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Radboud Universiteit Nijmegen, 13 september 2012, Promotor : Adema, G.J. Co-promotor : Nierkens, S., Contains fulltext : 98581.pdf (publisher's version ) (Open Access)

1 juni 2012, Item does not contain fulltext, INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by the formation of autoantibodies against nuclear components. Disturbed apoptosis and reduced clearance of apoptotic material have been assigned a role in the pathogenesis of SLE. During apoptosis, apoptotic blebs are formed, in which SLE autoantigens are clustered. In vitro, apoptotic blebs can induce maturation of dendritic cells (DC), which in turn can stimulate IL-17 production by T cells. Here, we investigated the effects of administration of apoptotic blebs, separate or in combination with dendritic cells, on disease progression and autoantibody production in lupus and normal mice. METHODS: A preparation of apoptotic blebs, with or without DC, was intravenously administered to MRL/lpr and CBA mice at weeks 7, 9, and 11 of age. T-cell responses against autoantigens present in blebs were examined by delayed type hypersensitivity reactions. Disease progression of the mice was evaluated by determining proteinuria and the titers of anti-DNA, anti-histone, and anti-nucleosome autoantibodies in plasma. RESULTS: Repeated administration of apoptotic blebs, with or without DC, had no effect on the course of proteinuria or on anti-DNA, anti-histone and anti-nucleosome autoantibody levels in MRL/lpr mice. Intravenous injections of apoptotic blebs resulted in a decrease in the DTH response towards s.c. administered blebs in MRL/lpr mice and in reduced anti-nucleosome antibody titers in CBA mice. These tolerizing effects were lost when apoptotic blebs were administered together with syngeneic DC after 2 hours of co-incubation. DISCUSSION AND CONCLUSIONS: In contrast to previous studies with apoptotic cells, and deviating from our in vitro findings with apoptotic blebs, we observed no stimulating effect of the administration of apoptotic blebs on disease progression in MRL/lpr lupus mice. The tolerogenic effects that were observed may be associated with rapid removal of i.v. administered

Item does not contain fulltext, The identification of responding patients early during treatment would improve the capability to develop effective new immunotherapies more rapidly. Here, we describe a bioassay that may link early T-cell-mediated immune responses to later clinical benefits. This bioassay rests upon the tenet of immunotherapy that tumor-specific effector T cells capable of invading peripheral tissue can recognize tumor antigens and exert cytotoxic functions there. To show its utility, we conducted a retrospective study of a large cohort of metastatic melanoma patients (n = 91) enrolled in dendritic cell (DC)-based vaccination protocols to examine a hypothesized correlation of posttreatment skin-infiltrating lymphocytes (SKIL) with overall survival (OS). Stringent immunologic criteria were defined to identify long-term survivors. The presence of tumor-associated antigen (TAA)-specific CD8(+) T cell populations within SKILs (criterion I) was highly predictive for long-term survival. Further restriction by selecting for the presence of TAA-specific CD8(+) T cells specifically recognizing tumor peptide (criterion II) was also associated with improved OS. Recognition of naturally processed antigen (criterion III) maximized the accuracy of the test, with a median OS of 24.1 versus 9.9 months (P = 0.001). Our results show that detailed characterization of SKILs can permit an accurate selection of metastatic melanoma patients who benefit most from DC-based vaccination. This simple and robust bioassay integrates multiple aspects of cellular functions that mediate effective immune responses, thereby offering an effective tool to rapidly identify patients who are responding to immunotherapy at an early stage of treatment. Cancer Res; 72(23); 6102-10. (c)2012 AACR.

1 februari 2012, Item does not contain fulltext, Neuroblastoma (NBL) is an aggressive malignancy of the sympathetic nervous system. Advanced-stage NBLs prove fatal in approximately 50% of patients within 5 years. Therefore, new treatment modalities are urgently needed. Immunotherapy is a treatment modality that can be combined with established forms of treatment. Administration of monoclonal antibodies or dendritic cell-based therapies alone can lead to favorable clinical outcomes in individual cancer patients; for example patients with melanoma, lymphoma and NBL. However, clinical benefit is still limited to a minority of patients, and further improvements are clearly needed. In this article, we review the most commonly used approaches to treat patients with NBL and highlight the prerequisites and opportunities of cell-based immunotherapy, involving both innate and adaptive immune-effector cells. Furthermore, we discuss the potential of the combined application of immunotherapy and novel tumor-targeted therapies for the treatment of both cancer in general and NBL in particular.

Item does not contain fulltext, PURPOSE: Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses. EXPERIMENTAL DESIGN: Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA. RESULTS: KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20 mug/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9 %, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients. CONCLUSIONS: We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.

1 mei 2012, Item does not contain fulltext, The induction of tumor-specific immune responses is largely dependent on the ability of dendritic cells (DCs) to present tumor-associated antigens to T lymphocytes. Therefore, we investigated the use of DC-associated promoter-driven genetic vaccines to specifically target DC in vivo. Restricted expression of vaccine-encoding genes in DC should enhance specificity and improves their safety for clinical applications. Hereto, 3-5 kb upstream sequences of the murine genes encoding CD11c, DC-SIGN, DC-STAMP and Langerin were isolated, characterized and subcloned into enhanced green fluorescent protein (EGFP) reporter constructs. Upon electroporation, EGFP was expressed in DC cell lines, but not in other cell lines, confirming DC-restricted promoter activity. When these promoters were cloned into a construct upstream of the gene for ovalbumin (OVA), it appeared that DC-STAMP promoter-driven expression of OVA (pDCSTAMP/OVA) in DC yielded the most efficient OVA-specific CD4+ and CD8+ T-cell responses in vitro. Administration of pDC-STAMP/OVA in vivo, using the tattoo gun vaccination system, evoked specific immune responses as evidenced in a mouse tumor model. Adoptively transferred pDC-STAMP/OVA-transfected DCs induced strong CD8+ T-cell proliferation in vivo. These experiments demonstrate that our DC-directed promoter constructs are potential tools to restrict antigen expression in DC and could be implemented to modulate DC function by the introduction of relevant proteins.

