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1.
Sci Immunol ; 6(61)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272227

RESUMO

Cytoplasmic double-stranded RNA is sensed by RIG-I-like receptors (RLRs), leading to induction of type I interferons (IFN-Is), proinflammatory cytokines, and apoptosis. Here, we elucidate signaling mechanisms that lead to cytokine secretion and cell death induction upon stimulation with the bona fide RIG-I ligand 5'-triphosphate RNA (3p-RNA) in tumor cells. We show that both outcomes are mediated by dsRNA-receptor families with RLR being essential for cytokine production and IFN-I-mediated priming of effector pathways but not for apoptosis. Affinity purification followed by mass spectrometry and subsequent functional analysis revealed that 3p-RNA bound and activated oligoadenylate synthetase 1 and RNase L. RNase L-deficient cells were profoundly impaired in their ability to undergo apoptosis. Mechanistically, the concerted action of translational arrest triggered by RNase L and up-regulation of NOXA was needed to deplete the antiapoptotic MCL-1 to cause intrinsic apoptosis. Thus, 3p-RNA-induced apoptosis is a two-step process consisting of RIG-I-dependent priming and an RNase L-dependent effector phase.


Assuntos
2',5'-Oligoadenilato Sintetase/imunologia , Endorribonucleases/imunologia , Neoplasias/imunologia , Receptores do Ácido Retinoico/imunologia , 2',5'-Oligoadenilato Sintetase/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Linhagem Celular Tumoral , Técnicas de Cocultura , Proteína DEAD-box 58/genética , Endorribonucleases/genética , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Ligantes , Camundongos , Receptores Imunológicos/genética
2.
Front Immunol ; 12: 595390, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995343

RESUMO

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/ß. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics.


Assuntos
Proteína DEAD-box 58/metabolismo , Genoma Viral , Mutação , Receptores Imunológicos/metabolismo , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral , Animais , Linhagem Celular , Genoma Viral/genética , Genoma Viral/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunomodulação , RNA Viral/genética , RNA Viral/imunologia
3.
Dig Dis Sci ; 66(4): 1349-1359, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32451758

RESUMO

BACKGROUND: The benefit of ischemic postconditioning (IPostC) might be the throttled inflow following cold ischemia. The current study investigated advantage and mechanisms of IPostC in healthy and fatty rat livers. METHODS: Male SD rats received a high-fat diet to induce fatty livers. Isolated liver perfusion was performed after 24 h ischemia at 4 °C as well as in vivo experiments after 90 min warm ischemia. The so-called follow-up perfusions served to investigate the hypothesis that medium from IPostC experiments is less harmful. Lactate dehydrogenase (LDH), transaminases, different cytokines, and gene expressions, respectively, were measured. RESULTS: Fatty livers showed histologically mild inflammation and moderate to severe fat storage. IPostC reduced LDH and TXB2 in healthy and fatty livers and increased bile flow. LDH, TNF-α, and IL-6 levels in serum decreased after warm ischemia + IPostC. The gene expressions of Tnf, IL-6, Ccl2, and Ripk3 were downregulated in vivo after IPostC. CONCLUSIONS: IPostC showed protective effects after ischemia in situ and in vivo in healthy and fatty livers. Restricted cyclic inflow was an important mechanism and further suggested involvement of necroptosis. IPostC represents a promising and easy intervention to improve outcomes after transplantation.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/patologia , Fígado Gorduroso/prevenção & controle , Pós-Condicionamento Isquêmico/métodos , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Fígado Gorduroso/etiologia , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações
4.
Cancer Immunol Immunother ; 69(10): 2101-2112, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32448983

RESUMO

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8+ T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4flox. In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.


Assuntos
Biomarcadores Tumorais/análise , Linfócitos T CD8-Positivos/imunologia , Fatores Reguladores de Interferon/fisiologia , Células Supressoras Mieloides/imunologia , Neoplasias Pancreáticas/imunologia , Microambiente Tumoral/imunologia , Animais , Apoptose , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Modelos Animais de Doenças , Humanos , Terapia de Imunossupressão , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
5.
Leukemia ; 34(4): 1017-1026, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31740809

RESUMO

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4+ and CD8+ T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3+ T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.


