Publications

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2022

• Küçükköse, E., Heesters, B. A., Villaudy, J., Verheem, A., Cercel, M., van Hal, S., Boj, S. F., Rinkes, I. H. M. B., Punt, C. J. A., Roodhart, J. M. L., Laoukili, J., Koopman, M., Spits, H., & Kranenburg, O. (2022). Modeling Resistance of Colorectal Peritoneal Metastases to Immune Checkpoint Blockade in Humanized Mice. Journal for ImmunoTherapy of Cancer, 10(12), e005345. https://doi.org/10.1136/jitc-2022-005345
  Background The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. Methods We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. Results PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)—containing B cells and juxtaposed T cells—and with expression of an interferon-γ signature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. Conclusions By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB.
• Tomris, I., Unione, L., Nguyen, L., Zaree, P., Bouwman, K. M., Liu, L., Li, Z., Fok, J. A., Carrasco, M. R., van der Woude, R., Kimpel, A. L. M., Linthorst, M. W., Verpalen, E. C. J. M., Caniels, T. G., Sanders, R. W., Heesters, B. A., Pieters, R. J., Jiménez-Barbero, J., Klassen, J. S., … de Vries, R. P. (2022). The SARS-CoV-2 Spike N-terminal Domain Engages 9-O-acetylated A2-8-Linked Sialic Acids (p. 2022.09.14.507904). bioRxiv. https://doi.org/10.1101/2022.09.14.507904
  SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD) which, in other coronaviruses, includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of functionality. To analyze gain or loss of glycan-binding in VoC, trimeric fluorescent NTD proteins were used. Binding properties were analyzed biochemically on Vero E6 cells and tissue samples. Unexpectedly, the SARS-CoV-2 Beta (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. This demonstrates plasticity for improved engagement to sialic acids, possibly under immunological pressure.

