
March 2021

Myeloid Ezh2 Deficiency Limits Atherosclerosis Development.

Front Immunol 2020 26;11:594603. Epub 2021 Jan 26.

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam Infection and Immunity, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

Macrophages define a key component of immune cells present in atherosclerotic lesions and are central regulators of the disease. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways in macrophages might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by polycomb repressive complex with EZH2 as the catalytic subunit. EZH2 is described to increase macrophage inflammatory responses by supressing the suppressor of cytokine signaling, . We previously showed that myeloid deletion of , an enzymes that in contrast to EZH2 removes repressive histone H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function and importance of EZH2 in macrophage inflammatory responses, we here studied the role of myeloid EZH2 in atherosclerosis. A myeloid-specific deficient mouse strain () was generated (LysM-cre+ x ) and bone marrow from or mice was transplanted to mice which were fed a high fat diet for 9 weeks to study atherosclerosis. Atherosclerotic lesion size was significantly decreased in transplanted mice compared to control. The percentage of macrophages in the atherosclerotic lesion was similar, however neutrophil numbers were lower in transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased in mice. Moreover, peritoneal foam cells showed a reduction in the inflammatory response with reduced production of nitric oxide, IL-6 and IL-12. In Conclusion, myeloid deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.
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http://dx.doi.org/10.3389/fimmu.2020.594603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871783PMC
January 2021

Neutrophil specific granule and NETosis defects in gray platelet syndrome.

Blood Adv 2021 Jan;5(2):549-564

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

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.
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http://dx.doi.org/10.1182/bloodadvances.2020002442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839360PMC
January 2021

Kindlin3-Dependent CD11b/CD18-Integrin Activation Is Required for Potentiation of Neutrophil Cytotoxicity by CD47-SIRPα Checkpoint Disruption.

Cancer Immunol Res 2021 Feb 22;9(2):147-155. Epub 2020 Dec 22.

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

The CD47-signal regulatory protein-alpha (SIRPα) immune checkpoint constitutes a therapeutic target in cancer, and initial clinical studies using inhibitors of CD47-SIRPα interactions in combination with tumor-targeting antibodies show promising results. Blockade of CD47-SIRPα interaction can promote neutrophil antibody-dependent cellular cytotoxicity (ADCC) toward antibody-opsonized targets. Neutrophils induce killing of antibody-opsonized tumor cells by a process identified as trogoptosis, a necrotic/lytic type of cancer cell death that involves trogocytosis, the antibody-mediated endocytic acquisition of cancer membrane fragments by neutrophils. Both trogocytosis and killing strictly depend on CD11b/CD18-(Mac-1)-mediated neutrophil-cancer cell conjugate formation, but the mechanism by which CD47-SIRPα checkpoint disruption promotes cytotoxicity has remained elusive. Here, by using neutrophils from patients with leukocyte adhesion deficiency type III carrying gene mutations, hence lacking the integrin-associated protein kindlin3, we demonstrated that CD47-SIRPα signaling controlled the inside-out activation of the neutrophil CD11b/CD18-integrin and cytotoxic synapse formation in a kindlin3-dependent fashion. Our findings also revealed a role for kindlin3 in trogocytosis and an absolute requirement in the killing process, which involved direct interactions between kindlin3 and CD18 integrin. Collectively, these results identified a dual role for kindlin3 in neutrophil ADCC and provide mechanistic insights into the way neutrophil cytotoxicity is governed by CD47-SIRPα interactions.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0491DOI Listing
February 2021

Comparison of the PU.1 transcriptional regulome and interactome in human and mouse inflammatory dendritic cells.

J Leukoc Biol 2020 Dec 2. Epub 2020 Dec 2.