Assuntos
Anticorpos Neutralizantes/farmacologia , Proteína DEAD-box 58/imunologia , Imunoterapia/métodos , Leucemia Mieloide Aguda/terapia , RNA de Cadeia Dupla/farmacologia , Receptores Virais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proteína DEAD-box 58/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Xenoenxertos , Humanos , Memória Imunológica/efeitos dos fármacos , Interferons/genética , Interferons/imunologia , Isoenxertos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/mortalidade , Camundongos , Receptores Virais/agonistas , Receptores Virais/genética , Indução de Remissão , Transdução de Sinais , Análise de Sobrevida , Resultado do Tratamento
6.
J Immunother Cancer ; 7(1): 349, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31843014

RESUMO

Following publication of the original article [1], the authors have reported that Fig. 2 and Additional file 1: Figure S1, S2 partially show red scripts.

7.
J Immunother Cancer ; 7(1): 288, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694706

RESUMO

BACKGROUND: The tumor microenvironment (TME) combines features of regulatory cytokines and immune cell populations to evade the recognition by the immune system. Myeloid-derived suppressor cells (MDSC) comprise populations of immature myeloid cells in tumor-bearing hosts with a highly immunosuppressive capacity. We could previously identify RIG-I-like helicases (RLH) as targets for the immunotherapy of pancreatic cancer inducing immunogenic tumor cell death and type I interferons (IFN) as key mediators linking innate with adaptive immunity. METHODS: Mice with orthotopically implanted KrasG12D p53fl/R172H Ptf1a-Cre (KPC) pancreatic tumors were treated intravenously with the RLH ligand polyinosinic-polycytidylic acid (poly(I:C)), and the immune cell environment in tumor and spleen was characterized. A comprehensive analysis of the suppressive capacity as well as the whole transcriptomic profile of isolated MDSC subsets was performed. Antigen presentation capability of MDSC from mice with ovalbumin (OVA)-expressing tumors was investigated in T cell proliferation assays. The role of IFN in MDSC function was investigated in Ifnar1-/- mice. RESULTS: MDSC were strongly induced in orthotopic KPC-derived pancreatic cancer, and frequencies of MDSC subsets correlated with tumor weight and G-CSF serum levels, whereas other immune cell populations decreased. Administration of the RLH-ligand induced a IFN-driven immune response, with increased activation of T cells and dendritic cells (DC), and a reduced suppressive capacity of both polymorphonuclear (PMN)-MDSC and monocytic (M)-MDSC fractions. Whole transcriptomic analysis confirmed an IFN-driven gene signature of MDSC, a switch from a M2/G2- towards a M1/G1-polarized phenotype, and the induction of genes involved in the antigen presentation machinery. Nevertheless, MDSC failed to present tumor antigen to T cells. Interestingly, we found MDSC with reduced suppressive function in Ifnar1-deficient hosts; however, there was a common flaw in immune cell activation, which was reflected by defective immune cell activation and tumor control. CONCLUSIONS: We provide evidence that the treatment with immunostimulatory RNA reprograms the TME of pancreatic cancer by reducing the suppressive activity of MDSC, polarizing myeloid cells into a M1-like state and recruiting DC. We postulate that tumor cell-targeting combination strategies may benefit from RLH-based TME remodeling. In addition, we provide novel insights into the dual role of IFN signaling in MDSC's suppressive function and provide evidence that host-intrinsic IFN signaling may be critical for MDSC to gain suppressive function during tumor development.