01 februari 2012, Item does not contain fulltext, Dendritic cells (DCs) are professional antigen presenting cells of the immune system that play a crucial role in initiating immune responses and maintaining self tolerance. Better understanding of the molecular basis of DC immunobiology is required to improve DC-based immunotherapies. We previously described the interaction of transcription factor LUMAN (also known as CREB3 or LZIP) with the DC-specific transmembrane protein DC-STAMP in DCs. Target genes of LUMAN and its role in DCs are currently unknown. In this study we set out to identify genes regulated by LUMAN in DCs using microarray analysis. Expression of a constitutively active form of LUMAN in mouse DC cell line D2SC/1 identified Apolipoprotein A4 (ApoA4) as its target gene. Subsequent validation experiments, bioinformatics-based promoter analysis, and silencing studies confirmed that ApoA4 is a true target gene of LUMAN in bone marrow-derived DCs (BMDCs).

1 augustus 2012, Item does not contain fulltext, Enteroviruses of the human enterovirus B species (HEV-Bs) (e.g., coxsackie B viruses [CVBs] and echoviruses) have been implicated as environmental factors that trigger/accelerate type 1 diabetes, but the underlying mechanism remains elusive. The aim of this study was to gain insight into the cytokines and chemokines that are produced by human pancreatic islets upon infection with CVBs. To this end, we studied the response of human islets of Langerhans upon mock or CVB3 infection. Using quantitative PCR, we showed that upon CVB3 infection, transcription of interferon (IFN), IFN-stimulated genes, and inflammatory genes was induced. Analysis of secreted cytokines and chemokines by Luminex technology confirmed production and secretion of proinflammatory cytokines (e.g., interleukin [IL]-6 and tumor necrosis factor-alpha) as well as various chemotactic proteins, such as IFN-gamma-induced protein 10, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and IL-8. Infection with other HEV-Bs induced similar responses, yet their extent depended on replication efficiency. Ultra violet-inactivated CVB3 did not induce any response, suggesting that virus replication is a prerequisite for antiviral responses. Our data represent the first comprehensive overview of inflammatory mediators that are secreted by human islets of Langerhans upon CVB infection and may shed light on the role of enteroviruses in type 1 diabetes pathogenesis.

1 januari 2012, Item does not contain fulltext, Immunotherapy studies in patients with melanoma have reported success in the expansion of tumour-specific effector T cells in vivo, but even in the presence of substantial numbers of functional T cells circulating in the blood, favourable clinical outcomes are scarce. This failure to induce robust clinical responses might be related to tumour-induced immune evasion, rendering the host tolerant to melanoma antigens. Immunosuppression in the tumour microenvironment mediated by regulatory T cells (Treg) is a dominant mechanism of tumour immune escape and is a major hurdle for tumour immunotherapy. Accumulation of Treg in melanoma is frequently recorded and the ratio of CD8-positive T cells versus Treg in the tumour microenvironment is predictive for survival of patients with melanoma. Hence, depletion of Treg seems to be a promising strategy for the enhancement of melanoma-specific immunity. Indeed, murine studies have shown that Treg depletion greatly increases the efficacy of immunotherapy. But despite the success of some strategies in depletion of Treg in patients, overall clinical efficacy has been disappointing. The lack of Treg specificity of the Treg depleting strategies applied so far imply that well-designed studies into dosage, timing, and administration regimens with more specific agents are urgently needed. Depletion of functional Treg from the tumour microenvironment as part of multifaceted immunotherapeutic treatments is a major challenge to induce clinically relevant immune responses against melanomas.

Item does not contain fulltext, CLEC9A is a recently discovered C-type lectin receptor involved in sensing necrotic cells. In humans, this receptor is selectively expressed by BDCA3(+) myeloid dendritic cells (mDCs), which have been proposed to be the main human cross-presenting mDCs and may represent the human homologue of murine CD8(+) DCs. In mice, it was demonstrated that antigens delivered with antibodies to CLEC9A are presented by CD8(+) DCs to both CD4(+) and CD8(+) T cells and induce antitumor immunity in a melanoma model. Here we assessed the ability of CLEC9A to mediate antigen presentation by human BDCA3(+) mDCs, which represent < 0.05% of peripheral blood leukocytes. We demonstrate that CLEC9A is only expressed on immature BDCA3(+) mDCs and that cell surface expression is lost after TLR-mediated maturation. CLEC9A triggering via antibody binding rapidly induces receptor internalization but does not affect TLR-induced cytokine production or expression of costimulatory molecules. More importantly, antigens delivered via CLEC9A antibodies to BDCA3(+) mDCs are presented by both MHC class I (cross-presentation) and MHC class II to antigen-specific T cells. We conclude that CLEC9A is a promising target for in vivo antigen delivery in humans to increase the efficiency of vaccines against infectious or malignant diseases.

