Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. Proper function of DCs is crucial to elicit an effective immune response against pathogens and to induce ...antitumor immunity. Different members of the nuclear receptor (NR) family of transcription factors have been reported to affect proper function of immune cells. Nur77 is a member of the NR4A subfamily of orphan NRs that is expressed and has a function within the immune system. We now show that Nur77 is expressed in different murine DCs subsets
and
, in human monocyte-derived DCs (moDCs) and in freshly isolated human BDCA1
DCs, but its expression is dispensable for DC development in the spleen and lymph nodes. We show, by siRNA-mediated knockdown of Nur77 in human moDCs and by using Nur77
murine DCs, that Nur77-deficient DCs have enhanced inflammatory responses leading to increased T cell proliferation. Treatment of human moDCs with 6-mercaptopurine, an activator of Nur77, leads to diminished DC activation resulting in an impaired capacity to induce IFNγ production by allogeneic T cells. Altogether, our data show a yet unexplored role for Nur77 in modifying the activation status of murine and human DCs. Ultimately, targeting Nur77 may prove to be efficacious in boosting or diminishing the activation status of DCs and may lead to the development of improved DC-based immunotherapies in, respectively, cancer treatment or treatment of autoimmune diseases.
Dendritic cells (DCs) play a central role in the immune system; they can induce immunity or tolerance depending on diverse factors in the DC environment. Pathogens, but also tissue damage, hormones, ...and vitamins, affect DC activation and maturation. In particular, glucocorticoids (GCs) are known for their immunosuppressive effect on DCs, creating tolerogenic DCs. GCs activate the type I nuclear receptor (NR) glucocorticoid receptor (GR), followed by induced expression of the transcription factor glucocorticoid-inducible leucine zipper (GILZ). GILZ has been shown to be necessary and sufficient for GC-induced tolerogenic DC generation. Recently, we have identified the DC-specific transcript (DC-SCRIPT) as an NR coregulator, suppressing type I steroid NRs estrogen receptor and progesterone receptor. In this study, we analyzed the effect of DC-SCRIPT on GR activity. We demonstrate that DC-SCRIPT coexists with GR in protein complexes and functions as a corepressor of GR-mediated transcription. Coexpression of DC-SCRIPT and GR is shown in human monocyte-derived DCs, and DC-SCRIPT knockdown enhances GR-dependent upregulation of GILZ mRNA expression in DCs. This demonstrates that DC-SCRIPT serves an important role in regulating GR function in DCs, corepressing GR-dependent upregulation of the tolerance-inducing transcription factor GILZ. These data imply that by controlling GR function and GILZ expression DC-SCRIPT is potentially involved in the balance between tolerance and immunity.
To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a ...tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in
(SCCNij185),
(SCCNij185), and
(UT-SCC-5 and UT-SCC-38).
mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and
mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.
Dendritic cell (DC)-based immunotherapy makes use of the DC's ability to direct the adaptive immune response toward activation or inhibition. DCs perform this immune orchestration in part by ...secretion of selected cytokines. The most potent anti-inflammatory cytokine interleukin-10 (IL-10) is under tight regulation, as it needs to be predominantly expressed during the resolution phase of the immune response. Currently it is not clear whether there is active suppression of IL-10 by DCs at the initial pro-inflammatory stage of the immune response. Previously, knockdown of the DC-specific transcription factor DC-SCRIPT has been demonstrated to mediate an extensive increase in IL-10 production upon encounter with pro-inflammatory immune stimuli. Here, we explored how DC-SCRIPT contributes to IL-10 suppression under pro-inflammatory conditions by applying chromatin immunoprecipitation sequencing analysis of DC-SCRIPT and the epigenetic marks H3K4me3 and H3K27ac in human DCs. The data showed binding of DC-SCRIPT to a GA-rich motif at H3K27ac-marked genomic enhancers that associated with genes encoding MAPK dual-specificity phosphatases (DUSPs). Functional studies revealed that upon knockdown of DC-SCRIPT, human DCs express much less DUSP4 and exhibit increased phosphorylation of the three major MAPKs (ERK, JNK, and p38). Enhanced ERK signaling in DC-SCRIPT-knockdown-DCs led to higher production of IL-10, which was reverted by rescuing DUSP4 expression. Finally, DC-SCRIPT-knockdown-DCs induced less IFN-γ and increased IL-10 production in naïve T cells, indicative for a more anti-inflammatory phenotype. In conclusion, we have delineated a new mechanism by which DC-SCRIPT allows DCs to limit IL-10 production under inflammatory conditions and potentiate pro-inflammatory Th1 responses. These insights may be exploited to improve DC-based immunotherapies.