8.
Front Immunol ; 10: 2162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632388

RESUMO

Ischemia reperfusion injury (IRI) of the kidney results in interferon regulatory factor 4 (IRF4)-mediated counter-regulation of the acute inflammatory response. Beyond that, IRF4 exerts important functions in controlling the cytokine milieu, T-cell differentiation, and macrophage polarization. The latter has been implicated in tissue remodeling. It therefore remains elusive what the role of IRF4 is in terms of long-term outcome following IRI. We hypothesized that an inability to resolve chronic inflammation in Irf4-/- mice would promote chronic kidney disease (CKD) progression. To evaluate the effects of IRF4 in chronic upon acute injury in vivo, a mouse model of chronic injury following acute IRI was employed. The expression of Irf4 increased within 10 days after IRI in renal tissue. Both mRNA and protein levels remained high up to 5 weeks upon IRI, suggesting a regulatory function in the chronic phase. Mice deficient in IRF4 display increased tubular cell loss and defective clearance of infiltrating macrophages. These phenomena were associated with increased expression of pro-inflammatory macrophage markers together with reduced expression of alternatively activated macrophage markers. In addition, IRF4-deficient mice showed defective development of alternatively activated macrophages. Hints of a residual M1 macrophage signature were further observed in human biopsy specimens of patients with hypertensive nephropathy vs. living donor specimens. Thus, IRF4 restricts CKD progression and kidney fibrosis following IRI, potentially by enabling M2 macrophage polarization and restricting a Th1 cytokine response. Deteriorated alternative macrophage subpopulations in Irf4-/- mice provoke chronic intrarenal inflammation, tubular epithelial cell loss, and renal fibrosis in the long course after IRI in mice. The clinical significance of these finding for human CKD remains uncertain at present and warrants further studies.


Assuntos
Suscetibilidade a Doenças , Fatores Reguladores de Interferon/genética , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/prevenção & controle , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Regeneração , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
9.
Mol Cancer Ther ; 18(12): 2343-2356, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515294

RESUMO

The pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen-specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.


Assuntos
Proteína DEAD-box 58/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Polifosfatos/uso terapêutico , RNA/metabolismo , Animais , Linhagem Celular Tumoral , Proteína DEAD-box 58/farmacologia , Humanos , Melanoma/patologia , Camundongos , Polifosfatos/farmacologia , Receptores Imunológicos , Transdução de Sinais , Transfecção
10.
Sci Rep ; 8(1): 8810, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29892077

RESUMO

Checkpoint molecules such as programmed death 1 (PD-1) dampen excessive T cell activation to preserve immune homeostasis. PD-1-specific monoclonal antibodies have revolutionized cancer therapy, as they reverse tumour-induced T cell exhaustion and restore CTL activity. Based on this success, deciphering underlying mechanisms of PD-1-mediated immune functions has become an important field of immunological research. Initially described for T cells, there is emerging evidence of unconventional PD-1 expression by myeloid as well as tumor cells, yet, with cell-intrinsic functions in various animal tumor models. Here, we describe positive PD-1 antibody staining of various murine immune and tumour cells that is, unlike for T cells, not the PD-1 receptor and restricted to cells with low forward scatter characteristics. Based on flow cytometry and various approaches, including two established murine anti-PD-1 antibody clones, CRISPR/Cas9 genome editing and confocal imaging, we describe a staining pattern assigned to a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies. Lack of PD-1 expression was further underlined by the analysis of PD-1 expression from B16-F10-derived 3D cultures and ex vivo tumours. Thus, our data provide multiple lines of evidence that PD-1 expression by non-T cells is unlikely to be the case and, taking recent data of PD-1 tumour cell-intrinsic functions into account, suggest that other antibody-mediated pathways might apply.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Nucleares/imunologia , Reações Cruzadas , Receptor de Morte Celular Programada 1/imunologia , Animais , Linhagem Celular , Citometria de Fluxo , Imunofluorescência , Camundongos Endogâmicos C57BL , Microscopia Confocal
11.
JCI Insight ; 3(5)2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29515025

RESUMO

Inflammatory bowel disease (IBD) is associated with enhanced levels of the IL-1 family cytokines IL-1ß and IL-18, which are activated by the Nlrp3 inflammasome. Here, we investigated the role of inflammasome-driven cytokine release on T cell polarization and DC differentiation in steady state and T cell transfer colitis. In vitro and in vivo data showed that IL-1ß induces Th17 polarization and increases GM­CSF production by T cells. Reduced IL-1ß levels in Nlrp3-/- mice correlated with enhanced FLT3L levels and increased frequency of tolerogenic CD103+ DC. In the T cell transfer colitis model, Nlrp3 deficiency resulted in lower IL­1ß levels, reduced Th17 immunity, and less severe colitis. Unaltered IL-18 levels in both mouse strains pointed toward Nlrp3-independent processing. Importantly, cohousing revealed that the gut microbiome had no impact on the observed Nlrp3-/- phenotype. This study demonstrates that NLRP3 acts as a molecular switch of intestinal homeostasis by shifting local immune cells toward an inflammatory phenotype via IL-1ß.