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Item does not contain fulltext, BACKGROUND: Nuclear receptors (NR), including the Androgen Receptor (AR) and the Vitamin D Receptor (VDR), play an important role in prostate cancer etiology. We recently found that DC-SCRIPT is a prognostic marker in breast cancer and a unique NR coregulator differentially regulating different classes of NRs. Here we investigated the importance of DC-SCRIPT in prostate cancer. METHODS: DC-SCRIPT mRNA expression was measured by qPCR. Immunohistochemistry was used to detect DC-SCRIPT protein expression. The functional effects of DC-SCRIPT on the transcriptional activity of AR and VDR were assessed by luciferase reporter assays and qPCR assays on well-known AR and VDR target genes. RESULTS: DC-SCRIPT mRNA was higher in normal than in corresponding malignant prostate tissue but could not be related to disease stage. DC-SCRIPT protein was found in morphologically normal prostate glands and in infiltrating immune cells. Strikingly, DC-SCRIPT protein expression was absent in malignant prostate epithelial tissue and prostate carcinoma cell lines. DC-SCRIPT protein expression appears to be lost prior to the basal cell marker HMW cytokeratin used in prostate carcinoma diagnostics. In addition, our data demonstrated that DC-SCRIPT repressed transcription mediated by wild-type and mutated AR while enhancing VDR mediated transcription. In addition, transient expression of DC-SCRIPT expression in prostate carcinoma cells strongly repressed cell growth. CONCLUSIONS: DC-SCRIPT is a key regulator of nuclear receptors AR and VDR that play an opposite role in prostate cancer etiology and loss of DC-SCRIPT may be involved in the onset of prostate cancer. Prostate 72:1708-1717, 2012. (c) 2012 Wiley Periodicals, Inc.

Contains fulltext : 95990.pdf (publisher's version ) (Closed access), The differences in function, location, and migratory pattern of conventional dendritic cells (cDC) and plasmacytoid DCs (pDC) not only point to specialized roles in immune responses but also signify additive and interdependent relationships required to clear pathogens. We studied the in vivo requirement of cross-talk between cDCs and pDCs for eliciting antitumor immunity against in situ released tumor antigens in the absence or presence of the Toll-like receptor (TLR) 9 agonist CpG. Previous data indicated that CpG boosted tumor-specific T-cell responses after in vivo tumor destruction and increased survival after tumor rechallenges. The present study shows that cDCs are indispensable for cross-presentation of ablation-released tumor antigens and for the induction of long-term antitumor immunity. Depletion of pDCs or applying this model in type I IFN receptor-deficient mice abrogated CpG-mediated responses. CD8alpha(+) cDCs and the recently identified merocytic cDCs were dependent on pDCs for CpG-induced upregulation of CD80. Moreover, DC transfer studies revealed that merocytic cDCs and CD8alpha(+) cDCs were most susceptible to pDC help and subsequently promoted tumor-free survival in a therapeutic setting. By transferring wild-type pDCs into TLR9-deficient mice, we finally showed that TLR9 expression in pDCs is sufficient to benefit from CpG as an adjuvant. These studies indicate that the efficacy of CpG in cancer immunotherapy is dependent on cross-talk between pDCs and specific subsets of cDCs. Cancer Res; 71(20); 6428-37. (c)2011 AACR.

Contains fulltext : 97521.pdf (publisher's version ) (Closed access), Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients. Several strategies have been employed to load DC with antigen, including peptide loading. To increase immunogenicity of peptides, major histocompatibility complex (MHC) class I binding affinity and stability of peptide-MHC complexes at the cell surface may be improved by modification of the amino acid sequence. In this study, we compared the capacity of DC loaded with wild-type versus modified gp100 peptides with higher binding affinities to induce an immune and clinical response in advanced melanoma patients. Metastatic HLA-A2.1(+) melanoma patients were vaccinated intravenously (on average 25 x 10(6) DC) and intradermally (on average 11 x 10(6) DC) with mature DC loaded with keyhole limpet hemocyanin (KLH) together with tyrosinase peptide and either wild-type (15 patients) or modified (12 patients) gp100 peptides. All vaccinated patients showed a pronounced proliferative T cell or humoral response against KLH. Gp100-specific T cell responses were monitored in post-treatment delayed type hypersensitivity (DTH) skin biopsies by tetramer and functional analysis. Antigen-specific T cells were found in 2 of 15 patients vaccinated with wild-type gp100-loaded DC, versus 1 of 12 patients vaccinated with modified peptide-loaded DC. These three patients also had the best clinical response, with long-term (>8 years) complete responses in two patients, one in each group. We conclude that vaccination with peptide-loaded DC can result in long-term clinical responses in a minority of metastatic melanoma patients, and that the use of modified as compared to wild-type gp100 peptides for DC loading does not result in a relevant enhanced immune responses.

Radboud Universiteit Nijmegen, 5 oktober 2011, Promotor : Adema, G.J., Contains fulltext : 91253.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, Effective antitumor immunotherapy requires the identification of suitable target Ags. Interestingly, many of the tumor Ags used in clinical trials are present in preparations of secreted tumor vesicles (exosomes). In this study, we compared T cell responses elicited by murine MCA101 fibrosarcoma tumors expressing a model Ag at different localizations within the tumor cell in association with secreted vesicles (exosomes), as a nonsecreted cell-associated protein, or as secreted soluble protein. Remarkably, we demonstrated that only the tumor-secreting vesicle-bound Ag elicited a strong Ag-specific CD8(+) T cell response, CD4(+) T cell help, Ag-specific Abs, and a decrease in the percentage of immunosuppressive regulatory T cells in the tumor. Moreover, in a therapeutic tumor model of cryoablation, only in tumors secreting vesicle-bound Ag could Ag-specific CD8(+) T cells still be detected up to 16 d after therapy. We concluded that the localization of an Ag within the tumor codetermines whether a robust immunostimulatory response is elicited. In vivo, vesicle-bound Ag clearly skews toward a more immunogenic phenotype, whereas soluble or cell-associated Ag expression cannot prevent or even delay outgrowth and results in tumor tolerance. This may explain why particular immunotherapies based on these vesicle-bound tumor Ags are potentially successful. Therefore, we conclude that this study may have significant implications in the discovery of new tumor Ags suitable for immunotherapy and that their location should be taken into account to ensure a strong antitumor immune response.