2021

• Krabbendam, L., Heesters, B. A., Kradolfer, C. M. A., Haverkate, N. J. E., Becker, M. a. J., Buskens, C. J., Bemelman, W. A., Bernink, J. H., & Spits, H. (2021). CD127+ CD94+ Innate Lymphoid Cells Expressing Granulysin and Perforin Are Expanded in Patients with Crohn’s Disease. Nature Communications, 12(1), 1–11. https://doi.org/10.1038/s41467-021-26187-x
  Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease.
• Heesters, B. A., van Megesen, K., Tomris, I., de Vries, R. P., Magri, G., & Spits, H. (2021). Characterization of Human FDCs Reveals Regulation of T Cells and Antigen Presentation to B Cells. Journal of Experimental Medicine. https://doi.org/10.1084/jem.20210790
  Stromal-derived follicular dendritic cells (FDC) are essential for germinal centers (GC), the site where B cells maturate their antibodies. FDC present native antigen to B cells and maintain a CXCL13 gradient to form the B cell follicle. Yet despite their essential role, the transcriptome of human FDC remains undefined. Using single-cell RNA sequencing and microarray, we provided the transcriptome of these enigmatic cells as a comprehensive resource. Key genes were validated by flow cytometry and microscopy. Surprisingly, marginal reticular cells (MRC) rather than FDC expressed B cell activating factor (BAFF). Furthermore, we found that human FDC expressed TLR4 and can alter antigen availability in response to pathogen-associated molecular patterns (PAMPs). High expression of PD-L1 and PD-L2 on FDC activated PD1 on T cells. In addition, we found expression of genes related to T cell regulation, such as HLA-DR, CD40 and others. These data suggest intimate contact between human FDC and T cells.
• Krabbendam, L., Heesters, B. A., Kradolfer, C. M. A., Spits, H., & Bernink, J. H. (2021). Identification of Human Cytotoxic ILC3s. European Journal of Immunology, 51(4), 811–823. https://doi.org/10.1002/eji.202048696
  Human ILCs are classically categorized into five subsets; cytotoxic CD127-CD94+ NK cells and non-cytotoxic CD127+CD94-, ILC1s, ILC2s, ILC3s, and LTi cells. Here, we identify a previously unrecognized subset within the CD127+ ILC population, characterized by the expression of the cytotoxic marker CD94. These CD94+ ILCs resemble conventional ILC3s in terms of phenotype, transcriptome, and cytokine production, but are highly cytotoxic. IL-15 was unable to induce differentiation of CD94+ ILCs toward mature NK cells. Instead, CD94+ ILCs retained RORγt, CD127 and CD200R1 expression and produced IL-22 in response to IL-15. Culturing non-cytotoxic ILC3s with IL-12 induced upregulation of CD94 and cytotoxic activity, effects that were not observed with IL-15 stimulation. Thus, human helper ILCs can acquire a cytotoxic program without differentiating into NK cells.
• Golebski, K., Layhadi, J. A., Sahiner, U., Steveling-Klein, E. H., Lenormand, M. M., Li, R. C. Y., Bal, S. M., Heesters, B. A., Vilà-Nadal, G., Hunewald, O., Montamat, G., He, F. Q., Ollert, M., Fedina, O., Lao-Araya, M., Vijverberg, S. J. H., der Zee, A.-H. M.-van, van Drunen, C. M., Fokkens, W. J., … Shamji, M. H. (2021). Induction of IL-10-producing Type 2 Innate Lymphoid Cells by Allergen Immunotherapy Is Associated with Clinical Response. Immunity, 0(0). https://doi.org/10.1016/j.immuni.2020.12.013
  The role of innate immune cells in allergen immunotherapy that confers immune tolerance to the sensitizing allergen is unclear. Here, we report a role of interleukin-10-producing type 2 innate lymphoid cells (IL-10+ ILC2s) in modulating grass-pollen allergy. We demonstrate that KLRG1+ but not KLRG1– ILC2 produced IL-10 upon activation with IL-33 and retinoic acid. These cells attenuated Th responses and maintained epithelial cell integrity. IL-10+ KLRG1+ ILC2s were lower in patients with grass-pollen allergy when compared to healthy subjects. In a prospective, double-blind, placebo-controlled trial, we demonstrated that the competence of ILC2 to produce IL-10 was restored in patients who received grass-pollen sublingual immunotherapy. The underpinning mechanisms were associated with the modification of retinol metabolic pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathways in the ILCs. Altogether, our findings underscore the contribution of IL-10+ ILC2s in the disease-modifying effect by allergen immunotherapy.
• van der Ploeg, E. K., Golebski, K., van Nimwegen, M., Fergusson, J. R., Heesters, B. A., Martinez-Gonzalez, I., Kradolfer, C. M. A., van Tol, S., Scicluna, B. P., de Bruijn, M. J. W., de Boer, G. M., Tramper-Stranders, G. A., Braunstahl, G.-J., van IJcken, W. F. J., Nagtegaal, A. P., van Drunen, C. M., Fokkens, W. J., Huylebroeck, D., Spits, H., … Bal, S. M. (2021). Steroid-Resistant Human Inflammatory ILC2s Are Marked by CD45RO and Elevated in Type 2 Respiratory Diseases. Science Immunology, 6(55). https://doi.org/10.1126/sciimmunol.abd3489
  Defiant ILC2s resist steroids Group 2 innate lymphoid cells (ILC2s) contribute to the inflammation associated with human allergic airway diseases, including asthma and chronic rhinosinusitis. Corticosteroid drugs are used to manage type 2 respiratory diseases, but steroid resistance may arise in the course of therapy. By comparing ILC2s from inflamed nasal polyps with blood ILC2s from healthy controls, van der Ploeg et al. observed that enhanced cytokine expression by nasal polyp ILC2s and loss of steroid responsiveness were both associated with ILC2s becoming CD45RO+ rather than CD45RA+ like most resting ILC2s. Expression of the CD45RO isoform by inflammatory ILC2s in blood was increased in patients with asthma and correlated with more severe airway disease. CD45RO is a candidate biomarker for human inflammatory ILC2s that correlates with acquisition of steroid resistance. Group 2 innate lymphoid cells (ILC2s) orchestrate protective type 2 immunity and have been implicated in various immune disorders. In the mouse, circulatory inflammatory ILC2s (iILC2s) were identified as a major source of type 2 cytokines. The human equivalent of the iILC2 subset remains unknown. Here, we identify a human inflammatory ILC2 population that resides in inflamed mucosal tissue and is specifically marked by surface CD45RO expression. CD45RO+ ILC2s are derived from resting CD45RA+ ILC2s upon activation by epithelial alarmins such as IL-33 and TSLP, which is tightly linked to STAT5 activation and up-regulation of the IRF4/BATF transcription factors. Transcriptome analysis reveals marked similarities between human CD45RO+ ILC2s and mouse iILC2s. Frequencies of CD45RO+ inflammatory ILC2 are increased in inflamed mucosal tissue and in the circulation of patients with chronic rhinosinusitis or asthma, correlating with disease severity and resistance to corticosteroid therapy. CD45RA-to-CD45RO ILC2 conversion is suppressed by corticosteroids via induction of differentiation toward an immunomodulatory ILC2 phenotype characterized by low type 2 cytokine and high amphiregulin expression. Once converted, however, CD45RO+ ILC2s are resistant to corticosteroids, which is associated with metabolic reprogramming resulting in the activation of detoxification pathways. Our combined data identify CD45RO+ inflammatory ILC2s as a human analog of mouse iILC2s linked to severe type 2 inflammatory disease and therapy resistance. An inflammatory CD45RO+ ILC2 population found in inflamed human nasal tissue is steroid resistant and linked to type 2 immunopathology. An inflammatory CD45RO+ ILC2 population found in inflamed human nasal tissue is steroid resistant and linked to type 2 immunopathology.

