RESUMO
T-cell development is a spatially and temporally regulated process, orchestrated by well-defined contributions of transcription factors and cytokines. Here, we identify the noncoding RNA miR-142 as an additional regulatory layer within murine thymocyte development and proliferation. MiR-142 deficiency impairs the expression of cell cycle-promoting genes in mature mouse thymocytes and early progenitors, accompanied with increased levels of cyclin-dependent kinase inhibitor 1B (Cdkn1b, also known as p27Kip1 ). By using CRISPR/Cas9 technology to delete the miR-142-3p recognition element in the 3'UTR of cdkn1b, we confirm that this gene is a novel target of miR-142-3p in vivo. Increased Cdkn1b protein expression alone however was insufficient to cause proliferation defects in thymocytes, indicating the existence of additional critical miR-142 targets. Collectively, we establish a key role for miR-142 in the control of early and mature thymocyte proliferation, demonstrating the multifaceted role of a single miRNA on several target genes.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , MicroRNAs/metabolismo , Timócitos/fisiologia , Regiões 3' não Traduzidas , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação Neoplásica da Expressão Gênica , Camundongos , MicroRNAs/genética , Processamento Pós-Transcricional do RNARESUMO
Imatinib (IM) has been described to modulate the function of dendritic cells and T lymphocytes and to affect the expression of antigen in CML cells. In our study, we investigated the effect of the tyrosine kinase inhibitors IM and nilotinib (NI) on antigen presentation and processing by analyzing the proteasomal activity in CML cell lines and patient samples. We used a biotinylated active site-directed probe, which covalently binds to the proteasomally active beta-subunits in an activity-dependent fashion. Additionally, we analyzed the cleavage and processing of HLA-A3/11- and HLA-B8-binding peptides derived from BCR-ABL by IM- or NI-treated isolated 20S immunoproteasomes using mass spectrometry. We found that IM treatment leads to a reduction in MHC-class I expression which is in line with the inhibition of proteasomal activity. This process is independent of BCR-ABL or apoptosis induction. In vitro digestion experiments using purified proteasomes showed that generation of epitope-precursor peptides was significantly altered in the presence of NI and IM. Treatment of the immunoproteasome with these compounds resulted in an almost complete reduction in the generation of long precursor peptides for the HLA-A3/A11 and -B8 epitopes while processing of the short peptide sequences increased. Treatment of isolated 20S proteasomes with serine-/threonine- and tyrosine-specific phosphatases induced a significant downregulation of the proteasomal activity further indicating that phosphorylation of the proteasome regulates its function and antigen processing. Our results demonstrate that IM and NI can affect the immunogenicity of malignant cells by modulating proteasomal degradation and the repertoire of processed T cell epitopes.
Assuntos
Antígenos de Neoplasias/imunologia , Benzamidas/farmacologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fragmentos de Peptídeos/imunologia , Piperazinas/farmacologia , Complexo de Endopeptidases do Proteassoma/imunologia , Pirimidinas/farmacologia , Sequência de Aminoácidos , Apresentação de Antígeno/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/imunologia , Proteína Fosfatase 2/metabolismoRESUMO
Dectin-1 is the major receptor for fungal ß-glucans. The activation of Dectin-1 leads to the up-regulation of surface molecules on dendritic cells (DCs) and cytokine secretion. Furthermore, Dectin-1 is important for the recruitment of leukocytes and the production of inflammatory mediators. Peroxisome proliferator-activated receptor-γ (PPAR-γ) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have modulatory effects on B-cell, T-cell, and DC function. In the present study, we analyzed the effects of troglitazone (TGZ), a high-affinity synthetic PPAR-γ ligand, on the Dectin-1-mediated activation of monocyte-derived human DCs. Dectin-1-mediated activation of DCs was inhibited by TGZ, as shown by down-regulation of costimulatory molecules and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation. Furthermore, TGZ inhibited the T-cell-stimulatory capacity of DCs. These effects were not due to a diminished expression of Dectin-1 or to a reduced phosphorylation of spleen tyrosine kinase; they were mediated by the inhibition of downstream signaling molecules such as mitogen-activated protein kinases and nuclear factor-κB. Furthermore, curdlan-mediated accumulation of caspase recruitment domain 9 (CARD9) in the cytosol was inhibited by TGZ. Our data demonstrate that the PPAR-γ ligand TGZ inhibits Dectin-1-mediated activation by interfering with CARD9, mitogen-activated protein kinase, and nuclear factor-κB signaling pathways. This confirms their important role as negative-feedback regulators of potentially harmful inflammatory responses.