Contains fulltext : 95914.pdf (publisher's version ) (Closed access), PURPOSE: Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the impact of three medications on blood Treg frequency in vaccinated cancer patients. EXPERIMENTAL DESIGN: To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox. RESULTS: In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2. CONCLUSIONS: We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs.

Contains fulltext : 97226.pdf (publisher's version ) (Open Access), Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds.

Contains fulltext : 97359.pdf (publisher's version ) (Closed access), Tyrosine kinase inhibitors (TKIs) as sorafenib are known to reduce the number of immunosuppressive regulatory T cells (Tregs) in the peripheral blood and thereby shifting the immune balance to a more stimulating setting. The effect of sorafenib on intratumoural Tregs is unclear but important for future combinations of TKIs and immunotherapy. We, therefore, evaluated the accumulation of regulatory T cells (Tregs, defined as, CD4(+)FoxP3(+)CD25(high)CD127(low)-cells) in blood, ascites, metastases and primary tumours of patients with renal cell carcinoma (RCC), and we explored the effect of neoadjuvant treatment with sorafenib 400 mg bid on intratumoural Tregs in 11 patients with RCC in comparison to 15 nontreated RCC patients. We found that immunosuppressive Tregs specifically accumulate in primary tumour, metastases and ascites of RCC-patients. Tumour infiltrating Tregs were functional. Neoadjuvant sorafenib treatment significantly reduced the percentage of tumour-infiltrating Tregs (mean 17.3% vs. 28.1%, p = 0.046). Diminished Treg accumulation at the tumour site adds to explain the clinical effectiveness of sorafenib treatment. This observation may have important implications for the use of sorafenib in combination with immunotherapy in patients with RCC, since the depletion of Tregs has been associated with enhanced responses on vaccine mediated immunotherapy.

Contains fulltext : 97823.pdf (publisher's version ) (Open Access), The use of probiotics as a food supplement has gained tremendous interest in the last few years as beneficial effects were reported in gut homeostasis and nutrient absorption but also in immunocompromised patients, supporting protection from colonization or infection with pathogenic bacteria or fungi. As a treatment approach for inflammatory bowel diseases, a suitable probiotic strain would ideally be one with a low immunogenic potential. Insight into the immunogenicities and types of T-cell responses induced by potentially probiotic strains allows a more rational selection of a particular strain. In the present study, the bacterial strains Bifidobacterium breve (NumRes 204), Lactobacillus rhamnosus (NumRes1), and Lactobacillus casei (DN-114 001) were compared concerning their capacity to induce inflammatory responses in terms of cytokine production by human and mouse primary immune cells. It was demonstrated that the B. breve strain induced lower levels of the proinflammatory cytokine gamma interferon (IFN-gamma) than the tested L. rhamnosus and L. casei strains. Both B. breve and lactobacilli induced cytokines in a Toll-like receptor 9 (TLR9)-dependent manner, while the lower inflammatory profile of B. breve was due to inhibitory effects of TLR2. No role for TLR4, NOD2, and C-type lectin receptors was apparent. In conclusion, TLR signaling is involved in the differentiation of inflammatory responses between probiotic strains used as food supplements.

Contains fulltext : 97133.pdf (publisher's version ) (Open Access), BACKGROUND: Regulatory T cells (Treg) play a crucial role in maintaining immune homeostasis and self-tolerance. The immune suppressive effects of Tregs should however be limited in case effective immunity is required against pathogens or cancer cells. We previously found that the Toll-like receptor 2 (TLR2) agonist, Pam3CysSK4, directly stimulated Tregs to expand and temporarily abrogate their suppressive capabilities. In this study, we evaluate the effect of Pam3CysSK4 and Legionella pneumophila, a natural TLR2 containing infectious agent, on effector T (Teff) cells and dendritic cells (DCs) individually and in co-cultures with Tregs. RESULTS: TLR2 agonists can directly provide a co-stimulatory signal inducing enhanced proliferation and cytokine production of naive CD4+ Teff cells. With respect to cytokine production, DCs appear to be most sensitive to low amounts of TLR agonists. Using wild type and TLR2-deficient cells in Treg suppression assays, we accordingly show that all cells (e.g. Treg, Teff cells and DCs) contributed to overcome Treg-mediated suppression of Teff cell proliferation. Furthermore, while TLR2-stimulated Tregs readily lost their ability to suppress Teff cell proliferation, cytokine production by Teff cells was still suppressed. Similar results were obtained upon stimulation with TLR2 ligand containing bacteria, Legionella pneumophila. CONCLUSIONS: These findings indicate that both synthetic and natural TLR2 agonists affect DCs, Teff cells and Treg directly, resulting in multi-modal modulation of Treg-mediated suppression of Teff cells. Moreover, Treg-mediated suppression of Teff cell proliferation is functionally distinct from suppression of cytokine secretion.

