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Supplementary Table 2 PDF file - 30K, Patient characteristics and historical controls

Supplementary Figure 2 PDF file - 46K, Supplementary Figure 2 Plasmacytoid DCs acquire CCL21-driven chemotactic ability upon activation

Supplementary Figure 1 PDF file - 34K, Supplementary Figure 1 CliniMACS-based pDC isolation is feasible

Supplementary Table 1 PDF file - 29K, Induration (mm) of the different injection sites in the DTH skin test

Gliomas appear to be highly immunosuppressive tumors, with a strong myeloid component. This includes MDSCs, which are a heterogeneous, immature myeloid cell population expressing myeloid markers Siglec-3 (CD33) and CD11b and lacking markers of mature myeloid cells including MHC II. Siglec-3 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and has been suggested to promote MDSC expansion and suppression. Siglecs form a recently defined family of receptors with potential immunoregulatory functions but only limited insight in their expression on immune regulatory cell subsets, prompting us to investigate Siglec expression on MDSCs. We determined the expression of different Siglec family members on monocytic-MDSCs (M-MDSCs) and polymorphnuclear-MDSCs (PMN-MDSCs) from blood of glioma patients and healthy donors, as well as from patient-derived tumor material. Furthermore, we investigated the presence of sialic acid ligands for these Siglecs on MDSCs and in the glioma tumor microenvironment. Both MDSC subsets express Siglec-3, -5, -7 and -9, with higher levels of Siglec-3, -7 and -9 on M-MDSCs and higher Siglec-5 levels on PMN-MDSCs. Similar Siglec expression profiles were found on MDSCs from healthy donors. Furthermore, the presence of Siglec-5 and -9 was also confirmed on PMN-MDSCs from glioma tissue. Interestingly, freshly isolated glioma cells predominantly expressed sialic acid ligands for Siglec-7 and -9, which was confirmed in situ. In conclusion, our data show a distinct Siglec expression profile for M- and PMN-MDSCs and propose possible sialic acid–Siglec interactions between glioma cells and MDSCs in the tumor microenvironment. Electronic supplementary material The online version of this article (10.1007/s00262-019-02332-w) contains supplementary material, which is available to authorized users.

Contains fulltext : 177820.pdf (Publisher’s version ) (Closed access) Sialic acid sugars cover the surface of dendritic cells (DCs) and have been suggested to impact several aspects of DC biology. Research into the role of sialic acids in DCs, however, is complicated by the limited number of tools available to modulate sialic acid expression. Here we report on a synthetic, fluorinated sialic acid mimetic, Ac53FaxNeu5Ac, which potently blocks sialic acid expression in human monocyte-derived DCs (moDCs). Sialic acid blockade enhanced the responsiveness of moDCs to Toll-like receptor (TLR) stimulation as measured by increased maturation marker expression and cytokine production. Consequently, the T-cell activation capacity of Ac53FaxNeu5Ac-treated moDCs was strongly increased. In addition to sialic acids, moDCs also expressed the sialic acid-binding immunoglobulin-like lectins (Siglecs) -3, -5, -7, -9 and -10, immune inhibitory receptors recognizing these sialic acids. Treatment with Ac53FaxNeu5Ac abrogated putative cis and trans interactions between sialic acids and Siglec-7/-9. Together, these data indicate that sialic acids limit the activation of moDCs via the TLR pathway, potentially by interacting with Siglec-7 or Siglec-9. Metabolic sialic acid blockade with Ac53FaxNeu5Ac could therefore potentially be used to generate more potent DC-based vaccines for induction of robust anti-viral or anti-tumor immune responses.