Assuntos
Cromanos/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Hipoglicemiantes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lectinas Tipo C , Ligantes , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , PPAR gama/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Quinase Syk , TroglitazonaRESUMO
NK cell anti-tumor reactivity is governed by a balance of activating and inhibitory receptors including various TNF receptor (TNFR) family members. Here we report that human tumor cells release a soluble form of the TNF family member Glucocorticoid-Induced TNFR-Related Protein (GITR) ligand (sGITRL), which can be detected in cell culture supernatants. Tumor-derived sGITRL concentration-dependently reduced NK cell cytotoxicity and IFN-gamma production, which could be overcome by neutralization of sGITRL using a GITR-Ig fusion protein. Although sGITRL did not induce apoptosis in NK cells, it diminished nuclear localized RelB, indicating that sGITRL negatively modulates NK cell NF-kappaB activity. Furthermore, we detected substantial levels of sGITRL in sera of patients with various malignancies, but not in healthy controls. Presence of sGITRL-containing patient serum in cocultures with tumor cells significantly reduced NK cell cytotoxicity and IFN-gamma production, which could again be restored by neutralization of sGITRL. The strong correlation of tumor incidence and elevated sGITRL levels indicates that sGITRL is released from cancers in vivo, leading to impaired NK cell immunosurveillance of human tumors. Our data suggest that determination of sGITRL levels might be implemented as a tumor marker in patients, and GITRL neutralization may be used to improve immunotherapeutic strategies relying on NK cell reactivity.
Assuntos
Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Inibidores do Fator de Necrose Tumoral , Fatores de Necrose Tumoral/metabolismo , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/imunologia , Humanos , Interferon gama/biossíntese , Células Matadoras Naturais/metabolismo , NF-kappa B/metabolismo , Neoplasias/metabolismo , Testes de Neutralização , Solubilidade , Fatores de Necrose Tumoral/sangueRESUMO
The Cancer Report from the World Health Organization states that in the year 2000 12% of all death cases worldwide were caused by cancer. In the western world, the cancer death rates are often devastating, being at about 25%. This fact stresses the urgency to find effective cures against malignant diseases. New approaches in the treatment of cancer focus on the development of immunotherapies to fight the disease. Besides other methods, the usage of tumor-specific RNA as part of vaccines is investigated lately. RNA, administered alone or used for transfection of dendritic cells, shows several advantages as a vaccine including feasibility, applicability, safeness, and effectiveness when it comes to the generation of immune responses. This review concentrates on results from in vitro experiments and recent trials using RNA vaccines to present an overview about this specific strategy.
Assuntos
Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , RNA/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Humanos , ImunoterapiaRESUMO
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by excessive granulopoiesis due to the formation of the constitutively active tyrosine kinase BCR-ABL. An effective drug against CML is imatinib mesylate, a tyrosine kinase inhibitor acting on Abl kinases, c-KIT, and platelet-derived growth factor receptor. Recently, a study revealed that patients treated with imatinib showed impaired CTL responses compared with patients treated with IFN-alpha, which might be due to a treatment-induced reduction in immunogenicity of CML cells or immunosuppressive effects. In our study, we found that inhibition of BCR-ABL leads to a down-regulation of immunogenic antigens on the CML cells in response to imatinib treatment, which results in the inhibition of CML-directed immune responses. By treating CML cells with imatinib, we could show that the resulting inhibition of BCR-ABL leads to a decreased expression of tumor antigens, including survivin, adipophilin, hTERT, WT-1, Bcl-x(L), and Bcl-2 in correlation to a decreased development of CML-specific CTLs. In contrast, this reduction in immunogenicity was not observed when a CML cell line resistant to the inhibitory effects of imatinib was used, but could be confirmed by transfection with specific small interfering RNA against BCR-ABL or imatinib treatment of primary CML cells.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Benzamidas , Células Dendríticas/imunologia , Regulação para Baixo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Ativação Linfocitária/efeitos dos fármacos , RNA Interferente Pequeno/genética , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GITRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-beta by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking GITR-GITRL interaction in cocultures of tumor cells and NK cells substantially increased cytotoxicity and IFN-gamma production of NK cells demonstrating that constitutive expression of GITRL by tumor cells diminishes NK cell antitumor immunity. GITRL-Ig fusion protein or cell surface-expressed GITRL did not induce apoptosis in NK cells, but diminished nuclear localized c-Rel and RelB, indicating that GITR might negatively modulate NK cell NF-kappaB activity. Taken together, our data indicate that tumor-expressed GITRL mediates immunosubversion in humans.
Assuntos
Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Receptores de Fator de Crescimento Neural/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fatores de Necrose Tumoral/fisiologia , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Clonagem Molecular , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Ligantes , Receptores de Fator de Crescimento Neural/genética , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Fatores de Necrose Tumoral/genéticaRESUMO
BACKGROUND: Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM. METHODS: The MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235). RESULTS: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs. CONCLUSIONS: Our results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.