Contains fulltext : 98097.pdf (publisher's version ) (Open Access), BACKGROUND: MicroRNAs (miRNAs) play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. RESULTS: To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs). Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs) based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. CONCLUSIONS: Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

Contains fulltext : 96310.pdf (publisher's version ) (Closed access), PURPOSE: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine. EXPERIMENTAL DESIGN: HLA-A2.1(+) melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 x 10 to 17 x 10 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with (51)Cr release assays or IFNgamma release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells. RESULTS: In 19 of 43 vaccinated patients, functional tumor antigen-specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen-specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies. CONCLUSION: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown.

Contains fulltext : 96778.pdf (publisher's version ) (Open Access), BACKGROUND: Dendritic cells (DCs) are the highly specialized antigen presenting cells of the immune system that play a key role in regulating immune responses. DCs can efficiently initiate immune responses or induce tolerance. Due to this dual function, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. Characterization of DC-specific genes, leading to better understanding of DC immunobiology, will help to guide their use in clinical settings. We previously identified DC-STAMP, a multi-membrane spanning protein preferentially expressed by DCs. DC-STAMP resides in the endoplasmic reticulum (ER) of immature DCs and translocates towards the Golgi compartment upon maturation. In this study we knocked down DC-STAMP in mouse bone marrow-derived DCs (mBMDCs) to determine its function. RESULTS: We demonstrate that DC-STAMP knock-down mBMDCs secrete less IL-6, IL-12, TNF-alpha and IL-10 while IL-1 production is enhanced. Moreover, LPS-matured DC-STAMP knock-down mBMDCs show impaired T cell activation potential and induction of Th1 responses in an alloreaction. CONCLUSIONS: We show that DC-STAMP plays an important role in cytokine production by mBMDCs following LPS exposure. Our results reveal a novel function of DC-STAMP in regulating DC-initiated immune responses.

Contains fulltext : 97777.pdf (publisher's version ) (Closed access), DEC-205 is a type I C-type lectin receptor (CLR) that is expressed on various APC subsets and has been suggested to bind necrotic and apoptotic cells. Here we study DEC-205 characteristics in plasmacytoid DCs (pDCs) obtained from healthy individuals and assess its ability to mediate antigen presentation by isolating sufficient numbers of pDCs from apheresis material obtained from stage III/IV melanoma patients. The results demonstrate that DEC-205 is expressed on human pDCs. Internalization of DEC-205 after antibody ligation is clathrin- and dynamin-dependent as it is blocked by hypertonic shock or by inhibition of dynamin activity. Antibody targeting to DEC-205 does not affect TLR-induced expression levels of co-stimulatory and MHC molecules, but clearly impairs TLR-induced IFN-alpha secretion by 40%. We observed that TLR-mediated signaling increases DEC-205 expression levels without affecting receptor internalization. Moreover, human pDCs retained the capacity to present antigens via DEC-205 following TLR activation.

Contains fulltext : 96386.pdf (publisher's version ) (Open Access), After infection with Borrelia species, the risk for developing Lyme disease varies significantly between individuals. Recognition of Borrelia by the immune system is mediated by pattern recognition receptors (PRRs), such as TLRs. While TLR2 is the main recognition receptor for Borrelia spp., little is known about the role of TLR1 and TLR6, which both can form functionally active heterodimers with TLR2. Here we investigated the recognition of Borrelia by both murine and human TLR1 and TLR6. Peritoneal macrophages from TLR1- and TLR6- gene deficient mice were isolated and exposed to Borrelia. Human PBMCs were stimulated with Borrelia with or without specific TLR1 and TLR6 blocking using specific antibodies. Finally, the functional consequences of TLR polymorphisms on Borrelia-induced cytokine production were assessed. Splenocytes isolated from both TLR1-/- and TLR6-/- mice displayed a distorted Th1/Th2 cytokine balance after stimulation with B.burgdorferi, while no differences in pro-inflammatory cytokine production were observed. In contrast, blockade of TLR1 with specific neutralizing antibodies led to decreased cytokine production by human PBMCs after exposure to B.burgdorferi. Blockade of human TLR6 did not lead to suppression of cytokine production. When PBMCs from healthy individuals bearing polymorphisms in TLR1 were exposed to B.burgdorferi, a remarkably decreased in vitro cytokine production was observed in comparison to wild-type controls. TLR6 polymorphisms lead to a minor modified cytokine production. This study indicates a dominant role for TLR1/TLR2 heterodimers in the induction of the early inflammatory response by Borrelia spirochetes in humans.

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.

Radboud Universiteit Nijmegen, 6 december 2010, Promotor : Punt, C.J.A. Co-promotores : Adema, G.J., Vries, I.J.M. de, Contains fulltext : 83250.pdf (publisher's version ) (Open Access)