Contains fulltext : 172472.pdf (Publisher’s version ) (Closed access) In children at risk for type 1 diabetes, innate immune activity is detected before seroconversion. Enterovirus infections have been linked to diabetes development, and a polymorphism (A946T) in the innate immune sensor recognizing enterovirus RNA, interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5, predisposes to disease. We hypothesized that the strength of innate antienteroviral responses is affected in autoimmune type 1 diabetes patients and linked to the A946T polymorphism. We compared induction of interferon-stimulated genes (ISGs) in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) in healthy individuals and diabetes patients upon stimulation with enterovirus, enterovirus-antibody complexes, or ligands mimicking infection in relation to the A946T polymorphism. Overall, PBMCs of diabetes patients and healthy donors showed comparable ISG induction upon stimulation. No differences were observed in DCs. Interestingly, the data imply that the magnitude of responses to enterovirus and enterovirus-antibody complexes in PBMCs is critically influenced by the A946T polymorphism and elevated in heterozygotes compared to TT homozygous individuals in autoimmune diabetes patients, but not healthy controls. These data imply an intrinsic difference in the responses to enterovirus and enterovirus-antibody complexes in diabetes patients carrying a TT risk genotype compared to heterozygotes that may influence control of enterovirus clearance.

Contains fulltext : 172329.pdf (Publisher’s version ) (Closed access) Derailment of immune responses can lead to autoimmune type 1 diabetes, and this can be accelerated or even induced by local stress caused by inflammation or infection. Dendritic cells (DCs) shape both innate and adaptive immune responses. Here, we report on the responses of naturally occurring human myeloid BDCA1(+) DCs towards differentially stressed pancreatic beta cells. Our data show that BDCA1(+) DCs in human pancreas-draining lymph node (pdLN) suspensions and blood-derived BDCA1(+) DCs both effectively engulf beta cells, thus mimicking physiological conditions. Upon uptake of enterovirus-infected, but not mock-infected cells, BDCA1(+) DCs induced interferon (IFN)-alpha/beta responses, co-stimulatory molecules and proinflammatory cytokines and chemokines. Notably, induction of stress in beta cells by ultraviolet irradiation, culture in serum-free medium or cytokine-induced stress did not provoke strong DC activation, despite efficient phagocytosis. DC activation correlated with the amount of virus used to infect beta cells and required RNA within virally infected cells. DCs encountering enterovirus-infected beta cells, but not those incubated with mock-infected or stressed beta cells, suppressed T helper type 2 (Th2) cytokines and variably induced IFN-gamma in allogeneic mixed lymphocyte reaction (MLR). Thus, stressed beta cells have little effect on human BDCA1(+) DC activation and function, while enterovirus-infected beta cells impact these cells significantly, which could help to explain their role in development of autoimmune diabetes in individuals at risk.

Background The aryl hydrocarbon receptor (AhR) is a nuclear receptor and transcriptional regulator with pleiotropic effects. The production of potent AhR ligands by Malassezia yeasts, such as indirubin, indolo[3,2-b]carbazole (ICZ), tryptanthrin and malassezin, has been associated with the pathogenesis of seborrhoeic dermatitis and pityriasis versicolor. Antigen-presenting cells in the skin can encounter microbes in the presence of these bioactive metabolites that could potentially modulate their function. Objectives To study the effects of the aforementioned naturally occurring ligands on AhR activation and Toll-like receptor (TLR)-induced maturation in human monocyte-derived dendritic cells (moDCs). Methods These indoles were screened for AhR activation capacity in moDCs employing CYP1A1 and CYP1B1 induction as read out and for their effects on the function of moDCs after TLR-ligand stimulation. Results Indirubin and ICZ were the most potent AhR ligands and were selected for subsequent experiments. Concurrent exposure of moDCs to indirubin or ICZ together with TLR agonists significantly augmented the AhR-mediated CYP1A1 and CYP1B1 gene expression. Additionally, mature DCs that were subsequently stimulated with AhR ligands showed increased AhR target gene expression. Moreover, these ligands limited TLR-induced phenotypic maturation (CD80, CD83, CD86, MHC II upregulation) of moDCs, reduced secretion of the inflammatory cytokines interleukin (IL)-6 and IL-12, and decreased their ability to induce alloreactive T-lymphocyte proliferation. Conclusions These results demonstrate that AhR agonists of yeast origin are able to inhibit moDC responses to TLR ligands and that moDCs can adapt through increased transcription of metabolizing enzymes such as CYP1A1 and CYP1B1.

