Granulocyte Function and Immunodeficiency

We have a unique research line related to the functional properties of human granulocytes, specifically dedicated to inborn granulocyte defects. Closely related to this we also perform phagocyte diagnostics, an activity that is also embedded within  Sanquin Diagnostics  and the Center for Immunodeficiency Amsterdam  (CIDA). In particular, we study very rare patient genetic defects, which affect the principal functions of phagocytes relevant for host defense, such as e.g. their motility and microbial killing. Amongst other things, our laboratory has in the past described, for the first time, mutations in the FERMT3 gene, encoding the integrin binding protein kindlin-3, that cause Leukocyte Adhesion Deficiency type III (LAD3) (Kuijpers et al. (2009) Blood 113:4740). Recently, we also described a novel immunodeficiency, related to mutations in the ARPC1B gene, encoding ArpC1b, a component of the Arp2/3 complex that regulates the branching of actin filaments (Kuijpers TW et al., 2016, J Allergy Clin Immunol., in press).

Furthermore, our studies with neutrophils from patients with defined genetic defects have provided important insights into the cytotoxic mechanisms employed by neutrophils to kill Candida sp., and this is relevant as persistent infections with Candida and other fungi are a serious threat for patients. Finally, we explore the potential for using granulocyte transfusions as a therapeutic method to combat fungal infections in neutropenic patients that are refractory to antibiotic treatment. Collectively, our studies in this area provide information about the genetic and cellular basis of phagocyte defects in patients with primary immunodeficiency, and, in doing so, also provide unique fundamental insights into the functioning of the human immune system.    

Immunoglobulins and Fc-receptor genetics and function

This line of research involves the functions of human immunoglobulins, including endogenous antibodies, therapeutic immunoglobulin preparations (IVIg), and therapeutic monoclonal antibodies. Our interest are specifically focused on the cellular effector functions of these immunoglobulins, such as for instance their capacity to promote phagocytosis or cytotoxicity. In this context we study the relevance and application of posttranslational modifications of immunoglobulins, including glycosylation. Furthermore, we are interested in the functional and genetic aspects of the human Fc-receptor (FcR) family, which is being studied in relationship to a variety of diseases, such as e.g. antibody-mediated autoimmunity (e.g. AIHA, ITP), allo-immunity (e.g. Sickle cell disease), Kawasaki disease, and also during monoclonal antibody therapy in cancer (see also below). We also study the contribution of other relevant immunoreceptor families (e.g. KIR and SIRP families) and their activating and inhibitory family members in relation to function in health and disease. This aims to provide insights into the function of immunoglobulins, FcR and other immunoreceptors, including their relevance for inter-individual variation in antibody/immune effector functions, and to provide a rational basis for the optimal application of immunoglobulins in various therapeutic settings. 

Monoclonal antibody therapy in cancer

Cancer therapeutic monoclonal antibodies, such as trastuzumab, cetuximab or rituximab, are widely used for treating defined types of metastatic and disseminated cancer. However, because their clinical efficacy is insufficient they are generally used still in combination with non-specific chemotherapeutics, which cause substantial morbidity and ironically are mutagenic themselves. So there is a pertinent medical need to improve the efficacy of cancer therapeutic antibodies. Cancer therapeutic antibodies act, at least in part, by opsonizing tumor cells for antibody-dependent cellular cytotoxicity (ADCC), which can be exerted by Fc-receptor expressing cells, including NK cells, macrophages and neutrophils. We have discovered that interactions between CD47 expressed on cancer cells and the inhibitory immunoreceptor SIRPa function as an immune checkpoint during phagocyte ADCC towards cancer cells (Zhao XW et al. (2011) PNAS 108:18342). In collaboration with Synthon Biopharmaceuticals we are developing therapeutics targeting the CD47-SIRPa axis, which are anticipated to potentiate the clinical efficacy of a variety of already available cancer therapeutic antibodies. Neutrophils are known to be important during ADCC against cancer cells in vivo, and we are also exploring the basic cellular cytotoxic mechanisms by which neutrophils kill antibody-opsonized cancer cells. We have already demonstrated that neutrophils employ a unique previously undescribed mechanism of tumor cell destruction and we are exploring this in further detail. This will be instrumental for identifying pathways and targets that can be exploited to improve antibody therapy against cancer.