2020

• Genzel, L., Adan, R., Berns, A., van den Beucken, J., Blokland, A., Boddeke, E. H. W. G. M., Bogers, W. M., Bontrop, R., Bulthuis, R., Bousema, T., Clevers, H., Coenen, T. C. J. J., van Dam, A.-M., Deen, P. M. T., van Dijk, K. W., Eggen, B. J. L., Elgersma, Y., Erdogan, I., Englitz, B., … Homberg, J. R. (2020). How the COVID-19 Pandemic Highlights the Necessity of Animal Research. Current Biology, 30(18), R1014–R1018. https://doi.org/10.1016/j.cub.2020.08.030
  Recently, a petition was offered to the European Commission calling for an immediate ban on animal testing. Although a Europe-wide moratorium on the use of animals in science is not yet possible, there has been a push by the non-scientific community and politicians for a rapid transition to animal-free innovations. Although there are benefits for both animal welfare and researchers, advances on alternative methods have not progressed enough to be able to replace animal research in the foreseeable future. This trend has led first and foremost to a substantial increase in the administrative burden and hurdles required to make timely advances in research and treatments for human and animal diseases. The current COVID-19 pandemic clearly highlights how much we actually rely on animal research. COVID-19 affects several organs and systems, and the various animal-free alternatives currently available do not come close to this complexity. In this Essay, we therefore argue that the use of animals is essential for the advancement of human and veterinary health.

2019

• Nagasawa, M., Heesters, B. A., Kradolfer, C. M. A., Krabbendam, L., Martinez-Gonzalez, I., de Bruijn, M. J. W., Golebski, K., Hendriks, R. W., Stadhouders, R., Spits, H., & Bal, S. M. (2019). KLRG1 and NKp46 Discriminate Subpopulations of Human CD117+CRTH2- ILCs Biased toward ILC2 or ILC3. Journal of Experimental Medicine, jem.20190490. https://doi.org/10.1084/jem.20190490
  Recently, human ILCs that express CD117 and CD127 but lack CRTH2 and NKp44 have been shown to contain precursors of ILC1, ILC2, and ILC3. However, these ILCs have not been extensively characterized. We performed an unbiased hierarchical stochastic neighbor embedding (HSNE) analysis of the phenotype of peripheral blood CD117+ ILCs, which revealed the presence of three major subsets: the first expressed NKp46, the second expressed both NKp46 and CD56, and the third expressed KLRG1, but not NKp46 or CD56. Analysis of their cytokine production profiles and transcriptome revealed that NKp46+ ILCs predominantly develop into ILC3s; some of them can differentiate into ILC1/NK-like cells, but they are unable to develop into ILC2s. In contrast, KLRG1+ ILCs predominantly differentiate into ILC2s. Single-cell cultures demonstrate that KLRG1+ ILCs can also differentiate into other ILC subsets depending on the signals they receive. Epigenetic profiling of KLRG1+ ILCs is consistent with the broad differentiation potential of these cells.
• Golebski, K., Ros, X. R., Nagasawa, M., van Tol, S., Heesters, B. A., Aglmous, H., Kradolfer, C. M. A., Shikhagaie, M. M., Seys, S., Hellings, P. W., van Drunen, C. M., Fokkens, W. J., Spits, H., & Bal, S. M. (2019). IL-1β, IL-23, and TGF-β Drive Plasticity of Human ILC2s towards IL-17-producing ILCs in Nasal Inflammation. Nature Communications, 10(1), 2162. https://doi.org/10.1038/s41467-019-09883-7
  Innate lymphoid cells (ILCs) play critical immunological roles including immune surveillance at mucosal sites. Here the authors show that during nasal inflammation pathogen-induced cytokine production guides the differentiation of ILCs.
• Willemze, R. A., Welting, O., van Hamersveld, P., Verseijden, C., Nijhuis, L. E., Hilbers, F. W., Meijer, S. L., Heesters, B. A., Folgering, J. H. A., Darwinkel, H., Blancou, P., Vervoordeldonk, M. J., Seppen, J., Heinsbroek, S. E. M., & de Jonge, W. J. (2019). Loss of Intestinal Sympathetic Innervation Elicits an Innate Immune Driven Colitis. Molecular Medicine, 25(1), 1. https://doi.org/10.1186/s10020-018-0068-8
  Both the parasympathetic and sympathetic nervous system exert control over innate immune responses. In inflammatory bowel disease, sympathetic innervation in intestinal mucosa is reduced. Our aim was to investigate the role of sympathetic innervation to the intestine on regulation of the innate immune responses.