Contains fulltext : 88377.pdf (Publisher’s version ) (Open Access) Previous studies have shown that enteroviral RNA can be detected in blood at the onset of type 1 diabetes (T1D). The infection may play a role in triggering T1D and genetic host factors may contribute to this process. We investigated (1) whether enterovirus is present at the onset of T1D in peripheral blood mononuclear cells (PBMC), plasma, throat, or stool, and (2) whether enteroviral presence is linked with HLA-DR type and/or polymorphisms in melanoma differentiation-associated gene 5 (MDA5) and 2'-5' oligoadenylate synthetase 1 (OAS1), factors of antiviral immunity. To this end, PBMC, plasma, throat, and stool samples from 10 T1D patients and 20 unrelated controls were tested for the presence of enteroviruses (RT-PCR), for HLA-DR type, and polymorphisms in MDA5 and OAS1. Enterovirus RNA was detected in PBMC of 4/10 T1D patients, but none of 20 controls. Plasma was positive in 2/10 T1D patients and none of 20 controls, suggesting that enteroviruses found at the onset of T1D are mainly present in PBMC. All throat samples from positive T1D patients were virus-negative and only 1 fecal sample was positive. The negative results for all throat and most stool samples argues against acute infection. Enterovirus presence was linked with HLA-DR4, but not with polymorphisms in MDA5 or OAS1. 01 februari 2010

OBJECTIVEType 1 diabetes is a chronic endocrine disorder in which enteroviruses, such as coxsackie B viruses and echoviruses, are possible environmental factors that can trigger or accelerate disease. The development or acceleration of type 1 diabetes depends on the balance between autoreactive effector T-cells and regulatory T-cells. This balance is particularly influenced by dendritic cells (DCs). The goal of this study was to investigate the interaction between enterovirus-infected human pancreatic islets and human DCs.RESEARCH DESIGN AND METHODSIn vitro phagocytosis of human or porcine primary islets or Min6 mouse insuloma cells by DCs was investigated by flow cytometry and confocal analysis. Subsequent innate DC responses were monitored by quantitative PCR and Western blotting of interferon-stimulated genes (ISGs).RESULTSIn this study, we show that both mock- and coxsackievirus B3 (CVB3)-infected human and porcine pancreatic islets were efficiently phagocytosed by human monocyte–derived DCs. Phagocytosis of CVB3-infected, but not mock-infected, human and porcine islets resulted in induction of ISGs in DCs, including the retinoic acid–inducible gene (RIG)-I–like helicases (RLHs), RIG-I, and melanoma differentiation–associated gene 5 (Mda5). Studies with murine Min6 insuloma cells, which were also efficiently phagocytosed, revealed that increased ISG expression in DCs upon encountering CVB-infected cells resulted in an antiviral state that protected DCs from subsequent enterovirus infection. The observed innate antiviral responses depended on RNA within the phagocytosed cells, required endosomal acidification, and were type I interferon dependent.CONCLUSIONSHuman DCs can phagocytose enterovirus-infected pancreatic cells and subsequently induce innate antiviral responses, such as induction of RLHs. These responses may have important consequences for immune homeostasis in vivo and may play a role in the etiology of type 1 diabetes.

Dendritic cells (DCs) play a central role in instructing antiviral immune responses. DCs, however, can become targeted by different viruses themselves. We recently demonstrated that human DCs can be productively infected with echoviruses (EVs), but not coxsackie B viruses (CVBs), both of which are RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. We now show that phagocytosis of CVB-infected, type I interferon-deficient cells induces an antiviral state in human DCs. Uptake of infected cells increased the expression of the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 as well as other interferon-stimulated genes and protected DCs against subsequent infection with EV9. These effects depended on recognition of viral RNA and could be mimicked by exposure to the synthetic double-stranded RNA analogue poly(I:C) but not other Toll-like receptor (TLR) ligands. Blocking endosomal acidification abrogated protection, suggesting a role for TLRs in the acquisition of an antiviral state in DCs. In conclusion, recognition of viral RNA rapidly induces an antiviral state in human DCs. This might provide a mechanism by which DCs protect themselves against viruses when attracted to an environment with ongoing infection.