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

Dendritic cells (DCs) are key immune modulators and are able to mount immune responses or tolerance. DC differentiation and activation imply a plethora of molecular and cellular responses, including transcriptional changes. PU.1 is a highly expressed transcription factor in DCs and coordinates relevant aspects of DC biology. Due to their role as immune regulators, DCs pose as a promising immunotherapy tool. However, some of their functional features, such as survival, activation, or migration, are compromised due to the limitations to simulate in vitro the physiologic DC differentiation process. A better knowledge of transcriptional programs would allow the identification of potential targets for manipulation with the aim of obtaining "qualified" DCs for immunotherapy purposes. Most of the current knowledge regarding DC biology derives from studies using mouse models, which not always find a parallel in human. In the present study, we dissect the PU.1 transcriptional regulome and interactome in mouse and human DCs, in the steady state or LPS activated. The PU.1 transcriptional regulome was identified by performing PU.1 chromatin immunoprecipitation followed by high-throughput sequencing and pairing these data with RNAsequencing data. The PU.1 interactome was identified by performing PU.1 immunoprecipitation followed by mass spectrometry analysis. Our results portray PU.1 as a pivotal factor that plays an important role in the regulation of genes required for proper DC activation and function, and assures the repression of nonlineage genes. The interspecies differences between human and mouse DCs are surprisingly substantial, highlighting the need to study the biology of human DCs.
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http://dx.doi.org/10.1002/JLB.6A1219-711RRRDOI Listing
December 2020

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

Front Immunol 2020 9;11:570963. Epub 2020 Oct 9.

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

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPα mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPα>LDLR) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.
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http://dx.doi.org/10.3389/fimmu.2020.570963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581795PMC
October 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

MKL1 deficiency results in a severe neutrophil motility defect due to impaired actin polymerization.

Blood 2020 06;135(24):2171-2181

Department of Blood Cell Research, Sanquin Research, Amsterdam University Medical Center (AUMC), University of Amsterdam, Amsterdam, The Netherlands.

Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.
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http://dx.doi.org/10.1182/blood.2019002633DOI Listing
June 2020

Activated neutrophils exert myeloid-derived suppressor cell activity damaging T cells beyond repair.

Blood Adv 2019 11;3(22):3562-3574

Department of Blood Cell Research, Sanquin Research, Amsterdam University Medical Center (AUMC), University of Amsterdam, Amsterdam, The Netherlands.

Myeloid-derived suppressor cells (MDSCs) have the capacity to suppress T-cell-mediated immune responses and impact the clinical outcome of cancer, infections, and transplantation settings. Although MDSCs were initially described as bone marrow-derived immature myeloid cells (either monocytic or granulocytic MDSCs), mature neutrophils have been shown to exert MDSC activity toward T cells in ways that remain unclear. In this study, we demonstrated that human neutrophils from both healthy donors and cancer patients do not exert MDSC activity unless they are activated. By using neutrophils with genetically well-defined defects, we found that reactive oxygen species (ROS) and granule-derived constituents are required for MDSC activity after direct CD11b-dependent interactions between neutrophils and T cells. In addition to these cellular interactions, neutrophils are engaged in the uptake of pieces of T-cell membrane, a process called trogocytosis. Together, these interactions led to changes in T-cell morphology, mitochondrial dysfunction, and adenosine triphosphate depletion, as indicated by electron microscopy, mass spectrometry, and metabolic parameters. Our studies characterize the different steps by which activated mature neutrophils induce functional T-cell nonresponsiveness and irreparable cell damage.
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http://dx.doi.org/10.1182/bloodadvances.2019031609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880908PMC
November 2019

IgA-Mediated Killing of Tumor Cells by Neutrophils Is Enhanced by CD47-SIRPα Checkpoint Inhibition.

Cancer Immunol Res 2020 01 5;8(1):120-130. Epub 2019 Nov 5.