2018

• Fergusson, J. R., Morgan, M. D., Bruchard, M., Huitema, L., Heesters, B. A., van Unen, V., van Hamburg, J. P., van der Wel, N. N., Picavet, D., Koning, F., Tas, S. W., Anderson, M. S., Marioni, J. C., Holländer, G. A., & Spits, H. (2018). Maturing Human CD127+ CCR7+ PDL1+ Dendritic Cells Express AIRE in the Absence of Tissue Restricted Antigens. Frontiers in Immunology, 9, 2902. https://doi.org/10.3389/fimmu.2018.02902
  Expression of the Autoimmune regulator (AIRE) outside of the thymus has long been suggested in both humans and mice, but the cellular source in humans has remained undefined. Here we identify AIRE expression in human tonsils and extensively analyzed these “extra-thymic AIRE expressing cells” (eTACs) using combinations of flow cytometry, CyTOF and single cell RNA-sequencing. We identified AIRE+ cells as dendritic cells (DCs) with a mature and migratory phenotype including high levels of antigen presenting molecules and costimulatory molecules, and specific expression of CD127, CCR7, and PDL1. These cells also possessed the ability to stimulate and re-stimulate T cells and displayed reduced responses to toll-like receptor (TLR) agonists compared to conventional DCs. While expression of AIRE was enriched within CCR7+CD127+ DCs, single-cell RNA sequencing revealed expression of AIRE to be transient, rather than stable, and associated with the differentiation to a mature phenotype. The role of AIRE in central tolerance induction within the thymus is well-established, however our study shows that AIRE expression within the periphery is not associated with an enriched expression of tissue-restricted antigens (TRAs). This unexpected finding, suggestive of wider functions of AIRE, may provide an explanation for the non-autoimmune symptoms of APECED patients who lack functional AIRE.

2017

• Garrison, B. S., Rybak, A. P., Beerman, I., Heesters, B. A., Mercier, F. E., Scadden, D. T., Bryder, D., Baron, R., & Rossi, D. J. (2017). ZFP521 Regulates Murine Hematopoietic Stem Cell Function and Facilitates MLL-AF9 Leukemogenesis in Mouse and Human Cells. Blood, 130(5), 619–624. https://doi.org/10.1182/blood-2016-09-738591
  ZFP521 regulates HSC self-renewal and differentiation.ZFP521 facilitates leukemogenesis in an MLL-AF9-mediated leukemia model. The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis, whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically up-regulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.
• Das, A., Heesters, B. A., Bialas, A., O’Flynn, J., Rifkin, I. R., Ochando, J., Mittereder, N., Carlesso, G., Herbst, R., & Carroll, M. C. (2017). Follicular Dendritic Cell Activation by TLR Ligands Promotes Autoreactive B Cell Responses. Immunity, 46(1), 106–119. https://doi.org/10.1016/j.immuni.2016.12.014
  A hallmark of autoimmunity in murine models of lupus is the formation of germinal centers (GCs) in lymphoid tissues where self-reactive B cells expand and differentiate. In the host response to foreign antigens, follicular dendritic cells (FDCs) maintain GCs through the uptake and cycling of complement-opsonized immune complexes. Here, we examined whether FDCs retain self-antigens and the impact of this process in autoantibody secretion in lupus. We found that FDCs took up and retained self-immune complexes composed of ribonucleotide proteins, autoantibody, and complement. This uptake, mediated through CD21, triggered endosomal TLR7 and led to the secretion of interferon (IFN) α via an IRF5-dependent pathway. Blocking of FDC secretion of IFN-α restored B cell tolerance and reduced the amount of GCs and pathogenic autoantibody. Thus, FDCs are a critical source of the IFN-α driving autoimmunity in this lupus model. This pathway is conserved in humans, suggesting that it may be a viable therapeutic target in systemic lupus erythematosus.
• Heesters, B. A., van der Poel, C. E., & Carroll, M. C. (2017). Follicular Dendritic Cell Isolation and Loading of Immune Complexes. Germinal Centers: Methods and Protocols, 105–112. https://doi.org/10.1007/978-1-4939-7095-7_9
  Follicular dendritic cells (FDCs) are stromal cells that are centrally located within B cell follicles of lymph nodes and other lymphoid organs such as the spleen. Due to their relative low abundance and difficulty to isolate, FDCs are still largely an enigma. Here we describe how to isolate FDCs for ex vivo cell culture, sorting by flow cytometry and how to load them in vivo or in vitro with immune complexes.

