RESUMO
Tumors create an immunosuppressive tumor microenvironment by altering protein expression, but also by changing their glycosylation status, like altered expression of sialoglycans. Sialoglycans are capped with sialic acid sugar residues and are recognized by Siglec immune receptors. Siglec-7 is an inhibitory immune receptor similar to PD-1, and is emerging as glycoimmune checkpoint exploited by cancer cells to evade the immune system. However, the exact cellular and molecular conditions required for Siglec-7-mediated immune cell inhibition remain largely unknown. Here, we report on the development of a chimeric Siglec-7 cell system that enables dissection of Siglec-7 signaling, rather than Siglec-7 binding. Antibody-induced clustering, sialic acid-containing polymers, and highly sialylated erythrocytes effectively induced Siglec-7 signaling, thereby validating functionality of this reporter system. Moreover, the system reveals tumor cell-dependent Siglec-7 signaling. Tumor-associated conditions important for Siglec-7 signaling were defined, such as Siglec-7 ligand expression levels, presence of the known Siglec-7 ligand CD43, and sialic acid availability for sialylation of glycans. Importantly, therapeutic targeting of the Siglec-7/sialic acid axis using a sialyltransferase inhibitor resulted in strong reduction of Siglec-7 signaling. In conclusion, using a newly established cellular tool, we defined a set of tumor-associated conditions that influence Siglec-7 signaling. Moreover, the system allows to assess the efficacy of novel cancer drugs interfering with the Siglec-7/sialic acid axis as immunotherapy to treat cancer.
Assuntos
Ácido N-Acetilneuramínico , Neoplasias , Humanos , Microambiente Tumoral , Ligantes , Neoplasias/terapia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
Loss of expression of the transcription regulator DC-SCRIPT (Zfp366) is a prominent prognostic event in estrogen receptor-positive breast cancer patients. Studying the inherent link between breast morphogenesis and tumorigenesis, we recently reported that DC-SCRIPT affects normal mammary branching morphogenesis and mammary epithelium homeostasis. Here we investigated the molecular mechanism involved in DC-SCRIPT mediated regulation of FGF2 induced mammary branching morphogenesis in a 3D organoid culture system. Our data show that the delayed mammary organoid branching observed in DC-SCRIPT-/- organoids cannot be compensated for by increasing FGF2 levels. Interestingly, FGFR1, the dominant FGF2 receptor, was expressed at a significantly lower level in basal epithelial cells of DC-SCRIPT deficient organoids relative to wildtype organoids. A potential link between DC-SCRIPT and FGFR1 was further supported by the predicted locations of the DC-SCRIPT DNA binding motif at the Fgfr1 gene. Moreover, ERK1/2 phosphorylation downstream of the FGFR1 pathway was decreased in basal epithelial cells of DC-SCRIPT deficient organoids. Altogether, this study shows a relationship between DC-SCRIPT and FGFR1 related pERK signaling in modulating the branching morphogenesis of mammary organoids in vitro.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glândulas Mamárias Animais/embriologia , Proteínas Nucleares/metabolismo , Organogênese , Organoides/embriologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Feminino , Sistema de Sinalização das MAP Quinases , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Organoides/citologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição/genéticaRESUMO
Mammary glands are unique organs in which major adaptive changes occur in morphogenesis and development after birth. Breast cancer is the most common cancer and a major cause of mortality in females worldwide. We have previously identified the loss of expression of the transcription regulator DC-SCRIPT (Zfp366) as a prominent prognostic event in estrogen receptor positive breast cancer patients. DC-SCRIPT affects multiple transcriptional events in breast cancer cells, including estrogen and progesterone receptor-mediated transcription, and promotes CDKN2B-related cell cycle arrest. As loss of DC-SCRIPT expression appears an early event in breast cancer development, we here investigated the role of DC-SCRIPT in mammary gland development using wild-type and DC-SCRIPT knockout mice. Mice lacking DC-SCRIPT exhibited severe breeding problems and showed significant growth delay relative to littermate wild-type mice. Subsequent analysis revealed that DC-SCRIPT was expressed in mouse mammary epithelium and that DC-SCRIPT deficiency delayed mammary gland morphogenesis in vivo. Finally, analysis of 3D mammary gland organoid cultures confirmed that loss of DC-SCRIPT dramatically delayed mammary organoid branching in vitro. The study shows for the first time that DC-SCRIPT deficiency delays mammary gland morphogenesis in vivo and in vitro. These data define DC-SCRIPT as a novel modulator of mammary gland development.