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

Therapeutic monoclonal antibodies (mAb), directed toward either tumor antigens or inhibitory checkpoints on immune cells, are effective in cancer therapy. Increasing evidence suggests that the therapeutic efficacy of these tumor antigen-targeting mAbs is mediated-at least partially-by myeloid effector cells, which are controlled by the innate immune-checkpoint interaction between CD47 and SIRPα. We and others have previously demonstrated that inhibiting CD47-SIRPα interactions can substantially potentiate antibody-dependent cellular phagocytosis and cytotoxicity of tumor cells by IgG antibodies both and IgA antibodies are superior in killing cancer cells by neutrophils compared with IgG antibodies with the same variable regions, but the impact of CD47-SIRPα on IgA-mediated killing has not been investigated. Here, we show that checkpoint inhibition of CD47-SIRPα interactions further enhances destruction of IgA antibody-opsonized cancer cells by human neutrophils. This was shown for multiple tumor types and IgA antibodies against different antigens, i.e., HER2/neu and EGFR. Consequently, combining IgA antibodies against HER2/neu or EGFR with SIRPα inhibition proved to be effective in eradicating cancer cells In a syngeneic model, the eradication of cancer cells was predominantly mediated by granulocytes, which were actively recruited to the tumor site by SIRPα blockade. We conclude that IgA-mediated tumor cell destruction can be further enhanced by CD47-SIRPα checkpoint inhibition. These findings provide a basis for targeting CD47-SIRPα interactions in combination with IgA therapeutic antibodies to improve their potential clinical efficacy in tumor patients.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0144DOI Listing
January 2020

Tissue-specific expression of IgG receptors by human macrophages ex vivo.

PLoS One 2019 15;14(10):e0223264. Epub 2019 Oct 15.

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

Recently it was discovered that tissue-resident macrophages derive from embryonic precursors, not only from peripheral blood monocytes, and maintain themselves by self-renewal. Most in-vitro studies on macrophage biology make use of in-vitro cultured human monocyte-derived macrophages. Phagocytosis of IgG-opsonized particles by tissue-resident macrophages takes place via interaction with IgG receptors, the Fc-gamma receptors (FcγRs). We investigated the FcγR expression on macrophages both in-vivo and ex-vivo from different human tissues. Upon isolation of primary human macrophages from bone marrow, spleen, liver and lung, we observed that macrophages from all studied tissues expressed high levels of FcγRIII, which was in direct contrast with the low expression on blood monocyte-derived macrophages. Expression levels of FcγRI were highly variable, with bone marrow macrophages showing the lowest and alveolar macrophages the highest expression. Kupffer cells in the liver were the only tissue-resident macrophages that expressed the inhibitory IgG receptor, FcγRIIB. This inhibitory receptor was also found to be expressed by sinusoidal endothelial cells in the liver. In sum, our immunofluorescence data combined with ex-vivo stainings of isolated macrophages indicated that tissue-resident macrophages are remarkably unique and different from monocyte-derived macrophages in their phenotypic expression of IgG receptors. Tissue macrophages show distinct tissue-specific FcγR expression patterns.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223264PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6793881PMC
March 2020

Neutrophils acquire antigen-presenting cell features after phagocytosis of IgG-opsonized erythrocytes.

Blood Adv 2019 06;3(11):1761-1773

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

Neutrophils are particularly well known for their antimicrobial function. Although historically they are regarded as strictly a phagocyte of the innate immune system, over time it has become clear that neutrophils are versatile cells with numerous functions including innate and adaptive immune regulation. We have previously described a role for human neutrophils in antibody-mediated red blood cell (RBC) clearance. Under homeostatic conditions, neutrophils do not take up RBCs. Yet, when RBCs are immunoglobulin G (IgG) opsonized, which can occur in alloimmunization or autoimmunization reactions, neutrophils can effectively phagocytose RBCs. In the present study, we show that human neutrophils acquire an antigen-presenting cell (APC) phenotype following RBC phagocytosis. Subsequent to RBC phagocytosis, neutrophils expressed major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40 and CD80. Moreover, in classical APCs, the respiratory burst is known to regulate antigen presentation. We found that the respiratory burst in neutrophils is reduced after IgG-mediated RBC phagocytosis. Additionally, following RBC phagocytosis, neutrophils were demonstrated to elicit an antigen-specific T-cell response, using tetanus toxoid (TT) as an antigen to elicit an autologous TT-specific CD4 T-cell response. Lastly, although the "don't eat me" signal CD47 is known to have a powerful restrictive role in the activation of immunity against RBCs in dendritic cells, CD47 does not seem to have a significant effect on the antigen-presenting function of neutrophils in this context. Overall, these findings reveal that besides their classical antimicrobial role, neutrophils show plasticity in their phenotype.
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http://dx.doi.org/10.1182/bloodadvances.2018028753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560339PMC
June 2019

Identification of genetic biomarkers for alloimmunization in sickle cell disease.