RESUMO
Human Dectin-1 (hDectin-1) is a member of the C-type lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In this study, we demonstrate that hDectin-1 is involved in the uptake and cross-presentation of cellular antigens. Furthermore, activation of monocyte-derived dendritic cells (MDCs) with toll-like receptor 3 (TLR3) ligand but not with TLR2 ligand or TLR7 ligand resulted in down-regulation of hDectin-1 expression and reduced phagocytosis of apoptotic tumor cells as well as presentation of pp65-derived T-cell epitopes upon engulfment of cytomegalovirus (CMV)-infected human foreskin fibroblasts.
Assuntos
Apresentação de Antígeno/imunologia , Apresentação Cruzada/imunologia , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Apoptose , Linhagem Celular , Cromo , Infecções por Citomegalovirus/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Fibroblastos/citologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Antígeno HLA-A2/imunologia , Humanos , Lectinas Tipo C , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fagocitose , Fenótipo , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Receptores Toll-Like/agonistasRESUMO
Dendritic cells (DCs) have the unique ability to efficiently present T-cell epitopes from exogenous antigens on MHC class I molecules, a process called cross-presentation. In our study we demonstrate that stimulation of monocyte-derived DCs with Toll-like receptor (TLR) ligands differentially affects the uptake and cross-presentation of cellular antigens. Activation of DCs with TLR3 or TLR4 but not with TLR2 or TLR7/8 ligands inhibited phagocytosis of apoptotic tumor cells and resulted in a reduced cross-presentation of pp65-derived T-cell epitopes on MHC class I molecules upon engulfment of cytomegalovirus (CMV)-infected fibroblasts. These results have an important impact on the understanding of the interactions between the immune system and pathogens and the development of vaccination strategies to treat malignant diseases.
Assuntos
Apresentação de Antígeno , Infecções por Citomegalovirus/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias/imunologia , Receptores Toll-Like/imunologia , Apoptose/imunologia , Linhagem Celular , Citomegalovirus/imunologia , Infecções por Citomegalovirus/terapia , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Monócitos/imunologia , Neoplasias/terapia , Fosfoproteínas/imunologia , Vacinação , Proteínas da Matriz Viral/imunologiaRESUMO
Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells of the immune system with the unique ability to initiate and maintain primary immune responses. In order to better characterize the functional and phenotypic features of DCs, a subtractive cDNA library to identify differentially expressed genes in monocyte-derived DCs (MDCs) was constructed. Using this approach, we found that the epithelial transcription factor ESE-3, which was previously shown to be exclusively expressed in cells of epithelial origin, is differentially expressed in MDCs. This was further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses. The expression of ESE-3 is up-regulated upon maturation of MDCs and inhibited by treating the cells with IL-10 or IFN-gamma. Knockdown experiments using siRNA suggest that ESE-3 plays an important role during MDC development. Our results might help to improve the phenotypic characterization of DCs and lead to a better understanding of the cellular mechanisms involved in antigen presentation and T-cell stimulation.
Assuntos
Apresentação de Antígeno/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Monócitos/imunologia , Fatores de Transcrição/imunologia , Apresentação de Antígeno/genética , Western Blotting/métodos , Diferenciação Celular/genética , Células Cultivadas , Células Dendríticas/citologia , Regulação da Expressão Gênica/genética , Humanos , Monócitos/citologia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição/genéticaRESUMO
Dendritic cells (DCs) play an important role in initiating and maintaining primary immune responses. However, mechanisms involved in the resolution of these responses are elusive. We analyzed the effects of 15d-PGJ2 and the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma ligand troglitazone (TGZ) on the immunogenicity of human monocyte-derived DCs upon stimulation with toll-like receptor (TLR) ligands. Activation of PPAR-gamma resulted in a reduced stimulation of DCs via the TLR ligands 2, 3, 4, and 7, characterized by down-regulation of costimulatory and adhesion molecules and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation and recruitment. MCP-1 (monocyte chemotactic protein-1) production was increased due to PPAR-gamma activation. Furthermore, TGZ-treated DCs showed a significantly reduced capacity to stimulate T-cell proliferation, emphasizing the inhibitory effect of PPAR-gamma activation on TLR-induced DC maturation. Western blot analyses revealed that these inhibitory effects on TLR-induced DC activation were mediated via inhibition of the NF-kappaB and mitogen-activated protein (MAP) kinase pathways while not affecting the PI3 kinase/Akt signaling. Our data demonstrate that inhibition of the MAP kinase and NF-kappaB pathways is critically involved in the regulation of TLR and PPAR-gamma-mediated signaling in DCs.