Assuntos
Diferenciação Celular/imunologia , Colite/imunologia , Células Dendríticas/imunologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Colo/citologia , Colo/imunologia , Colo/patologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Cadeias alfa de Integrinas/imunologia , Cadeias alfa de Integrinas/metabolismo , Interleucina-1beta/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Células Th17/transplante
12.
Mitochondrion ; 40: 1-12, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28935446

RESUMO

Western lifestyle-associated malnutrition causes steatosis that may progress to liver inflammation and mitochondrial dysfunction has been suggested as a key factor in promoting this disease. Here we have molecularly, biochemically and biophysically analyzed mitochondria from steatotic wild type and immune-compromised mice fed a Western diet (WD) - enriched in saturated fatty acids (SFAs). WD-mitochondria demonstrated lipidomic changes, a decreased mitochondrial ATP production capacity and a significant sensitivity to calcium. These changes preceded hepatocyte damage and were not associated with enhanced ROS production. Thus, WD-mitochondria do not promote steatohepatitis per se, but demonstrate bioenergetic deficits and increased sensitivity to stress signals.


Assuntos
Fígado Gorduroso/patologia , Hepatócitos/patologia , Mitocôndrias/fisiologia , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Dieta/métodos , Modelos Animais de Doenças , Ácidos Graxos/administração & dosagem , Metabolismo dos Lipídeos , Camundongos , Mitocôndrias/metabolismo
13.
Proc Natl Acad Sci U S A ; 114(49): 12994-12999, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29150554

RESUMO

IL-22 has been identified as a cancer-promoting cytokine that is secreted by infiltrating immune cells in several cancer models. We hypothesized that IL-22 regulation would occur at the interface between cancer cells and immune cells. Breast and lung cancer cells of murine and human origin induced IL-22 production from memory CD4+ T cells. In the present study, we found that IL-22 production in humans is dependent on activation of the NLRP3 inflammasome with the subsequent release of IL-1ß from both myeloid and T cells. IL-1 receptor signaling via the transcription factors AhR and RORγt in T cells was necessary and sufficient for IL-22 production. In these settings, IL-1 induced IL-22 production from a mixed T helper cell population comprised of Th1, Th17, and Th22 cells, which was abrogated by the addition of anakinra. We confirmed these findings in vitro and in vivo in two murine tumor models, in primary human breast and lung cancer cells, and in deposited expression data. Relevant to ongoing clinical trials in breast cancer, we demonstrate here that the IL-1 receptor antagonist anakinra abrogates IL-22 production and reduces tumor growth in a murine breast cancer model. Thus, we describe here a previously unrecognized mechanism by which cancer cells induce IL-22 production from memory CD4+ T cells via activation of the NLRP3 inflammasome and the release of IL-1ß to promote tumor growth. These findings may provide the basis for therapeutic interventions that affect IL-22 production by targeting IL-1 activity.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Interleucina-1beta/fisiologia , Interleucinas/biossíntese , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultivo Condicionados , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamassomos/metabolismo , Interleucinas/metabolismo , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transplante de Neoplasias , Transdução de Sinais , Carga Tumoral , Interleucina 22
14.
Front Immunol ; 8: 2000, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29403480