Contains fulltext : 167769.pdf (Publisher’s version ) (Closed access) BACKGROUND: Gliomas are primary brain tumors that are associated with a poor prognosis. The introduction of new treatment modalities (including immunotherapy) for these neoplasms in the last 3 decades has resulted in only limited improvement in survival. Gliomas are known to create an immunosuppressive microenvironment that hampers the efficacy of (immuno)therapy. One component of this immunosuppressive environment is the myeloid-derived suppressor cell (MDSC). METHODS: We set out to analyze the presence and activation state of MDSCs in blood (n = 41) and tumor (n = 20) of glioma patients by measuring S100A8/9 and arginase using flow cytometry and qPCR. Inhibition of T cell proliferation and cytokine production after stimulation with anti-CD3/anti-CD28 coated beads was used to measure in vitro MDSC suppression capacity. RESULTS: We report a trend toward a tumor grade-dependent increase of both monocytic (M-) and polymorphonuclear (PMN-) MDSC subpopulations in the blood of patients with glioma. M-MDSCs of glioma patients have increased levels of intracellular S100A8/9 compared with M-MDSCs in healthy controls (HCs). Glioma patients also have increased S100A8/9 serum levels, which correlates with increased arginase activity in serum. PMN-MDSCs in both blood and tumor tissue demonstrated high expression of arginase. Furthermore, we assessed blood-derived PMN-MDSC function and showed that these cells have potent T cell suppressive function in vitro. CONCLUSIONS: These data indicate a tumor grade-dependent increase of MDSCs in the blood of patients with a glioma. These MDSCs exhibit an increased activation state compared with MDSCs in HCs, independent of tumor grade.

Contains fulltext : 51866.pdf (Publisher’s version ) (Closed access) Coxsackie B viruses (CVB) and Echoviruses (EV) form a single species; Human enterovirus B (HeV-B), within the genus Enterovirus. Although HeV-B infections are usually mild or asymptomatic, they can cause serious acute illnesses. In addition, HeV-B infections have been associated with chronic immune disorders, such as type 1 diabetes mellitus and chronic myocarditis/dilated cardiomyopathy. It has therefore been suggested that these viruses may trigger an autoimmune process. Here, we demonstrate that human dendritic cells (DCs), which play an essential role in orchestration of the immune response, are productively infected by EV, but not CVB strains, in vitro. Infection does not result in DC activation or the induction of antiviral immune responses. Instead, EV infection rapidly impedes Toll-like receptor-mediated production of cytokines and upregulation of maturation markers, and ultimately causes loss of DC viability. These results describe for the first time the effect of EV on the function and viability of human DCs and suggest that infection of DCs in vivo can impede regulation of immune responses.

Contains fulltext : 153143.pdf (Publisher’s version ) (Closed access) Myeloid-derived suppressor cells (MDSCs), defined as CD33-positive major histocompatibility complex class II-negative cells, are increased in a variety of human tumors and are associated with immunosuppression. Myeloid-derived suppressor cells can be further subdivided into CD14-positive monocytic MDSC and CD15-positive granulocytic MDSC (polymorphonuclear MDSC) subpopulations. Here we analyzed MDSC subsets in the blood and tumor tissue of patients with glioma, including the most malignant variant, glioblastoma multiforme (GBM). CD33-positive major histocompatibility complex class II-negative MDSCs in blood from 21 patients with glioma and 12 healthy individuals were phenotyped and quantified by flow cytometry. Myeloid populations of the monocytic MDSC and polymorphonuclear MDSC phenotypes were both significantly increased in the blood of patients with GBM versus healthy controls. The myeloid activation markers CD80 and PD-L1 could not be detected on either of these MDSC subsets; CD124, CD86, and CD40 were detected at similar levels on MDSCs in patients with glioma and healthy donors. By contrast, in tumor cell suspensions, the MDSC population consisted almost exclusively of CD15-positive cells. Immunohistochemistry confirmed infiltration of CD15-positive major histocompatibility complex class II-negative cells in glioma tissue samples. These data support a role for cells with an MDSC phenotype in the blood and tumor microenvironment of patients with GBM.