Assuntos
Proteínas de Ligação a DNA/genética , Glândulas Mamárias Animais/metabolismo , Morfogênese/genética , Proteínas Nucleares/genética , Organoides/metabolismo , Fatores de Transcrição/genética , Animais , Técnicas de Cultura de Células/métodos , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ligação a DNA/deficiência , Células Epiteliais/metabolismo , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Organoides/citologia , Organoides/crescimento & desenvolvimento , Fatores de Transcrição/deficiênciaRESUMO
The balance between tolerance and immunity is important for the outcome of an infection or cancer, and dendritic cells (DCs) are key regulators of this balance. DC-specific transcript (DC-SCRIPT) is a protein expressed by DCs and has been demonstrated to suppress both TLR-mediated expression of IL-10 and glucocorticoid receptor-mediated transcription of glucocorticoid-induced leucine zipper (GILZ). Because GILZ is known to promote IL-10 production, we investigated whether these two processes are linked. Dual-knockdown and inhibition experiments demonstrated that neither GILZ nor glucocorticoid receptor play a role in TLR-induced IL-10 production after DC-SCRIPT knockdown. The NF-κB pathway is another route involved in IL-10 production after DC activation. Strikingly, inhibition of NF-κB led to a decreased TLR-mediated IL-10 production in DC-SCRIPT knockdown DCs. Moreover, DC-SCRIPT knockdown DCs showed enhanced phosphorylation, acetylation, and IL10 enhancer binding of the NF-κB subunit p65. These data demonstrate that besides nuclear receptor regulation, DC-SCRIPT also modulates activation of NF-κBp65 after TLR activation in human DCs.
Assuntos
Proteínas de Transporte/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interleucina-10/biossíntese , Fator de Transcrição RelA/metabolismo , Proteínas de Transporte/genética , Elementos Facilitadores Genéticos , Ativação Enzimática , Técnicas de Inativação de Genes , Humanos , Interleucina-10/genética , Fosforilação , Ligação Proteica , Interferência de RNA , Receptores de Glucocorticoides/metabolismo , Receptores Toll-Like/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Breast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERα) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERα and as a strong and independent prognostic marker in ESR1 (ERα gene)-positive breast cancer patients. In this study, we further investigated the molecular mechanism on how DC-SCRIPT inhibits breast cancer cell growth. DC-SCRIPT mRNA levels from 190 primary ESR1-positive breast tumors were related to global gene expression, followed by gene ontology and pathway analysis. The effect of DC-SCRIPT on breast cancer cell growth and cell cycle arrest was investigated using novel DC-SCRIPT-inducible MCF7 breast cancer cell lines. Genome-wide expression profiling of DC-SCRIPT-expressing MCF7 cells was performed to investigate the effect of DC-SCRIPT on cell cycle-related gene expression. Findings were validated by real-time PCR in a cohort of 1,132 ESR1-positive breast cancer patients. In the primary ESR1-positive breast tumors, DC-SCRIPT expression negatively correlated with several cell cycle gene ontologies and pathways. DC-SCRIPT expression strongly reduced breast cancer cell growth in vitro, breast tumor growth in vivo, and induced cell cycle arrest. In addition, in the presence of DC-SCRIPT, multiple cell cycles related genes were differentially expressed including the tumor suppressor gene CDKN2B. Moreover, in 1,132 primary ESR1-positive breast tumors, DC-SCRIPT expression also correlated with CDKN2B expression. Collectively, these data show that DC-SCRIPT acts as a novel regulator of CDKN2B and induces cell cycle arrest in ESR1-positive breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Proteínas de Transporte/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Receptor alfa de Estrogênio/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Células MCF-7 , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/biossínteseRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Zíper de Leucina/genética , Receptores de Glucocorticoides/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Técnicas de Silenciamento de Genes , Humanos , Tolerância Imunológica/genética , Imunoprecipitação , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de RNA , Receptores de Glucocorticoides/genética , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas de Transporte/fisiologia , Citocinas/biossíntese , Células Dendríticas/imunologia , Receptores Toll-Like/fisiologia , Biomarcadores/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Células Cultivadas , Citocinas/genética , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-10/biossíntese , Interleucina-4/fisiologia , Simulação de Dinâmica Molecular , Monócitos/imunologia , Monócitos/metabolismo , RNA Mensageiro/biossínteseRESUMO
BACKGROUND: Adjuvants are key for effective vaccination against cancer and chronic infectious diseases. Saponin-based adjuvants (SBAs) are unique among adjuvants in their ability to induce robust cell-mediated immune responses in addition to antibody responses. Recent preclinical studies revealed that SBAs induced cross-presentation and lipid bodies in otherwise poorly cross-presenting CD11b+ murine dendritic cells (DCs). METHOD: Here, we investigated the response of human DC subsets to SBAs with RNA sequencing and pathway analyses, lipid body induction visualized by laser scanning microscopy, antigen translocation to the cytosol, and antigen cross-presentation to CD8+ T cells. RESULTS: RNA sequencing of SBA-treated conventional type 1 DC (cDC1) and type 2 DC (cDC2) subsets uncovered that SBAs upregulated lipid-related pathways in CD11c+ CD1c+ cDC2s, especially in the CD5- CD163+ CD14+ cDC2 subset. Moreover, SBAs induced lipid bodies and enhanced endosomal antigen translocation into the cytosol in this particular cDC2 subset. Finally, SBAs enhanced cross-presentation only in cDC2s, which requires the CD163+ CD14+ cDC2 subset. CONCLUSIONS: These data thus identify the CD163+ CD14+ cDC2 subset as the main SBA-responsive DC subset in humans and imply new strategies to optimize the application of saponin-based adjuvants in a potent cancer vaccine.
Assuntos
Apresentação Cruzada , Saponinas , Humanos , Animais , Camundongos , Linfócitos T CD8-Positivos , Adjuvantes Imunológicos/farmacologia , Células Dendríticas , Saponinas/farmacologiaRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Transformação Celular Neoplásica/genética , Próstata/patologia , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Receptores de Calcitriol/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Masculino , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Receptores de Calcitriol/metabolismoRESUMO
Dendritic cell-specific transmembrane protein (DC-STAMP) has been first identified as an EST in a cDNA library of human monocyte-derived dendritic cells (DC). DC-STAMP is a multimembrane spanning protein that has been implicated in skewing haematopoietic differentiation of bone marrow cells towards the myeloid lineage, and in cell fusion during osteoclastogenesis and giant cell formation. To gain molecular insight in how DC-STAMP exerts its function, DC-STAMP interacting proteins were identified in a yeast-2-hybrid analysis. Herein, we report that amplified in osteosarcoma 9 (OS9) physically interacts with DC-STAMP, and that both proteins colocalize in the endoplasmic reticulum in various cell lines, including immature DC. OS9 has previously been implicated in ER-to-Golgi transport and transcription factor turnover. Interestingly, we now demonstrate that toll-like receptor (TLR)-induced maturation of DC leads to the translocation of DC-STAMP from the ER to the Golgi while OS9 localization is unaffected. Applying TLR-expressing CHO cells we could confirm ER-to-Golgi translocation of DC-STAMP following TLR stimulation and demonstrated that the DC-STAMP/OS9 interaction is involved in this process. Collectively, the data indicate that OS9 is critically involved in the modulation of ER-to-Golgi transport of DC-STAMP in response to TLR triggering, suggesting a novel role for OS9 in myeloid differentiation and cell fusion.