RESUMO

Volcanic ash is a heterogeneous mineral dust that is typically composed of a mixture of amorphous (glass) and crystalline (mineral) fragments. It commonly contains an abundance of the crystalline silica (SiO2) polymorph cristobalite. Inhalation of crystalline silica can induce inflammation by stimulating the NLRP3 inflammasome, a cytosolic receptor complex that plays a critical role in driving inflammatory immune responses. Ingested material results in the assembly of NLRP3, ASC, and caspase-1 with subsequent secretion of the interleukin-1 family cytokine IL-1ß. Previous toxicology work suggests that cristobalite-bearing volcanic ash is minimally reactive, calling into question the reactivity of volcanically derived crystalline silica, in general. In this study, we target the NLRP3 inflammasome as a crystalline silica responsive element to clarify volcanic cristobalite reactivity. We expose immortalized bone marrow-derived macrophages of genetically engineered mice and primary human peripheral blood mononuclear cells (PBMCs) to ash from the Soufrière Hills volcano as well as representative, pure-phase samples of its primary componentry (volcanic glass, feldspar, cristobalite) and measure NLRP3 inflammasome activation. We demonstrate that respirable Soufrière Hills volcanic ash induces the activation of caspase-1 with subsequent release of mature IL-1ß in a NLRP3 inflammasome-dependent manner. Macrophages deficient in NLRP3 inflammasome components are incapable of secreting IL-1ß in response to volcanic ash ingestion. Cellular uptake induces lysosomal destabilization involving cysteine proteases. Furthermore, the response involves activation of mitochondrial stress pathways leading to the generation of reactive oxygen species. Considering ash componentry, cristobalite is the most reactive pure-phase with other components inducing only low-level IL-1ß secretion. Inflammasome activation mediated by inhaled ash and its potential relevance in chronic pulmonary disease was further evidenced in PBMC using the NLRP3 small-molecule inhibitor CP-456,773 (CRID3, MCC950). Our data indicate the functional activation of the NLRP3 inflammasome by volcanic ash in murine and human macrophages in vitro. Cristobalite is identified as the apparent driver, thereby contesting previous assertions that chemical and structural imperfections may be sufficient to abrogate the reactivity of volcanically derived cristobalite. This is a novel mechanism for the stimulation of a pro-inflammatory response by volcanic particulate and provides new insight regarding chronic exposure to environmentally occurring particles.

15.
Cancer Lett ; 381(1): 259-68, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-26968250

RESUMO

Pancreatic cancer is one of the most aggressive malignancies and has been considered poorly immunogenic for decades. However, this characterization might be over-simplistic. A more sophisticated approach is needed in order to develop new treatment strategies. In this review, we will focus on T cell exhaustion as a phenomenon of immune failure that is a useful paradigm to characterize immunosuppressive effects. Cancer creates an environment of constant antigen exposure and inflammation. In this setting, T cells transform into a differentiation state that has been termed T cell exhaustion, which is characterized by upregulation of inhibitory receptors, resulting in loss of effector function. The discovery of receptor-mediated immune checkpoints, which prevent uncontrolled T cell reactions, led to the development of a new class of antibodies termed checkpoint inhibitors. Unprecedented results in patients with metastatic melanoma and lung cancer have renewed interest in the immunotherapy of other solid tumor entities, including pancreatic cancer. Data on the efficacy of checkpoint inhibitors in pancreatic cancer are still sparse and indicate limited efficacy as single agents. Combination of checkpoint inhibitors with other immune-activating strategies or cytotoxic drugs might be a way to overcome therapy resistance in the treatment of pancreatic cancer.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Animais , Vacinas Anticâncer/uso terapêutico , Humanos , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Microambiente Tumoral
16.
Dtsch Arztebl Int ; 112(48): 809-15, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26667979

RESUMO

BACKGROUND: A number of new drugs for tumor immunotherapy have been approved in the past few years. They work by activating T cells to combat tumors. METHODS: This review is based on publications on recently approved T-cell-activating drugs that were retrieved by a selective search in PubMed. RESULTS: Randomized, controlled trials of "checkpoint" inhibitors, i.e., inhibitory antibodies for use against tumors, have shown that the cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor ipilimumab can prolong the survival of patients with advanced melanoma by 2 to 4 months. No data on median overall survival are yet available for the two programmed-death-1 (PD-1) inhibitors pembrolizumab und nivolumab; the endpoint "tumor response" was achieved in 24% and 32% of patients receiving these drugs, respectively. Grade 3 or 4 adverse effects occurred in 50% of patients receiving ipilimumab and in 12 to 13% of those taking either of the two PD-1-inhibitors. Nivolumab prolonged the median survival of patients with metastatic non-small-cell lung cancer from 6 to 9 months. In refractory or recurrent Philadelphia-chromosome-negative pre-B acute lymphoblastic leukemia (pre-B-ALL), treatment with the bispecific antibody construct blinatumomab led to complete remission in 43% of the patients, while grade 3, 4 or 5 toxicities occurred in 83%. CONCLUSION: T-cell-directed strategies have been established as a new pillar of treatment in medical oncology. As these drugs have frequent and severe adverse effects, therapeutic decision-making will have to take account not only of the predicted prolongation of survival, but also of the potential for an impaired quality of life while the patient is under treatment.