Myeloid-Derived Suppressor Cells 

Myeloid-derived suppressor cells (MDSC) have been identified as a population of myeloid cells, either from the monocytic or granulocytic lineage, that can suppress tumor-specific T cell responses. They have been characterized as a unique population of immune cells in cancer tissue and in the peripheral blood of cancer patients. So far, most studies have studied the properties and function of mouse MDSC and these have indicated that such cells do indeed play a significant role in restricting anti-cancer T cell-mediated immunity in vivo. We are primarily interested in the role of human MDSC and the mechanism(s) by which they suppress CD4 and CD8 T cell responses. This is expected to reveal relevant pathways and potential therapeutic targets for promoting T cells-mediated immunity in cancer patients.   

Primary immunodeficiencies

The phagocyte laboratory has helped to clarify certain defects in human host defense. Kuijpers’ group showed for the first time that CARD9 is essential in neutrophil defense against fungi, and found neutrophils from patients with degranulation defects (FHL-5) have impaired bactericidal activity towards Gram-negative bacteria in particular. The consequences of a actin polymerization defect in neutrophils led to the discovery of a novel PID (ARPC1B deficiency), and another patient with a similar polymerization defect was recently identified suffering from a different genetic disorder (MKL1 deficiency). These very rare human ‘knock-out’ disease models contribute hugely to understand the often unexpected role of certain proteins in host defense. These patients serve to elucidate often unidentified mechanisms of disease by in-depth in-vitro and in-vivo studies, and - at the same time – help to make decisions about clinical treatment of patients suffering of these specific or similar clinical syndromes. One of these treatment options consist of the temporary use of granulocyte transfusions, an often forgotten modality that may save lives when correctly initiated in time. The indications to use such transfusions are being investigated to date.

Multi-Omics

Construction of the epigenome and transcriptome of myeloid progenitors and mature neutrophils from the bone marrow as member of the EU Blueprint consortium was applied to compare with that of neutrophils from blood - both under steady state conditions and after G-CSF/dexamethasone mobilization for granulocyte transfusion purposes. At Sanquin we have recently mapped the proteome of these different neutrophil cell types onto the transcriptomes together with the dept of Plasma Proteins. This insight into the developmental processes during neutrophil differentiation has provided an excellent basis to compare normal conditions with well-characterized phagocyte defects, such as granular defects or the defective Neutrophil Extracellular Trap (NET) formation in genetically well-defined patients. These studies are directed to unravel the mechanisms behind the various functional aspects of phagocytes as potential drug targets to manipulate such functions specifically for treatment of inflammatory disease, being autoreactive or infection-induced hyperinflammation.   

Complement activation (together with the Complement Research group)

Characterization of complement activation and  regulation, e.g. by factor H (FH) and its related proteins (FHRs), with the complement group at the dept of Immunopathology has led to the characterization of FHRs. We have developed test reagents for accurately measuring for the first time the precise levels of FH and FHR1-5 protein levels in blood, both in health and different disease conditions. This has led to the finding that FH is a critical factor in the inherent risk for bacterial infections, meningococcal disease in particular. To date, levels of FH and FHR1-5 studied in various bacterial diseases and malaria are linked to the genetics at the CFH-CFHR gene cluster and show several novel associations. Apart from these studies, the identification of a novel antibody approach to treat (exaggerated) complement-mediated toxicity and disease – without increasing the risk of infection, ought to be highlighted. The potential as ‘biological’ in human disease may outcompete current use of C5-blocking antibodies as a much safer and more effective alternative.