Assuntos
Células Dendríticas/metabolismo , Retículo Endoplasmático/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células CHO , Cricetinae , Cricetulus , Células Dendríticas/citologia , Células Dendríticas/imunologia , Retículo Endoplasmático/metabolismo , Humanos , Lectinas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
Tumor-derived peptides are used frequently as antigen (Ag) source in dendritic cell (DC) therapy in cancer patients. An alternative is to load DC with tumor-associated Ag (TAA)-encoding RNA. RNA-loading obviates prior knowledge of CTL and Th epitopes in the Ag. Multiple epitopes for many HLA alleles (both MHC class I and class II) are encoded by the RNA and loading is independent of the patient's HLA make-up. Herein, we determined the optimal conditions for mRNA-electroporation of monocyte-derived DC for clinical application in relation to different maturation cocktails. The data demonstrate that TAA carcinoembryonic antigen, gp100 and tyrosinase are expressed already 30 min after electroporation with the encoding mRNA. Moreover, gp100-specific CTL are activated by gp100 mRNA-electroporated DC. Importantly, we show here that the presence of polyinosinic-polycytidylic acid [poly(I:C)] in the maturation cocktail prevents effective protein expression of the electroporated mRNA as well as subsequent CTL recognition. This effect of poly(I:C) correlates with the induction of IFN-induced genes and innate anti-viral effector molecules in DC. Together these data show that electroporation of mature DC with TAA-encoding mRNA is attractive for use in DC vaccination protocols in cancer patients, but protein expression should be tested for each maturation cocktail.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Antígenos de Neoplasias/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Indutores de Interferon/farmacologia , Poli I-C/farmacologia , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Células Dendríticas/transplante , Eletroporação , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/imunologia , RNA Mensageiro/genética , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Antígeno gp100 de MelanomaRESUMO
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 in vitro and ex vivo, 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.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Animais , Células Cultivadas , Citocinas/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
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.
RESUMO
Dendritic cell-specific transcript (DC-SCRIPT) is a putative DC zinc (Zn) finger-type transcription factor described recently in humans. Here, we illustrate that DC-SCRIPT is highly conserved in evolution and report the initial characterization of the murine ortholog of DC-SCRIPT, which is also preferentially expressed in DC as shown by real-time quantitative polymerase chain reaction, and its distribution resembles that of its human counterpart. Studies undertaken in human embryonic kidney 293 cells depict its nuclear localization and reveal that the Zn finger domain of the protein is mainly responsible for nuclear import. The human and the mouse genes are located in syntenic chromosomal regions and exhibit a similar genomic organization with numerous common transcription factor-binding sites in their promoter region, including sites for many factors implicated in haematopoiesis and DC biology, such as Gfi, GATA-1, Spi-B, and c-Rel. Taken together, these data show that DC-SCRIPT is well-conserved in evolution and that the mouse homologue is more than 80% homologous to the human protein. Therefore, mouse models can be used to elucidate the function of this novel DC marker.
Assuntos
Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas Nucleares/química , Proteínas Repressoras/química , Fatores de Transcrição/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Animais Recém-Nascidos , Sítios de Ligação/genética , Proteínas de Transporte , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 5/genética , Sequência Conservada , Proteínas de Ligação a DNA/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Dedos de Zinco/fisiologiaRESUMO
Recently, we described the molecular identification of dendritic cell-specific TrAnsMembrane protein (DC-STAMP), a multimembrane-spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC-STAMP (mDC-STAMP) as well as the characterization of the DC-STAMP protein. The results demonstrate that mDC-STAMP is over 90% homologous to hDC-STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC-STAMP expression is enhanced by interleukin-4 and down-regulated upon DC maturation. Analysis of differently tagged DC-STAMP proteins further demonstrates that hDC-STAMP and mDC-STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC-STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC-STAMP-green fluorescent protein fusion protein. These data imply that DC-STAMP may exert its effect in the ER.