Dendritic cells (DCs) are the professional APCs of the immune system that dictate the type and course of an immune response. Molecular understanding of DC biology is important for the design of ...DC-based immunotherapies and optimal clinical applications in vaccination settings. Previously, we isolated and characterized the cDNA-encoding dendritic cell-specific transcript (DC-SCRIPT; also known as ZNF366). DC-SCRIPT mRNA expression in the immune system was confined to DCs and was reported to be an early hallmark of DC differentiation. In this study, we demonstrate IL-4 to be the dominant factor for DC-SCRIPT expression in human monocyte-derived DCs. In addition, to our knowledge, we show for the first time endogenous DC-SCRIPT protein expression in human DCs both in vitro and in situ. DC-SCRIPT protein is detected early upon differentiation of monocytes into DCs and is also present in multiple freshly isolated DC subsets. Maturation of DCs with TLR ligands further increased DC-SCRIPT mRNA expression, suggesting a role in DC maturation. Indeed, small interfering RNA-mediated knockdown of DC-SCRIPT affected the cytokine response upon TLR stimulation. These DCs displayed enhanced IL-10 and decreased IL-12 production, compared with wild-type DCs. Silencing of IL-10 in DC-SCRIPT knockdown DCs rescued IL-12 expression, suggesting a primary role for DC-SCRIPT in the regulation of IL-10 production.
BackgroundTumor ablation techniques, like cryoablation, are successfully used in the clinic to treat tumors. The tumor debris remaining in situ after ablation is a major antigen depot, including ...neoantigens, which are presented by dendritic cells (DCs) in the draining lymph nodes to induce tumor-specific CD8+ T cells. We have previously shown that co-administration of adjuvants is essential to evoke strong in vivo antitumor immunity and the induction of long-term memory. However, which adjuvants most effectively combine with in situ tumor ablation remains unclear.Methods and resultsHere, we show that simultaneous administration of cytidyl guanosyl (CpG) with saponin-based adjuvants following cryoablation affects multifunctional T-cell numbers and interleukin (IL)-1 induced polymorphonuclear neutrophil recruitment in the tumor draining lymph nodes, relative to either adjuvant alone. The combination of CpG and saponin-based adjuvants induces potent DC maturation (mainly CpG-mediated), antigen cross-presentation (mainly saponin-based adjuvant mediated), while excretion of IL-1β by DCs in vitro depends on the presence of both adjuvants. Most strikingly, CpG/saponin-based adjuvant exposed DCs potentiate antigen-specific T-cell proliferation resulting in multipotent T cells with increased capacity to produce interferon (IFN)γ, IL-2 and tumor necrosis factor-α in vitro. Also in vivo the CpG/saponin-based adjuvant combination plus cryoablation increased the numbers of tumor-specific CD8+ T cells showing enhanced IFNγ production as compared with single adjuvant treatments.ConclusionsCollectively, these data indicate that co-injection of CpG with saponin-based adjuvants after cryoablation induces an increased amount of tumor-specific multifunctional T cells. The combination of saponin-based adjuvants with toll-like receptor 9 adjuvant CpG in a cryoablative setting therefore represents a promising in situ vaccination strategy.
•Nuclear receptor mRNA was analyzed in plasmacytoid and conventional dendritic cells.•pDC and cDC express the same NR repertoire in vitro and in vivo.•DC Maturation affects NR expression in a subset ...specific manner.•NR mRNA levels in pDCs and cDCs vary between lymphoid organs.•Our data indicate that hormones and vitamins differentially affect pDCs and cDCs.