Biomarker studies in infection versus inflammation

The identification of neutrophil- and lymphocyte-derived biomarkers to discriminate bacterial and viral infection from each other and from (non-infectious) inflammation fits with the need to find (indirect) evidence for neutrophil activation and their involvement in certain infections and inflammatory processes. Although RNA transcripts have been shown to discriminate     between bacterial and viral disease, the use of easier and more practical proteins tests are needed to really initiate a better tool-kit to disseminate between the different disease categories that so often mimic each other at the initial clinical presentation of a febrile patient at the clinic. As part of a European consortium, we have studied a large series of biomarkers and have now selected a handful of plasma protein markers – mostly derived from leukocytic subsets –  to become potentially validated as truly discriminating parameters.

Induced pluripotent stem cells (iPSCs)

The study of maturational processes in neutrophils during development in human iPSCs differentiating into neutrophils at the dept of Cell Biology. Manipulation of primary neutrophils (knockdown, transfection) is impossible, being an end-stage differentiated cell with a strong tendency to spontaneous cell death. Primary cell culture from CD34+ HSCs or iPSCs are being explored to manipulate neutrophils prior to the myeloid commitment. We have been successful in generating functional ‘primary’ neutrophils from iPSCs. We are in the process of generating iPSCs from patient cells (focusing on organelle biogenesis defects) for comparison of the functional and proteomics data to unravel the impact during development to myeloid progenitors and their mature neutrophil stage.

IgG and the IgG receptors

The study of IgG interactions with IgG receptors and the consequence of IgG binding for cellular effector functions of the various innate immune cells. We have genotyped the FCGR2/3 locus in many healthy controls and carefully characterized the variation within this gene cluster (including the selective expression of FCGR2C-ORF in a proportion of individuals). Having genotyped individuals throughout the world covering many different ethnicities, we have now generated a global representation of FCGR2/3 genotypes. This genotypic variation has been used to carefully investigate the various glycoforms of IgG molecules on their binding to the classic Fc-gamma receptors for IgG (FcgRs). The impact of different IgG binding patterns was studied at the level of cellular effector functions of FcgR-mediated activation of NK cells, neutrophils and macrophages, which were unexpectedly vary from what we had learnt from our single-receptor affinity profiles. The results of these studies is currently translated into the development of novel intravenous IgG product (IVIG) products.  

Key publications

Prof Timo van den Berg 

• Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody-dependent cellular cytotoxicity towards cancer cells. Treffers LW, Zhao XW, van der Heijden J, Nagelkerke SQ, van Rees DJ, Gonzalez P, Geissler J, Verkuijlen P, van Houdt M, de Boer M, Kuijpers TW, van den Berg TK, Matlung HL. Eur J Immunol. 2018 Feb;48(2):344-354
• The CD47-SIRPα signaling axis as an innate immune checkpoint in cancer. Matlung HL, Szilagyi K, Barclay NA, van den Berg TK. Immunol Rev. 2017 Mar;276(1):145-164.  Review.
• Combined immunodeficiency with severe inflammation and allergy caused by ARPC1B deficiency. Kuijpers TW, Tool ATJ, van der Bijl I, de Boer M, van Houdt M, de Cuyper IM, Roos D, van Alphen F, van Leeuwen K, Cambridge EL, Arends MJ, Dougan G, Clare S, Ramirez-Solis R, Pals ST, Adams DJ, Meijer AB, van den Berg TK. J Allergy Clin Immunol. 2017 Jul;140(1):273-277.e10. 
• CD47-signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction. Zhao XW, van Beek EM, Schornagel K, Van der Maaden H, Van Houdt M, Otten MA, Finetti P, Van Egmond M, Matozaki T, Kraal G, Birnbaum D, van Elsas A, Kuijpers TW, Bertucci F, van den Berg TK. Proc Natl Acad Sci U S A. 2011 Nov 8;108(45):18342-7