Assuntos
Células Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
CC chemokine ligand 18/dendritic cell-chemokine 1 (CCL18/DC-CK1) is a CC chemokine, preferentially expressed by DC, which acts as a chemoattractant for naive T cells and mantle zone B cells. Applying a newly developed CCL18/DC-CK1 sandwich enzyme-linked immunosorbent assay, we demonstrate that DC secrete high amounts of CCL18/DC-CK1 and that this expression can be increased by interleukin-10. High levels of CCL18/DC-CK1 were also detected in human serum (average of 88 ng/ml). Moreover, elevated CCL18/DC-CK1 levels were detected in synovial fluid from rheumatoid arthritis patients and in drain fluid (average of 254 ng/ml and 122 ng/ml, respectively). Immunoprecipitation experiment using anti-CCL18/DC-CK1 monoclonal antibodies revealed a protein of 6-7 kDa in serum and drain fluid that was indistinguishable from recombinant CCL18/DC-CK1 on Western blot and in re-aggregation assays. The concentration of CCL18/DC-CK1 found in human serum is in the same order of magnitude as was previously reported to completely inhibit CCL11/eotaxin-induced CC chemokine receptor 3 (CCR3) activation and consequent migration of eosinophils. CCL18/DC-CK1 may therefore function as an agonist (for naive T and B cells) and as an antagonist for CCR3-expressing leukocytes such as eosinophils.
Assuntos
Anticorpos Monoclonais/imunologia , Artrite Reumatoide/imunologia , Quimiocinas CC/imunologia , Células Dendríticas/imunologia , Interleucina-10/imunologia , Especificidade de Anticorpos , Líquidos Corporais/imunologia , Células Cultivadas , Quimiocinas CC/sangue , Quimiocinas CC/metabolismo , Quimiotaxia de Leucócito/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Leucócitos/imunologia , Receptores CCR3 , Receptores de Quimiocinas/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Neuroblastoma (NBL) is a childhood malignancy of the sympathetic nervous system. For high-risk NBL patients, the mortality rate is still over 50%, despite intensive multimodal treatment. Anti-GD2 monoclonal antibody (mAB) in combination with systemic cytokine immunotherapy has shown clinical efficacy in high-risk NBL patients. Targeted therapy using histone deacetylase inhibitors (HDACi) is currently being explored in cancer treatment and already shows promising results. Using our recently developed transplantable TH-MYCN NBL model, we here report that the HDAC inhibitor Vorinostat synergizes with anti-GD2 mAb therapy in reducing NBL tumor growth. Further mechanistic studies uncovered multiple mechanisms for the observed synergy, including Vorinostat-induced specific NBL cell death and upregulation of the tumor antigen GD2 on the cell surface of surviving NBL cells. Moreover, Vorinostat created a permissive tumor microenvironment (TME) for tumor-directed mAb therapy by increasing macrophage effector cells expressing high levels of Fc-receptors (FcR) and decreasing the number and function of myeloid-derived suppressor cells (MDSC). Collectively, these data imply further testing of other epigenetic modulators with immunotherapy and provide a strong basis for clinical testing of anti-GD2 plus Vorinostat combination therapy in NBL patients.