Br J Haematol 2019 09 5;186(6):887-899. Epub 2019 Jun 5.

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

Most sickle cell disease (SCD) patients rely on blood transfusion as their main treatment strategy. However, frequent blood transfusion poses the risk of alloimmunization. On average, 30% of SCD patients will alloimmunize while other patient groups form antibodies less frequently. Identification of genetic markers may help to predict which patients are at risk to form alloantibodies. The aim of this study was to evaluate whether genetic variations in the Toll-like receptor pathway or in genes previously associated with antibody-mediated conditions are associated with red blood cell (RBC) alloimmunization in a cohort of SCD patients. In this case-control study, cases had a documented history of alloimmunization while controls had received ≥20 RBC units without alloantibody formation. We used a customized single nucleotide polymorphism (SNP) panel to genotype 690 SNPs in 275 (130 controls, 145 cases) patients. Frequencies were compared using multiple logistic regression analysis. In our primary analysis, no SNPs were found to be significantly associated with alloimmunization after correction for multiple testing. However, in a secondary analysis with a less stringent threshold for significance we found 19 moderately associated SNPs. Among others, SNPs in TLR1/TANK and MALT1 were associated with a higher alloimmunization risk, while SNPs in STAM/IFNAR1 and STAT4 conferred a lower alloimmunization risk.
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http://dx.doi.org/10.1111/bjh.15998DOI Listing
September 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

Glutaminyl cyclase is an enzymatic modifier of the CD47- SIRPα axis and a target for cancer immunotherapy.

Nat Med 2019 04 4;25(4):612-619. Epub 2019 Mar 4.

Division of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRPα expressed on myeloid cells. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.
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http://dx.doi.org/10.1038/s41591-019-0356-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025889PMC
April 2019

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

Myeloid immune-checkpoint inhibition enters the clinical stage.

Nat Rev Clin Oncol 2019 05;16(5):275-276

Division of Stem Cell Transplantation and Immunotherapy, Department of Internal Medicine II, Christian-Albrechts-University, Kiel, Germany.

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http://dx.doi.org/10.1038/s41571-018-0155-3DOI Listing
May 2019

IgG Glyco-Engineering to Improve IVIg Potency.

Front Immunol 2018 23;9:2442. Epub 2018 Oct 23.

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

Intravenous immunoglobulins (IVIg) are used in the treatment of different autoimmune and inflammatory diseases, such as immune thrombocytopenia and hemolytic anemia. One of the modes of action of IVIg is preventing phagocytosis of autoantibody-opsonized blood cells by saturation of the Fc-gamma receptors of macrophages in spleen and liver. IgG contains a fixed glycan, which is in most cases biantennary, at the asparagine residue at position 297 in the Fc tail. This glycan consists of a core structure of N-acetyl glucosamine (GlcNAc) and mannose groups, variably extended with core fucose, bisecting GlcNAc as well as terminal galactose and sialic acid. This structural glycan influences the binding affinity of IgG to Fc-gamma receptors. By glyco-engineering, we generated monoclonal IgG antibodies with different Fc-tail glycans and tested both their opsonizing and blocking capacity in a phagocytosis assay of IgG-opsonized erythrocytes with human monocyte-derived macrophages. In contrast to a lack of effect in opsono-phagocytosis, these IgG glycovariants differentially inhibited the uptake of opsonized erythrocytes. The level of bisecting GlcNAc and galactosylation had unexpectedly larger impact than core fucosylation, and suggest that targeted modifications different from the core fucose may well improve the immunomodulating efficacy of IVIg treatment.
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http://dx.doi.org/10.3389/fimmu.2018.02442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206079PMC
September 2019

Immune Effector Functions of Human IgG2 Antibodies against EGFR.