Dendritic cells (DC) play a central role in the immune system. They can either induce immunity or promote tolerance. The DC family is generally comprised of two functionally distinct DC subsets. Conventional dendritic cells (cDC) are the classical antigen presenting cells; plasmacytoid dendritic cells (pDC) are the main producers of type I interferons thereby serving innate immunity. Upon activation DCs are able to present antigen and stimulate T cells. The immune modulatory functions of DCs largely depend on the recognition of soluble cues. Besides pathogen derived cues, recent data indicate that the tissue micro-environment, i.e. of the gut and skin affects cDC function. Many of these micro-environmental factors are ligands for the nuclear receptor (NR) family of transcription regulators known to affect immunity and tolerance. Whether pDC function is also influenced by tissue derived cues, like hormones, vitamins and metabolic products, is largely unknown. Here, we investigated the NR expression profile of murine pDCs and cDCs. We assessed the mRNA levels of 19 NRs of in vitro derived as well as ex vivo isolated DCs from four different lymphoid tissues. We observed that cDCs and pDCs expressed the same repertoire of NRs. Expression levels, however, differed between the two subsets, especially upon maturation of DCs. These data imply that NR ligands do impact pDC function and that their activity might be regulated in a DC-specific manner.
•Myeloid cells are crucial for cGAS/STING-mediated antitumor immunity.•Signal mediators dsDNA and cGAMP are propagated from tumor to immune cells in the TME.•Multiple intercellular cGAS/STING ...propagation routes exist.•Signal propagation routes can be distinguished based on proximity of cells.•Insight into intercellular cGAS/STING signaling provides novel therapeutic targets.
Cyclic GMP-AMP synthase (cGAS), second messenger 2′3′-cyclic GMP-AMP (cGAMP) and stimulator of interferon genes (STING) are fundamental for sensing cytoplasmic double stranded DNA. Radiotherapy treatment induces large amounts of nuclear and mitochondrial DNA damage and results in the presence of DNA fragments in the cytoplasm, activating the cGAS/STING pathway. Triggering of the cGAS/STING pathway in the tumor microenvironment (TME) results in the production of type I interferons (IFNs). Type I IFNs are crucial for an effective antitumor defense, with myeloid cells as key players. Many questions remain on how these myeloid cells are activated and in which cells (tumor versus myeloid) in the TME the signaling pathway is initiated. The significance of cGAS/STING signaling in the onco-immunology field is being recognized, emphasized by the frequent occurrence of mutations in or silencing of genes in this pathway. We here review several mechanisms of cGAS/STING signal propagation in the TME, focusing on tumor cells and myeloid cells. Cell-cell contact-dependent interactions facilitate the transfer of tumor-derived DNA and cGAMP. Alternatively, transport routes via the extracellular space such as extracellular vesicles, and channel-mediated cGAMP transfer to and from the extracellular space contribute to propagation of cGAS/STING signal mediators DNA and cGAMP. Finally, we discuss regulation of extracellular cGAMP. Altogether, we provide a comprehensive overview of cGAS/cGAMP/STING signal propagation from tumor to myeloid cells in the TME, revealing novel targets for combinatorial treatment approaches with conventional anticancer therapies like radiotherapy.
Dendritic cells (DCs) are widely used in DC-based immunotherapies because of their capacity to steer immune responses. So far treatment success is limited and more functional knowledge on how DCs ...initiate and stably drive specific responses is needed. Many intrinsic and extrinsic factors contribute to how DCs skew the immune response towards immunity or tolerance. The origin and type of DC, its maturation status, but also factors they encounter in the in vitro or in vivo microenvironment they reside in during differentiation and maturation affect this balance. Treatment success of DC vaccines will, therefore, also depend on the presence of these factors during the process of vaccination. Identification and further knowledge of natural and pharmacological compounds that modulate DC differentiation and function towards a specific response may help to improve current DC-based immunotherapies. This review focuses on factors that could improve the efficacy of DC vaccines in (pre-)clinical studies to enhance DC-based immunotherapy, with a particular emphasis on compounds acting on prostanoid or nuclear receptor families.
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.
We demonstrate that DC-STAMP knock-down mBMDCs secrete less IL-6, IL-12, TNF-α 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.
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.
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