Contains fulltext : 119153.pdf (Publisher’s version ) (Open Access) Vaccination against cancer by using dendritic cells has for more than a decade been based on dendritic cells generated ex vivo from monocytes or CD34(+) progenitors. Here, we report on the first clinical study of therapeutic vaccination against cancer using naturally occurring plasmacytoid dendritic cells (pDC). Fifteen patients with metastatic melanoma received intranodal injections of pDCs activated and loaded with tumor antigen-associated peptides ex vivo. In vivo imaging showed that administered pDCs migrated and distributed over multiple lymph nodes. Several patients mounted antivaccine CD4(+) and CD8(+) T-cell responses. Despite the limited number of administered pDCs, an IFN signature was observed after each vaccination. These results indicate that vaccination with naturally occurring pDC is feasible with minimal toxicity and that in patients with metastatic melanoma, it induces favorable immune responses. Cancer Res; 73(3); 1063-75. (c)2012 AACR.

Contains fulltext : 87889.pdf (Publisher’s version ) (Open Access) Dendritic cells (DCs) are professional antigen-presenting cells that provide a link between innate and adaptive immunity. Multiple DC subsets exist and their activation by microorganisms occurs through binding of conserved pathogen-derived structures to so-called pattern recognition receptors (PRRs). In this study we analyzed the expression of PRRs responding to viral RNA in human monocyte-derived DCs (moDCs) under steady-state or pro-inflammatory conditions. We found that mRNA and protein levels for most PRRs were increased under pro-inflammatory conditions, with the most pronounced increases in the RIG-like helicase (RLH) family. Additionally, freshly isolated human plasmacytoid DCs (pDCs) displayed significantly higher levels of TLR7, RIG-I, MDA5 and PKR as compared to myeloid DCs and moDCs. Finally, we demonstrate for the first time that cross-talk between TLR-matured or virus-stimulated pDCs and moDCs leads to a type I interferon-dependent antiviral state in moDCs. This antiviral state was characterized by enhanced RLH expression and protection against picornavirus infection. These findings might represent a novel mechanism by which pDCs can preserve the function and viability of myeloid DCs that are attracted to a site with ongoing infection, thereby optimizing the antiviral immune response.

Viral infection of mammalian cells triggers the synthesis and secretion of type I interferons (i.e. IFN-alpha/beta), which induce the transcription of genes that cause cells to adopt an antiviral state. Many viruses have adapted mechanisms to evade IFN-alpha/beta-mediated responses. The leader protein of mengovirus, a picornavirus, has been implicated as an IFN-alpha/beta antagonist. Here, we show that the leader inhibits the transcription of IFN-alpha/beta and that both the presence of a zinc finger motif in its N-terminus and phosphorylation of threonine-47 are required for this function. Transcription of IFN-alpha/beta genes relies on the activity of a number of transcription factors, including interferon regulatory factor 3 (IRF-3). We show that the leader interferes with the transactivation activity of IRF-3 by interfering with its dimerization. Accordingly, mutant viruses with a disturbed leader function were impaired in their ability to suppress IFN-alpha/beta transcription in vivo. By consequence, the leader mutant viruses had an impaired ability to replicate and spread in normal mice but not in IFNAR-KO mice, which are incapable of mounting an IFN-alpha/beta-dependent antiviral response. These results suggest that the leader, by suppressing IRF3-mediated IFN-alpha/beta production, plays an important role in replication and dissemination of mengovirus in its host.

Enteroviruses (e.g. Coxsackie B viruses, CVBs) have been implicated as environmental factors in type 1 diabetes, but the underlying mechanism(s) remain elusive.

Enteroviruses haven been associated with autoimmune type 1 diabetes development, however the possible underlying mechanism remains unclear. Dendritic cells (DCs) are the major antigen-presenting cells (APCs) and initiate immune responses against pathogens, but also prevent (auto-)immune responses harmful to the host. We hypothesize that enterovirus- infected β-cells that are phagocytosed by DCs can unleash the immune system and eventuate in β-cell-autoimmunity in genetically susceptible individuals. Therefore we investigate the interaction between primary BDCA1+ DCs and enterovirus- infected pancreatic β-cells.