Mol Cancer Ther 2019 01 3;18(1):75-88. Epub 2018 Oct 3.

Section for Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Kiel, Germany.

Three FDA-approved epidermal growth factor receptor (EGFR) antibodies (cetuximab, panitumumab, necitumumab) are clinically available to treat patients with different types of cancers. Interestingly, panitumumab is of human IgG2 isotype, which is often considered to have limited immune effector functions. Unexpectedly, our studies unraveled that human IgG2 antibodies against EGFR mediated effective CDC when combined with another noncross-blocking EGFR antibody. This second antibody could be of human IgG1 or IgG2 isotype. Furthermore, EGFR antibodies of human IgG2 isotype were highly potent in recruiting myeloid effector cells such as M1 macrophages and PMN for tumor cell killing by ADCC. Tumor cell killing by PMN was more effective with IgG2 than with IgG1 antibodies if tumor cells expressed lower levels of EGFR. Additionally, lower expression levels of the "don't eat me" molecule CD47 on tumor cells enabled ADCC also by M2 macrophages, and improved PMN and macrophage-mediated ADCC. A TCGA enquiry revealed broadly varying CD47 expression levels across different solid tumor types. Together, these results demonstrate that human IgG2 antibodies against EGFR can promote significant Fc-mediated effector functions, which may contribute to their clinical efficacy. The future challenge will be to identify clinical situations in which myeloid effector cells can optimally contribute to antibody efficacy.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0341DOI Listing
January 2019

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Cell Rep 2018 09;24(10):2784-2794

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.
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http://dx.doi.org/10.1016/j.celrep.2018.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326331PMC
September 2018

Inherited p40phox deficiency differs from classic chronic granulomatous disease.

J Clin Invest 2018 08 6;128(9):3957-3975. Epub 2018 Aug 6.

Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.
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http://dx.doi.org/10.1172/JCI97116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6118590PMC
August 2018

Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis.

Cell Rep 2018 06;23(13):3946-3959.e6

Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, the Netherlands. Electronic address:

Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions.
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http://dx.doi.org/10.1016/j.celrep.2018.05.082DOI Listing
June 2018

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

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

Platelets 2019 19;30(3):368-379. Epub 2018 Mar 19.

a Department of Blood Cell Research , Sanquin Research and Landsteiner Laboratory, Academic Medical Centre (AMC), University of Amsterdam (UvA) , Amsterdam , The Netherlands.

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to Mirasol PRT or PSL, and proves to be a methodology suitable to phenotype platelets in an unbiased manner, in various physiological contexts.
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http://dx.doi.org/10.1080/09537104.2018.1447658DOI Listing
April 2019

A false-carrier state for the c.579G>A mutation in the NCF1 gene in Ashkenazi Jews.

J Med Genet 2018 03 13;55(3):166-172. Epub 2018 Jan 13.

The Danek Gertner Institute of Human Genetics, Sheba Medical Center, Ramat Gan, Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Background: Mutations in the gene that encodes p47, a subunit of the NADPH oxidase complex, cause chronic granulomatous disease (CGD). In Kavkazi Jews, a c.579G>A (p.Trp193Ter) mutation in is frequently found, leading to CGD. The same mutation is found in about 1% of Ashkenazi Jews, although Ashkenazi CGD patients with this mutation have never been described.

Methods: We used Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), gene scan analysis and Ion Torrent Next Generation Sequencing for genetic analysis, and measured NADPH oxidase activity and p47 expression.

Results: In an Ashkenazi couple expecting a baby, both parents were found to be heterozygotes for this mutation, as was the fetus. However, segregation analysis in the extended family was consistent with the fetus inheriting both carrier alleles from the parents. MLPA indicated four complete genes in the fetus and three in each parent. Gene sequencing confirmed these results. Analysis of fetal leucocytes obtained by cordocentesis revealed substantial oxidase activity with three different assays, which was confirmed after birth. In six additional Ashkenazi carriers of the c.579G>A mutation, we found five individuals with three complete genes of which one was mutated (like the parents), and one individual with in addition a fusion gene of with a pseudogene.