Contains fulltext : 88281.pdf (publisher's version ) (Closed access), PURPOSE: The success of cancer immunotherapy depends on the balance between effector T cells and suppressive immune regulatory mechanisms within the tumor microenvironment. In this study we investigated whether transient monoclonal antibody-mediated depletion of CD25(high) regulatory T cells (Treg) is capable of enhancing the immunostimulatory efficacy of dendritic cell vaccines. EXPERIMENTAL DESIGN: Thirty HLA-A2.1(+) metastatic melanoma patients were vaccinated with mature dendritic cells pulsed with tumor peptide and keyhole limpet hemocyanin (KLH). Half of the patients were pretreated with daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor alpha-chain (CD25), either four or eight days before dendritic cell vaccinations. Clinical and immunologic parameters were determined. RESULTS: Daclizumab efficiently depleted all CD25(high) immune cells, including CD4(+)FoxP3(+)CD25(high) cells, from the peripheral blood within four days of administration. Thirty days after administration, daclizumab was cleared from the circulation and all CD25(+) cells reappeared. The presence of daclizumab during dendritic cell vaccinations prevented the induction of specific antibodies in vivo but not the presence of antigen-specific T cells. Daclizumab, however, did prevent these CD25(+) T cells from acquiring effector functions. Consequently, significantly less patients pretreated with daclizumab developed functional, vaccine-specific effector T cells and antibodies compared with controls. Daclizumab pretreatment had no significant effect on progression-free survival compared with the control group. CONCLUSIONS: Although daclizumab depleted the CD4(+)FoxP3(+)CD25(high) Tregs from the peripheral circulation, it did not enhance the efficacy of the dendritic cell vaccine. Residual daclizumab functionally suppressed de novo induced CD25(+) effector cells during dendritic cell vaccinations. Our results indicate that for immunotherapeutic benefit of transient Treg depl

Contains fulltext : 88009.pdf (publisher's version ) (Closed access), BACKGROUND: Nuclear receptors, including estrogen receptor (ER), progesterone receptor (PR)-B, peroxisome proliferator-activated receptor gamma, and retinoic acid receptor alpha, have been implicated in breast cancer etiology and progression. We investigated the role of dendritic cell-specific transcript (DC-SCRIPT) as coregulator of these nuclear receptors and as a prognostic factor in breast cancer. METHODS: The effect of DC-SCRIPT on the transcriptional activity of nuclear receptors was assessed by luciferase reporter assays. DC-SCRIPT expression in normal and tumor tissue from breast cancer patients was analyzed by polymerase chain reaction and immunohistochemistry. The prognostic value of tumor DC-SCRIPT mRNA expression was assessed in three independent cohorts of breast cancer patients: a discovery group (n = 47) and a validation group (n = 97) (neither of which had received systemic adjuvant therapy) and in a tamoxifen-treated validation group (n = 68) by using a DC-SCRIPT to porphobilinogen deaminase transcript ratio cutoff of 0.15 determined in the discovery group. Univariate and multivariable Cox proportional hazards model analyses were performed. All statistical tests were two-sided. RESULTS: DC-SCRIPT suppressed ER- and PR-mediated transcription in a ligand-dependent fashion, whereas it enhanced the retinoic acid receptor alpha- and peroxisome proliferator-activated receptor gamma-mediated transcription. In breast tissue samples from nine patients, DC-SCRIPT mRNA was expressed at lower levels in the tumor than in the corresponding normal tissue (P = .010). Patients in the discovery group with high tumor DC-SCRIPT mRNA levels (66%) had a longer disease-free interval than those with a low DC-SCRIPT mRNA level (34%) (hazard ratio [HR] of recurrence for high vs low DC-SCRIPT level = 0.23, 95% confidence interval [CI] = 0.06 to 0.93, P = .039), which was confirmed in the validation group (HR of recurrence = 0.50, 95% CI = 0.26 to 0.95, P = .034). This prognos

01 april 2010, Contains fulltext : 88553.pdf (publisher's version ) (Closed access), Stem cells are widely studied to enable their use in tissue repair. However, differences in function and differentiation potential exist between distinct stem cell populations. Whether those differences are due to donor variation, cell culture, or intrinsic properties remains elusive. Therefore, we compared 3 cell lines isolated from 3 different niches using the Affymetrix Exon Array platform: the cord blood-derived neonatal unrestricted somatic stem cell (USSC), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and adult adipose tissue-derived stem cells (AdAS). While donor variation was minimal, large differences between stem cells of different origin were detected. BM-MSC and AdAS, outwardly similar, are more closely related to each other than to USSC. Interestingly, USSC expressed genes involved in the cell cycle and in neurogenesis, consistent with their reported neuronal differentiation capacity. The BM-MSC signature indicates that they are primed toward developmental processes of tissues and organs derived from the mesoderm and endoderm. Remarkably, AdAS appear to be highly enriched in immune-related genes. Together, the data suggest that the different mesenchymal stem cell types have distinct gene expression profiles, reflecting their origin and differentiation potential. Furthermore, these differences indicate a demand for effective differentiation protocols tailored to each stem cell type.

Contains fulltext : 89613.pdf (publisher's version ) (Closed access), Regulatory T cells (Tregs) accumulate in tumors and can contribute to the dismal immune responses observed in these tumors. We reported that the percentage of tumor infiltrating Tregs is strongly correlated with the WHO grade of the brain tumor. We now report on the clinical follow-up of this patient cohort (n=83). Subgroup analyses in patients with glioblastomas (n=29) showed a moderate, not significant, inverted association between Tregs and survival. We further show that Tregs in glioblastomas, in contrast to other tumor infiltrating effector lymphocytes, highly express the CCR4 chemokine receptor. Moreover, the CCR4 ligand CCL22 is secreted by glioblastomas and may explain the specific Treg accumulation seen in these tumors.