2016

• Claessen, F. M. A. P., Heesters, B. A., Chan, J. J., Kachooei, A. R., & Ring, D. (2016). A Meta-Analysis of the Effect of Corticosteroid Injection for Enthesopathy of the Extensor Carpi Radialis Brevis Origin. The Journal of Hand Surgery, 41(10), 988–998. https://doi.org/10.1016/j.jhsa.2016.07.097
  Purpose The null hypothesis that there is no effect of corticosteroid injection on visual analog scale for pain in patients with enthesopathy of the extensor carpi radialis brevis (eECRB) origin 6 months after treatment was tested. Our secondary hypotheses were that there is no effect of corticosteroid injection on pain intensity at 1 and 3 months after treatment; that there is no effect of corticosteroid injection on grip strength at 1, 3, and 6 months after treatment; and that there is no effect of corticosteroid injection on Disabilities of the Arm, Shoulder, and Hand scores at 1, 3 and 6 months after treatment. Methods EMBASE, PubMed Publisher, MEDLINE, OvidSP, Web of Science, Google Scholar, and the Cochrane Central were searched for relevant studies. Studies were eligible if there was (1) a description of corticosteroid injection treatment for eECRB; (2) randomized placebo injection–controlled trials with at least 10 adults included with eECRB; (3) a full-text article available with data describing the mean differences between the corticosteroid and the control groups and the outcome measures used; and (4) follow-up of at least 1 month. In total, 7 randomized controlled trials comparing the effect of corticosteroid injection with a placebo injection on symptoms of eECRB were included in our meta-analysis. Results We found no difference in pain intensity 6 months after injection of corticosteroids or placebo. Pain intensity was slightly, but significantly, lower 1 month, but not 3 months, after steroid injection. There were no significant differences in grip strength or Disabilities of the Arm, Shoulder, and Hand score at any time point. Conclusions This meta-analysis showed that there is no difference in pain intensity between corticosteroid injection and placebo 6 months after injection. We interpret the weight of evidence to date as suggesting that corticosteroid injections are neither meaningfully palliative nor disease modifying when used to treat eECRB.
• Heesters, B. A., van der Poel, C. E., Das, A., & Carroll, M. C. (2016). Antigen Presentation to B Cells. Trends in Immunology, 37(12), 844–854. https://doi.org/10.1016/j.it.2016.10.003
  Unlike T cells that recognize digested peptides, B cells recognize their cognateantigen in its native form. The B cell receptor used in recognition can also besecreted to bind to antigens and initiate multiple effector functions such asphagocytosis, complement activation, or neutralization of receptors. While Bcells can interact with soluble antigens, it is now clear that the presentation ofmembrane-bound antigen plays a crucial role in B cell activation, and in partic-ular during affinity-maturation, the process during which high-affinity B cells areselected. In this review we discuss how native antigen is presented to B cellsand its impact at several stages of B cell responses.
• Heesters, B. A., & Carroll, M. C. (2016). The Role of Dendritic Cells in S. Pneumoniae Transport to Follicular Dendritic Cells. Cell Reports, 16(12), 3130–3137. https://doi.org/10.1016/j.celrep.2016.08.049
  Affinity-mature B cells require cognate antigen, retained by follicular dendritic cells (FDCs), for clonal selection within germinal centers. Studies on how FDCs in lymphoid tissues acquire antigen have relied primarily on model protein antigens. To examine delivery of intact bacteria to FDCs, we used inactivated Streptococcus pneumonia (SP). We found that both medullary macrophages and a subset of SIGN-R1-positive dendritic cells (DCs) in the lymph node capture SP from the draining afferent lymphatics. The presence of DCs is required for initial complement activation, opsonization of the bacteria, and efficient transport of SP to FDCs. Moreover, we observed a major role for transport of bacteria to FDCs by naive B cells via a CD21-dependent pathway. We propose a mechanism by which efficient transport of SP to FDCs is dependent on DCs for initial binding and activation of complement and either direct transport to FDCs or transfer to naive B cells.