Conclusion: These results point to the existence of a 'false-carrier' state in Ashkenazi Jews and have wide implications regarding pre-pregnancy screening in this and other population groups.
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http://dx.doi.org/10.1136/jmedgenet-2017-105022DOI Listing
March 2018

The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages.

Blood Adv 2017 Jan 16;1(5):293-305. Epub 2017 Jan 16.

Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.

Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan; however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages.
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http://dx.doi.org/10.1182/bloodadvances.2016001321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744037PMC
January 2017

Human and murine splenic neutrophils are potent phagocytes of IgG-opsonized red blood cells.

Blood Adv 2017 Jun 26;1(14):875-886. Epub 2017 May 26.

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

Red blood cell (RBC) clearance is known to occur primarily in the spleen, and is presumed to be executed by red pulp macrophages. Erythrophagocytosis in the spleen takes place as part of the homeostatic turnover of RBCs to remove old RBCs. It can be strongly promoted by immunoglobulin G (IgG) opsonization of RBCs, a condition that can occur as a consequence of autoantibody or alloantibody formation. The purpose of our study was to investigate which phagocytes are involved in IgG-mediated RBC clearance in the human spleen. We developed a highly specific in vitro assay to monitor RBC phagocytosis in total human splenocytes. Surprisingly, we have found that whereas homeostatic clearance of RBCs is primarily a task for splenic macrophages, neutrophils and, to a lesser extent, also monocytes can be a major factor in clearance of IgG-opsonized RBCs. Erythrophagocytosis by neutrophils is strongly dependent on the degree of opsonization of the RBCs. Additionally, the process is enhanced after blocking the "do not eat me" signal CD47 on the opsonized RBCs, which binds signal regulatory protein α, a myeloid inhibitory receptor that restricts phagocytosis. Moreover, RBCs isolated from autoimmune hemolytic anemia patients, opsonized by auto-IgGs, were shown to be readily phagocytosed by neutrophils. Finally, priming of neutrophils by inflammatory mediators such as tumor necrosis factor α and lipopolysaccharide further increases the magnitude of erythrophagocytosis. Collectively, our data suggest that neutrophils contribute significantly to the phagocytosis of antibody-opsonized RBCs, especially under inflammatory conditions. This indicates a hereto unanticipated contribution of neutrophils in RBC phagocytosis, especially under pathological conditions such as alloimmunization or autoimmunization.
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http://dx.doi.org/10.1182/bloodadvances.2017004671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737592PMC
June 2017

Increased circulating interleukin-8 in patients with resistance to thyroid hormone receptor α.

Endocr Connect 2017 Nov;6(8):731-740

Department of Endocrinology and MetabolismAcademic Medical Center, Amsterdam, The Netherlands

Innate immune cells have recently been identified as novel thyroid hormone (TH) target cells in which intracellular TH levels appear to play an important functional role. The possible involvement of TH receptor alpha (TRα), which is the predominant TR in these cells, has not been studied to date. Studies in TRα mice suggest a role for this receptor in innate immune function. The aim of this study was to determine whether TRα affects the human innate immune response. We assessed circulating interleukin-8 concentrations in a cohort of 8 patients with resistance to TH due to a mutation of TRα (RTHα) and compared these results to healthy controls. In addition, we measured neutrophil and macrophage function in one of these RTHα patients (mutation D211G). Circulating interleukin-8 levels were elevated in 7 out of 8 RTHα patients compared to controls. These patients harbor different mutations, suggesting that this is a general feature of the syndrome of RTHα. Neutrophil spontaneous apoptosis, bacterial killing, NAPDH oxidase activity and chemotaxis were unaltered in cells derived from the RTHαD211G patient. RTHα macrophage phagocytosis and cytokine induction after LPS treatment were similar to results from control cells. The D211G mutation did not result in clinically relevant impairment of neutrophil or pro-inflammatory macrophage function. As elevated circulating IL-8 is also observed in hyperthyroidism, this observation could be due to the high-normal to high levels of circulating T found in patients with RTHα.
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http://dx.doi.org/10.1530/EC-17-0213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5670275PMC
November 2017

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