1 december 2010, Item does not contain fulltext, BACKGROUND: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have demonstrated that vaccination of autologous ex vivo cultured DCs results in the induction of tumor-specific immune responses in cancer patients, which correlates with clinical response. Optimization of antigen loading is one of the possibilities for further improving the efficacy of DC vaccination. Theoretically, transfection of DCs with RNA encoding a tumor-specific antigen may induce a broader immune response as compared to the most widely used technique of peptide pulsing. PATIENTS AND METHODS: In this clinical study, RNA transfection was compared with peptide pulsing as an antigen loading strategy for DC vaccination. Patients with resectable liver metastases of colorectal cancer were vaccinated intravenously and intradermally 3 times weekly with either carcinoembryogenic antigen (CEA)-derived HLA-A2 binding peptide-loaded or CEA mRNA electroporated DCs prior to surgical resection of the metastases. All DCs were loaded with keyhole limpet hemocyanin (KLH) as a control protein. Evaluation of vaccine-induced immune reactivity consisted of T-cell proliferative responses and B-cell antibody responses against KLH in peripheral blood. CEA reactivity was determined in T-cell cultures of biopsies of post-treatment delayed type hypersensitivity skin tests. RESULTS: Sixteen patients were included. All patients showed T-cell responses against KLH upon vaccination. CEA peptide-specific T-cells were detected in 8 out of 11 patients in the peptide group, but in none of the 5 patients in the RNA group. CONCLUSION: In our study, DC CEA mRNA transfection was not superior to DC CEA peptide pulsing in the induction of a tumor-specific immune response in colorectal cancer patients.

Contains fulltext : 87723.pdf (publisher's version ) (Open Access), The etiology of the autoimmune disease systemic lupus erythematosus is not known, but aberrant apoptosis and/or insufficient clearance of apoptotic material have been assigned a pivotal role. During apoptosis, nucleosomes and several endogenous danger-associated molecular patterns are incorporated in blebs. Recent data indicate that apoptotic blebs induce maturation of myeloid dendritic cells, resulting in IL-17 production by T cells. In this review we summarize current knowledge on the role of dendritic cells in the pathogenesis of systemic lupus erythematosus with special emphasis on the uptake of apoptotic blebs by dendritic cells, and the subsequent induction of Th17 cells.

01 oktober 2010, Contains fulltext : 87675.pdf (publisher's version ) (Open Access), Dendritic cells (DCs) are central players of the immune response. To date, DC-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, predominantly with ex vivo-cultured monocyte-derived DCs (moDCs). However, the extensive culture period and compounds required to differentiate them into DCs may negatively affect their immunological potential. Therefore, it is attractive to consider alternative DC sources, such as blood DCs. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions. Besides scavenging pathogens and presenting antigens, DCs secrete cytokines, all of which is vital for both the acquired and the innate immune system. These immunological functions relate to Toll-like receptors (TLRs) expressed by DCs. TLRs recognize pathogen-derived products and subsequently provoke DC maturation, antigen presentation and cytokine secretion. However, not every TLR is expressed on each DC subset nor causes the same effects when activated. Considering the large amount of clinical trials using DC-based immunotherapy for cancer patients and the decisive role of TLRs in DC maturation, this review summarizes TLR expression in different DC subsets in relation to their function. Emphasis will be given to the therapeutic potential of TLR-matured DC subsets for DC-based immunotherapy.

1 juli 2010, Contains fulltext : 88017.pdf (publisher's version ) (Closed access), Dendritic cells (DCs) are the professional antigen-presenting cells (APC) which efficiently prime the immune response or induce tolerance. We recently identified Dendritic Cell Specific TrAnsMembrane Protein (DC-STAMP), a novel 470 amino acid protein preferentially expressed by dendritic cells. Previously we demonstrated that DC-STAMP re-localizes towards the Golgi upon DC maturation. To identify proteins that interact with DC-STAMP, a yeast-2-hybrid analysis was performed. Here, we report a physically interacting partner of DC-STAMP in the endoplasmic reticulum (ER), called LUMAN (also known as CREB3 or LZIP). LUMAN was previously described as an ER-resident transcription factor with unknown function. It is activated in a process called regulated intramembrane proteolysis (RIP), which involves translocation to the Golgi and subsequent proteolytic cleavage. The proteolytically activated form of the protein then translocates to the nucleus. Our data indicate that DC-STAMP plays an important role in the modulation of LUMAN activation. Moreover, we demonstrate that LUMAN is endogenously expressed by DC and becomes activated by RIP upon DC maturation induced by various different stimuli. These data define LUMAN/DC-STAMP as a novel regulatory circuit in DC.

1 februari 2010, Contains fulltext : 87686.pdf (publisher's version ) (Closed access), Previously, we demonstrated that several TLRs are expressed on cord blood-derived USSC. Stimulation of USSC with TLR agonists resulted in a marked increase of IL-6 and IL-8 production. Interestingly, TNF was undetectable after TLR stimulation, which appeared to be a result of an inactivated TNF promoter in USSC. Here, we elaborate this study by demonstrating that although USSC do not produce TNF, they are susceptible to TNF stimulation, resulting in NF-kappaB translocation and cytokine production. Additionally, we compared different stem cell sources for their ability to produce TNF. Interestingly, we found that the TNF promoter in BM-MSC is inactivated as well. Like USSC, they are able to respond to TNF stimulation, but they are not able to produce TNF, even not after LPS stimulation. This limited cytokine response in combination with the well-studied immunosuppressive properties of MSC makes these cells ideal for immune-suppressive treatment modalities such as graft-versus-host disease.

1 juli 2010, Contains fulltext : 87311.pdf (publisher's version ) (Closed access), The receptor for IgA, FcalphaRI or CD89, is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. These functions critically depend on the associated FcR gamma-chain; however, some biological functions, like receptor internalization, are solely mediated by FcalphaRI alpha-chain. Little is known as to how FcalphaRI regulates these processes and the FcalphaRI intracellular domain does not contain recognized signalling motifs. We searched for associating proteins and identified c-Jun activating binding protein 1 (JAB1) as a binding partner specifically for FcalphaRI. We found increased FcalphaRI surface expression after ectopic expression of JAB1 as well as diminished protein levels of total FcR gamma-chain levels after JAB1 knock-down. These data functionally link JAB1 with controlling protein expression levels of FcalphaRI-FcR gamma-chain protein complex.