2015

• Langereis, M. A., Bakkers, M. J. G., Deng, L., Padler-Karavani, V., Vervoort, S. J., Hulswit, R. J. G., van Vliet, A. L. W., Gerwig, G. J., de Poot, S. A. H., Boot, W., van Ederen, A. M., Heesters, B. A., van der Loos, C. M., van Kuppeveld, F. J. M., Yu, H., Huizinga, E. G., Chen, X., Varki, A., Kamerling, J. P., & de Groot, R. J. (2015). Complexity and Diversity of the Mammalian Sialome Revealed by Nidovirus Virolectins. Cell Reports, 11(12), 1966–1978. https://doi.org/10.1016/j.celrep.2015.05.044
  Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue- and cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function.
• Mooster, J. L., Le Bras, S., Massaad, M. J., Jabara, H., Yoon, J., Galand, C., Heesters, B. A., Burton, O. T., Mattoo, H., Manis, J., & Geha, R. S. (2015). Defective Lymphoid Organogenesis Underlies the Immune Deficiency Caused by a Heterozygous S32I Mutation in I\kappaBα. Journal of Experimental Medicine, 212(2), 185–202. https://doi.org/10.1084/jem.20140979
  Patients with ectodermal dysplasia with immunodeficiency (ED-ID) caused by mutations in the inhibitor of NF-κB α (IκBα) are susceptible to severe recurrent infections, despite normal T and B cell numbers and intact in vitro lymphocyte function. Moreover, the outcome of hematopoietic stem cell transplantation (HSCT) in these patients is poor despite good engraftment. Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID. Strikingly, the mice lacked lymph nodes, Peyer’s patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling. Lymphotoxin β receptor (LTβR)–driven induction of chemokines and adhesion molecules mediated by both canonical and noncanonical NF-κB pathways was impaired, and levels of p100 were markedly diminished in the mutant. IκBα mutant\textrightarrow Rag2-/-, but not WT\textrightarrow IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs. Defective architectural cell function explains the immunodeficiency and poor outcome of HSCT in patients with IκBα deficiency and suggests that correction of this niche is critical for reconstituting their immune function.
• Heesters, B. A. (2015). Antigen Dynamics of Follicular Dendritic Cells [PhD thesis]. Harvard University & Utrecht University.
  Stromal-derived follicular dendritic cells (FDCs) are a major depot for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate and high-affinity antibody production takes place. Historically, FDCs have been characterized as ‘accessory’ cells that passively support germinal center (GC) responses. However, our recent observations suggest that FDCs actively shape humoral immunity. In this dissertation, we discuss recent findings concerning the antigen acquisition and retention functions of FDCs, and relevant implications for protective immunity. We found that FDCs acquired complement-coated immune complexes (ICs) from noncognate B cells via complement receptors 1 and 2 (CD35 and CD21, respectively) and rapidly internalized them by an actin-dependent pathway. ICs were retained intact within a nondegradative cycling compartment and were displayed periodically on the cell surface where they were accessible to antigen-specific B cells. Furthermore, we discuss how FDCs are involved in persistence of Human Immunodeficiency Virus (HIV) in people on antiretroviral therapy (ART). Treatment with ART substantially reduces viral load and limits disease progression in subjects with HIV infection. Despite the success of ART, it does not cure HIV and discontinuation of treatment results in viral rebound. Growing evidence implicates lymph nodes (LN) as a major site for continued infection of CD4 T cells, but the cell sources of a persistent reservoir within LN remain unclear. We found that human FDC isolated from subjects on ART retain infectious HIV via binding to complement receptor 2 (CD21) within a cycling compartment and transmit infectious virus to CD4 T cells in vitro. Importantly, treatment of the HIV+ FDC with a soluble complement receptor 2 (sCD21-Ig) purges the FDC of HIV virions and prevents transmission of infectious virus in vitro. These results provide evidence that reservoirs are not restricted to infected cells and provide a method to purge one reservoir not targeted by conventional therapy. Our results suggest that sCD21-Ig could be a potential component of new therapeutic strategies to achieve functional cure or viral eradication in ART-treated HIV-infected humans. Finally, we show S. pneumonia binds only in the medulla of the LN and that a subset of dendritic cells (DC), besides B cells) are required for the transport of S. pneumoniae to FDC, independent of macrophages. A robust humoral immune response and germinal center formation requires transport of antigen to the FDC. A better understanding of this process can contribute to improvement of future vaccine design.
• Heesters, B. A., Lindqvist, M., Vagefi, P. A., Scully, E. P., Schildberg, F. A., Altfeld, M., Walker, B. D., Kaufmann, D. E., & Carroll, M. C. (2015). Follicular Dendritic Cells Retain Infectious HIV in Cycling Endosomes. PLoS Pathogens, 11(12), e1005285. https://doi.org/10.1371/journal.ppat.1005285
  Despite the success of antiretroviral therapy (ART), it does not cure Human Immunodeficiency Virus (HIV) and discontinuation results in viral rebound. Follicular dendritic cells (FDC) are in direct contact with CD4+ T cells and they retain intact antigen for prolonged periods. We found that human FDC isolated from patients on ART retain infectious HIV within a non-degradative cycling compartment and transmit infectious virus to uninfected CD4 T cells in vitro. Importantly, treatment of the HIV+ FDC with a soluble complement receptor 2 purges the FDC of HIV virions and prevents viral transmission in vitro. Our results provide an explanation for how FDC can retain infectious HIV for extended periods and suggest a therapeutic strategy to achieve cure in HIV-infected humans.