RESUMO
The aim of this study was to evaluate the tolerability of intratumoral administered recombinant human interleukin-12 (rhIL-12) in patients with head and neck squamous cell carcinoma. Six patients were treated once a week at two dose levels of 100 or 300 ng/kg, respectively, up to 24 weeks. The primary end point was to assess the toxicity and safety of intratumoral injected rhIL-12 in head and neck squamous cell carcinoma patients; the pharmacokinetics and pharmacodynamics of rhIL-12 and any evidence of antitumor effect were also determined. Toxicity was mild, with prolonged grade 4 lymphopenia observed in only one patient. No dose-limiting toxicities occurred. In all six patients, the rhIL-12 was detectable in plasma within 30 min. Significant reductions in absolute number of peripheral blood lymphocytes and all lymphocyte subsets, especially cytotoxic T cells and natural killer cells, were observed that were maximal between 12 and 24 h. Maximal plasma concentrations of IFN-gamma and IL-10 were detected after 12 h. A real-time semiquantitative PCR analysis in peripheral blood mononuclear cells showed a mean increase of mRNA encoding IFN-gamma of 2.2 times relative to the pretreatment sample. An unexpected, significant decrease of 80% in T-bet mRNA, a T-helper 1 transcription factor, was detected after 12 h, with normalization after 48-72 h. No complete or partial responses were observed. In one patient, a 40% regression of a tumor lesion was noted. In conclusion, rhIL-12 at these dose levels and schedule was well tolerated and resulted in measurable immunological responses.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Interleucina-12/uso terapêutico , Leucócitos Mononucleares/metabolismo , Proteínas Recombinantes/uso terapêutico , Adulto , Idoso , Área Sob a Curva , Citocinas/biossíntese , Feminino , Humanos , Interferon gama/sangue , Interleucina-10/sangue , Leucócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
The objective of this Phase II study was to evaluate the pharmacodynamic and immune effects of intratumorally administered recombinant human interleukin-12 (IL-12) on regional lymph nodes, primary tumor, and peripheral blood. Ten previously untreated patients with head and neck squamous cell carcinoma were injected in the primary tumor two to three times, once/week, at two dose levels of 100 or 300 ng/kg, before surgery. We compared these patients with 20 control (non-IL-12-treated) patients. Toxicity was high, with unexpected dose-limiting toxicities at the 300 ng/kg dose level. Dose-dependent plasma IFN-gamma and IL-10 increments were detected. These cytokine levels were higher after the first injection than after the subsequent injections. A rapid, transient reduction in lymphocytes, monocytes, and all lymphocyte subsets, especially natural killer cells, was observed, due to a redistribution to the lymph nodes. In the enlarged lymph nodes of the IL-12-treated patients, a higher percentage of natural killer cells and a lower percentage of T-helper cells were found compared with control patients. The same pattern was detected in the infiltrate in the primary tumor. Real-time semiquantitative PCR analysis of peripheral blood mononuclear cells in the peripheral blood showed a transient decrease of T-bet mRNA. Interestingly, the peripheral blood mononuclear cells in the lymph nodes showed a 128-fold (mean) increase of IFN-gamma mRNA. A switch from the Th2 to a Th1 profile in the lymph nodes compared with the peripheral blood occurred in the IL-12-treated patients. In conclusion, in previously untreated head and neck squamous cell carcinoma patients, recombinant human IL-12 intratumorally showed dose-limiting toxicities at the dose level of 300 ng/kg and resulted in measurable immunological responses locoregionally at both dose levels.
Assuntos
Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/terapia , Interleucina-12/administração & dosagem , Interleucina-12/farmacocinética , Linfonodos/patologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacocinética , Células Th1/citologia , Adulto , Idoso , Área Sob a Curva , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Humanos , Interferon gama/sangue , Interleucina-10/sangue , Cinética , Leucócitos Mononucleares/metabolismo , Linfonodos/efeitos dos fármacos , Subpopulações de Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Proteínas Recombinantes/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Resultado do TratamentoRESUMO
Topical application of the vitamin D (VitD) analog calcipotriol is a highly effective standard treatment modality of psoriatic skin lesions. However, the immune modulatory effects of the treatment are incompletely understood. VitD is well known to induce tolerogenic responses in conventional dendritic cells (cDCs). Plasmacytoid DCs (pDCs) comprise a specialized, naturally occurring DC subset known to be important in autoimmune diseases including psoriasis. pDCs from the blood rapidly infiltrate psoriatic skin and are key to the initiation of the immune-mediated pathogenesis of the disease. We now demonstrate that pDCs express various proteins of the VitD receptor (VDR) pathway, including the VitD-metabolizing enzymes Cyp27B1 and Cyp24A1, and that VDR is transcriptionally active in pDCs. Moreover, VitD impairs the capacity of murine and human pDCs to induce T-cell proliferation and secretion of the T-helper 1 cytokine IFNγ. The inhibitory effect of VitD is dependent on the expression of the VDR in the DCs. This study demonstrates that VitD signaling can act as a natural inhibitory mechanism on both cDCs and pDCs, which may instigate the development of VitD-based therapeutic applications for psoriasis and other inflammatory skin diseases.