RU Radboud Universiteit Nijmegen, 23 juni 2010, Promotores : Galama, J.M.D., Adema, G.J. Co-promotor : Kuppeveld, F.J.M. van, Contains fulltext : 77574.pdf (publisher's version ) (Open Access), Type 1 diabetes mellitus (T1D, insulin-dependent diabetes mellitus) is an endocrine autoimmune disorder in which the insulin-producing beta-cells in the pancreas are gradually destroyed. Enterovirus infections (in particular coxsackievirus and echovirus) have been implicated in the development of T1D. We set out to investigate the interplay between enteroviruses, beta-cells and dendritic cells (DCs) which are the most important antigen-presenting cells. We demonstrated that human DCs can be productively infected with echoviruses (EV) but not coxsackie B viruses (CVB). Productive EV infection results in impaired DC function and causes loss of DC viability. In addition, we showed that primary CVB-infected human pancreatic islets (and also CVB-infected Min6 cells, a mouse insuloma cell line resembling pancreatic beta cells) are efficiently phagocytosed by DC. Uptake of these infected cells resulted in DC activation as indicated by the production of interferon (IFN)-alpha/beta, cytokines with strong immunomodulatory and antiviral functions, and the expression of interferon-stimulated genes. The observed innate antiviral responses depended on RNA within the phagocytosed cells, required endosomal acidification and were type I interferon (IFN)-dependent. We propose a model in which enteroviruses may trigger or accelerate T1D upon infection of pancreatic beta-cells. These infected cells can be engulfed by DCs, which subsequently become activated and thereby may mediate autoimmune reactions, possibly resulting in T1D.

01 juni 2010, Contains fulltext : 87791.pdf (publisher's version ) (Open Access), Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.

1 februari 2010, 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.

1 mei 2010, Contains fulltext : 89763.pdf (publisher's version ) (Closed access), OBJECTIVE: Type 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 METHODS: In 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). RESULTS: In 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. CONCLUSIONS: Human 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.

Contains fulltext : 87604.pdf (publisher's version ) (Closed access), BACKGROUND: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses in cancer patients, but so far its clinical efficacy is limited. Recent evidence supports an immunogenic effect of cytotoxic chemotherapy. Pre-clinical data indicate that the combination of chemotherapy and immunotherapy may result in an enhanced anti-cancer activity. Most studies have focused on the immunogenic aspect of chemotherapy-induced cell death, but only few studies have investigated the effect of chemotherapeutic agents on the effector lymphocytes of the immune system. METHODS: Here we investigated the effect of treatment with oxaliplatin and capecitabine on non-specific and specific DC vaccine-induced adaptive immune responses. Stage III colon cancer patients receiving standard adjuvant oxaliplatin/capecitabine chemotherapy were vaccinated at the same time with keyhole limpet haemocyanin (KLH) and carcinoembryonic antigen (CEA)-peptide pulsed DCs. RESULTS: In 4 out of 7 patients, functional CEA-specific T-cell responses were found at delayed type hypersensitivity (DTH) skin testing. In addition, we observed an enhanced non-specific T-cell reactivity upon oxaliplatin administration. KLH-specific T-cell responses remained unaffected by the chemotherapy, whereas B-cell responses were diminished. CONCLUSION: The results strongly support further testing of the combined use of specific anti-tumour vaccination with oxaliplatin-based chemotherapy.

1 juni 2010, Contains fulltext : 88086.pdf (publisher's version ) (Closed access), DC are professional APCs that initiate and regulate adaptive immune responses by interacting with naive and memory T cells. Chemokines released by DC play an essential role in T cell recruitment and in the maintenance of antigen-specific T cell-DC conjugates. Here, we characterized the expression of the T cell-attracting chemokine CXCL16 by murine DC. We demonstrate that through alternative RNA splicing, DC not only express the previously characterized transmembrane CXCL16 isoform, which can be cleaved from the cell surface, but also a novel isoform lacking the transmembrane and cytoplasmic domains. Transfection of HEK293 cells shows that this novel isoform, termed CXCL16v, is not expressed on the cell membrane but is secreted as a protein of approximately 10 kDa. Quantitative PCR demonstrates that CXCL16v is broadly expressed in lymphoid and nonlymphoid tissues resembling the tissue distribution of DC. Indeed, CXCL16v mRNA is expressed significantly by spleen DC and BM-DC. Moreover, we show that mature DC have increased CXCL16v mRNA levels and express transmembrane and soluble CXCL16 proteins. Finally, we show that CXCL16v specifically attracts cells expressing the chemokine receptor CXCR6. Our data demonstrate that mature DC express secreted, transmembrane, and cleaved CXCL16 isoforms to recruit and communicate efficiently with CXCR6(+) lymphoid cells.

RU Radboud Universiteit Nijmegen, 25 juni 2009, Promotores : Hoogerbrugge, P.M., Adema, G.J. Co-promotor : Vries, I.J.M. de, Contains fulltext : 74418.pdf (publisher's version ) (Open Access)

RU Radboud Universiteit Nijmegen, 23 april 2009, Promotor : Adema, G.J. Co-promotores : Nierkens, S., Sutmuller, R.P.M., Contains fulltext : 80368.pdf (Publisher’s version ) (Open Access)