2014

• Zhao, F., Heesters, B. A., Chiu, I., Gao, Y., Shi, J., Zhou, N., Carroll, M. C., & Xu, B. (2014). L-Rhamnose-containing Supramolecular Nanofibrils as Potential Immunosuppressive Materials. Organic & Biomolecular Chemistry, 12(35), 6816–6819. https://doi.org/10.1039/c4ob01362j
  An l-rhamnose-based hydrogelator self-assembles to form nanofibrils, which, in contrast to the properties of monomeric l-rhamnose, suppress the antibody response of mice to phycoerythrin (PE), a fluorescent protein antigen. As the first example of the supramolecular assemblies of a saccharide to suppress immunity, this work illustrates a new approach of immunomodulation.
• Woodruff, M. C., Heesters, B. A., Herndon, C. N., Groom, J. R., Thomas, P. G., Luster, A. D., Turley, S. J., & Carroll, M. C. (2014). Trans-Nodal Migration of Resident Dendritic Cells into Medullary Interfollicular Regions Initiates Immunity to Influenza Vaccine. Journal of Experimental Medicine, 211(8), 1611–1621. https://doi.org/10.1084/jem.20132327
  Dendritic cells (DCs) are well established as potent antigen-presenting cells critical to adaptive immunity. In vaccination approaches, appropriately stimulating lymph node-resident DCs (LNDCs) is highly relevant to effective immunization. Although LNDCs have been implicated in immune response, their ability to directly drive effective immunity to lymph-borne antigen remains unclear. Using an inactive influenza vaccine model and whole node imaging approaches, we observed surprising responsiveness of LNDC populations to vaccine arrival resulting in a transnodal repositioning into specific antigen collection sites within minutes after immunization. Once there, LNDCs acquired viral antigen and initiated activation of viral specific CD4(+) T cells, resulting in germinal center formation and B cell memory in the absence of skin migratory DCs. Together, these results demonstrate an unexpected stimulatory role for LNDCs where they are capable of rapidly locating viral antigen, driving early activation of T cell populations, and independently establishing functional immune response.
• Heesters, B. A., Das, A., Chatterjee, P., & Carroll, M. C. (2014). Do Follicular Dendritic Cells Regulate Lupus-Specific B Cells? Molecular Immunology, 62(2), 283–288. https://doi.org/10.1016/j.molimm.2014.02.010
  The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. In this review we describe recently published results in which a B cell receptor-knock-in mouse strain specific for nucleolar self-antigens was bred with mice deficient in complement C4 and discuss the implications for the lupus field. Absence of C4 leads to a breakdown in the elimination of autoreactive B cell clones at the transitional stage. This is characterized by a relative increase in their response to a range of stimuli, entrance into follicles and a greater propensity to form self-reactive germinal centers. In this review, a model is proposed in which, in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells and follicular dendritic cells, resulting in secretion of Type I interferon. This allows for the maturation and activation of self-reactive B cell clones leading to increased spontaneous formation of germinal centers and subsequent generation of autoantibodies.
• Heesters, B. A., Myers, R. C., & Carroll, M. C. (2014). Follicular Dendritic Cells: Dynamic Antigen Libraries. Nature Reviews Immunology, 14(7), 495–504. https://doi.org/10.1038/nri3689
  Follicular dendritic cells (FDCs) are essential for high-affinity antibody production and for the development of B cell memory. Historically, FDCs have been characterized as ’accessory’ cells that passively support germinal centre (GC) responses. However, recent observations suggest that FDCs actively shape humoral immunity. In this Review, we discuss recent findings concerning the antigen acquisition and retention functions of FDCs, and relevant implications for protective immunity. Furthermore, we describe the roles of FDCs within GCs in secondary lymphoid organs and discuss FDC development within this dynamic environment. Finally, we discuss how a better understanding of FDCs could facilitate the design of next-generation vaccines.

2013

• Chiu, I. M., Heesters, B. A., Ghasemlou, N., Von Hehn, C. A., Zhao, F., Tran, J., Wainger, B., Strominger, A., Muralidharan, S., Horswill, A. R., & others. (2013). Bacteria Activate Sensory Neurons That Modulate Pain and Inflammation. Nature, 501(7465), 52–57. https://doi.org/10.1038/nature12479
  Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host–pathogen interactions.
• Heesters, B. A., Chatterjee, P., Kim, Y.-A., Gonzalez, S. F., Kuligowski, M. P., Kirchhausen, T., & Carroll, M. C. (2013). Endocytosis and Recycling of Immune Complexes by Follicular Dendritic Cells Enhances B Cell Antigen Binding and Activation. Immunity, 38(6), 1164–1175. https://doi.org/10.1016/j.immuni.2013.02.023
  Stromal-derived follicular dendritic cells (FDCs) are a major reservoir for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate. A long-standing question is how FDCs retain antigen in its native form for extended periods and how they display it to specific B cells. Here we found that FDCs acquired complement-coated immune complexes (ICs) from noncognate B cells via complement receptors 1 and 2 (CD35 and CD21, respectively) and rapidly internalized them by an actin-dependent pathway. ICs were retained intact within a nondegradative cycling compartment and were displayed periodically on the cell surface where they were accessible to antigen-specific B cells. This would explain how antigens are protected from damage and retained over long periods of time, while remaining accessible for B cells.
• Woodruff, M. C., Herndon, C. N., Heesters, B. A., & Carroll, M. C. (2013). Contextual Analysis of Immunological Response through Whole-Organ Fluorescent Imaging. Lymphatic Research and Biology, 11(3), 121–127. https://doi.org/10.1089/lrb.2013.0011
  Background: As fluorescent microscopy has developed, significant insights have been gained into the establishment of immune response within secondary lymphoid organs, particularly in draining lymph nodes. While established techniques such as confocal imaging and intravital multi-photon microscopy have proven invaluable, they provide limited insight into the architectural and structural context in which these responses occur. To interrogate the role of the lymph node environment in immune response effectively, a new set of imaging tools taking into account broader architectural context must be implemented into emerging immunological questions. Methods and Results: Using two different methods of whole-organ imaging, optical clearing and three-dimensional reconstruction of serially sectioned lymph nodes, fluorescent representations of whole lymph nodes can be acquired at cellular resolution. Using freely available post-processing tools, images of unlimited size and depth can be assembled into cohesive, contextual snapshots of immunological response. Through the implementation of robust iterative analysis techniques, these highly complex three-dimensional images can be objectified into sortable object data sets. These data can then be used to interrogate complex questions at the cellular level within the broader context of lymph node biology. Conclusions: By combining existing imaging technology with complex methods of sample preparation and capture, we have developed efficient systems for contextualizing immunological phenomena within lymphatic architecture. In combination with robust approaches to image analysis, these advances provide a path to integrating scientific understanding of basic lymphatic biology into the complex nature of immunological response.