Prof Taco Kuijpers

• Tuijnenburg P, Lango Allen H, Burns SO, Greene D, Jansen MH, Staples E, Stephens J, Carss KJ, Biasci D, Baxendale H, Thomas M, Chandra A, Kiani-Alikhan S, Longhurst HJ, Seneviratne SL, Oksenhendler E, Simeoni I, de Bree GJ, Tool ATJ, van Leeuwen EMM, Ebberink EHTM, Meijer AB, Tuna S, Whitehorn D, Brown M, Turro E, Thrasher AJ, Smith KGC, Thaventhiran JE, Kuijpers TW; NIHR-BioResource – Rare Diseases Consortium. Loss of function NFKB1 variants are the most common monogenic cause of CVID in Europeans. J Allergy Clin Immunol. 2018 Feb 22. [Epub ahead of print]
• Hiemstra IH, van Hamme JL, Janssen MH, van den Berg TK, Kuijpers TW. Dexamethasone promotes granulocyte mobilization by prolonging the half-life of granulocyte-colony-stimulating factor in healthy donors for granulocyte transfusions. Transfusion. 2017; 57:674-84.
• Kuijpers TW, Tool ATJ, van der Bijl I, de Boer M, van Houdt M, de Cuyper IM, Roos D, van Alphen F, van Leeuwen K, Cambridge EL, Arends MJ, Dougan G, Clare S, Ramirez-Solis R, Pals ST, Adams DJ, Meijer AB, van den Berg TK. Combined immunodeficiency with severe inflammation and allergy caused by ARPC1B deficiency. J Allergy Clin Immunol. 2017 Jul;140(1):273-277.e10.
• Astle WJ, Elding H, Jiang T, Allen D, Ruklisa D, Mann AL, Mead D, Bouman H, Riveros-Mckay F, Kostadima MA, Lambourne JJ, Sivapalaratnam S, Downes K, Kundu K, Bomba L, Berentsen K, Bradley JR, Daugherty LC, Delaneau O, Freson K, Garner SF, Grassi L, Guerrero J, Haimel M, Janssen-Megens EM, Kaan A, Kamat M, Kim B, Mandoli A, Marchini J, Martens JH, Meacham S, Megy K, O'Connell J, Petersen R, Sharifi N, Sheard SM, Staley JR, Tuna S, van der Ent M, Walter K, Wang SY, Wheeler E, Wilder SP, Iotchkova V, Moore C, Sambrook J, Stunnenberg HG, Di Angelantonio E, Kaptoge S, Kuijpers TW, Carrillo-de-Santa-Pau E, Juan D, Rico D, Valencia A, Chen L, Ge B, Vasquez L, Kwan T, Garrido-Martín D, Watt S, Yang Y, Guigo R, Beck S, Paul DS, Pastinen T, Bujold D, Bourque G, Frontini M, Danesh J, Roberts DJ, Ouwehand WH, Butterworth AS, Soranzo N. The allelic landscape of human blood cell trait variation and links to common complex disease. Cell. 2016; 167:1415-29.e19.
• Chen L, Ge B, Casale FP, Vasquez L, Kwan T, Garrido-Martín D, Watt S, Yan Y, Kundu K, Ecker S, Datta A, Richardson D, Burden F, Mead D, Mann AL, Fernandez JM, Rowlston S, Wilder SP, Farrow S, Shao X, Lambourne JJ, Redensek A, Albers CA, Amstislavskiy V, Ashford S, Berentsen K, Bomba L, Bourque G, Bujold D, Busche S, Caron M, Chen SH, Cheung W, Delaneau O, Dermitzakis ET, Elding H, Colgiu I, Bagger FO, Flicek P, Habibi E, Iotchkova V, Janssen-Megens E, Kim B, Lehrach H, Lowy E, Mandoli A, Matarese F, Maurano MT, Morris JA, Pancaldi V, Pourfarzad F, Rehnstrom K, Rendon A, Risch T, Sharifi N, Simon MM, Sultan M, Valencia A, Walter K, Wang SY, Frontini M, Antonarakis SE, Clarke L, Yaspo ML, Beck S, Guigo R, Rico D, Martens JH, Ouwehand WH, Kuijpers TW, Paul DS, Stunnenberg HG, Stegle O, Downes K, Pastinen T, Soranzo N. Genetic drivers of epigenetic and transcriptional variation in human immune cells. Cell. 2016; 167:1398-414.e24