2012

• Langereis, M. A., Zeng, Q., Heesters, B. A., Huizinga, E. G., & de Groot, R. J. (2012). The Murine Coronavirus Hemagglutinin-Esterase Receptor-Binding Site: A Major Shift in Ligand Specificity through Modest Changes in Architecture. PLoS Pathogens, 8(1), e1002492. https://doi.org/10.1371/journal.ppat.1002492
  The hemagglutinin-esterases (HEs), envelope glycoproteins of corona-, toro- and orthomyxoviruses, mediate reversible virion attachment to O-acetylated sialic acids (O-Ac-Sias). They do so through concerted action of distinct receptor-binding ("lectin") and receptor-destroying sialate O-acetylesterase ("esterase") domains. Most HEs target 9-O-acetylated Sias. In one lineage of murine coronaviruses, however, HE esterase substrate and lectin ligand specificity changed dramatically as these viruses evolved to use 4-O-acetylated Sias instead. Here we present the crystal structure of the lectin domain of mouse hepatitis virus (MHV) strain S HE, resolved both in its native state and in complex with a receptor analogue. The data show that the shift from 9-O- to 4-O-Ac-Sia receptor usage primarily entailed a change in ligand binding topology and, surprisingly, only modest changes in receptor-binding site architecture. Our findings illustrate the ease with which viruses can change receptor-binding specificity with potential consequences for host-, organ and/or cell tropism, and for pathogenesis.

2011

• Gonzalez, S. F., Degn, S. E., Pitcher, L. A., Woodruff, M., Heesters, B. A., & Carroll, M. C. (2011). Trafficking of B Cell Antigen in Lymph Nodes. Annual Review of Immunology, 29(1), 215–233. https://doi.org/10.1146/annurev-immunol-031210-101255
  The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology (1). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones (2). The experimental demonstration by Nossal & Lederberg (3) that B lymphocytes bear receptors for a single antigen raised the central question of where B lymphocytes encounter antigen. This question has remained mostly unanswered until recently. Advances in techniques such as multiphoton intravital microscopy (4, 5) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation.

2010

• Heesters, B. A. (2010). Complement Evasion Strategies of Bacteria, Viruses, Fungi and Parasites [Master's thesis]. Utrecht University.
  The human immune system consists of multiple subsystems and cascades to recognize and eliminate pathogenic intruders. The complement system is a vast and important component of this protective machinery. However, numerous of pathogens have developed ways to evade the complement system through a range of mechanisms, often by exploiting host regulators. Understanding these processes gives insight in the pathology of infectious and inflammatory diseases, eventually resulting in novel therapeutic treatments. This thesis gives an overview of the complement system and it’s host regulators. In addition the differences and similarities between evasion strategies and mechanisms of pathogens from different domains will be given.

In the News

Twenty-six groundbreaking research projects awarded via the Open Competition Domain Science – XS NWO website news, November 2022

Inflammation Network with keynote lecture from Balthasar Heesters on follicular dendritic cells.
Aarhus University news, August 2022

Infection and Immunology Seminar with Balthasar Heesters: “Follicular dendritic cells; neglected regulators”.
Karolinska news, October 2021

Veni grants for 17 UvA and AMC researchers.
University of Amsterdam news, July 2017

Infectiebacterie prikkelt zelf de pijnzenuw. (Dutch)
NRC Handelsblad, August 2013

Can bacteria use pain to tamp down the immune system?
Chicago Tribune, August 2013