Assuntos
Imunoterapia/métodos , Terapia de Alvo Molecular/métodos , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Medicina Baseada em Evidências , Humanos , Ipilimumab , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Resultado do Tratamento
17.
Oncoimmunology ; 4(10): e1029698, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26504669

RESUMO

The RIG-I-like helicase melanoma differentiation-associated protein 5 (MDA5) is an innate immune receptor for double-stranded viral RNA (dsRNA) that, upon activation, induces a Type I interferon (IFN)-driven immune response. In the present study, we demonstrate that human und murine pancreatic cancer cells express functional MDA5 and are highly sensitive to MDA5-induced cell death. Activation of MDA5 by cytosolic delivery of the synthetic dsRNA analog poly(I:C) led to phosphorylation of the transcription factor IRF3, IFNß production and upregulation of MHC-I expression. MDA5 signaling also induced tumor cell apoptosis via the intrinsic pathway and sensitized tumor cells toward extrinsic, Fas-mediated apoptosis. Systemic treatment of orthotopic pancreatic cancer-bearing mice with the MDA5 ligand resulted in activated CD8+ T cell tumor infiltration, an increased frequency of tumor antigen-specific CD8+ T cells and an immunogenic cytokine milieu in the tumor microenvironment. These effects were paralleled by MDA5-induced pronounced tumor cell death in situ and significantly prolonged survival in two different mouse models for pancreatic cancer, an immunotherapeutic response dependent on CD8+ T cells. Treated mice were further protected from subsequent tumor challenge. In summary, we identified MDA5 as a novel therapeutic target for overcoming apoptosis resistance and tumor-mediated immunosuppression in pancreatic cancer. MDA5 ligands link innate with adaptive immune mechanisms for effective tumor control.

18.
J Natl Cancer Inst ; 107(8)2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26105028

RESUMO

BACKGROUND: Adoptive T cell transfer (ACT) is currently under investigation for the treatment of metastatic cancer. Recent evidence suggests that the coinhibitory PD-1-PD-L1 axis plays a major role in ACT failure. We hypothesized that a new fusion receptor reverting PD-1-mediated inhibition into CD28 costimulation may break peripheral tolerance. METHODS: Different PD-1-CD28 fusion receptor constructs were created and retrovirally transduced into primary T cell receptor transgenic murine CD8(+) T cells specific for ovalbumin (OT-1). Cytokine release, proliferation, cytotoxicity, and tumor recognition were analyzed in vitro. Antitumor efficacy and mode of action were investigated in mice bearing subcutaneous tumors induced with the pancreatic carcinoma cell line Panc02 expressing the model antigen ovalbumin (Panc-OVA). For antitumoral efficacy, six to eight mice per group were used. All statistical tests are two-sided. RESULTS: Transduction of the PD-1-CD28 receptor constructs mediated enhanced cytokine release, T cell proliferation, and T cell-induced lysis of target tumor cells. The PD-1-CD28 receptor function was dependent on two of the CD28-signaling motifs and IFN-γ release. Treatment of mice with established Panc-OVA tumors with fusion receptor-transduced OT-1 T cells mediated complete tumor regression. Mice rejecting the tumor were protected upon subsequent rechallenge with either ovalbumin-positive or -negative tumors, indicative of a memory response and epitope spreading in nine of 11 mice vs none of the six naïve mice (P < .001). Treatment efficacy was associated with accumulation of IFN-γ-producing T cells and an increased ratio of CD8(+) T cells to immunosuppressive myeloid-derived suppressor cells in the tumors. CONCLUSIONS: Transduction of T cells with this new PD-1-CD28 receptor has the potential of breaking the PD-1-PD-L1-immunosuppressive axis in ACT.


Assuntos
Transferência Adotiva/métodos , Antígeno B7-H1/imunologia , Antígenos CD28/imunologia , Linfócitos T CD8-Positivos/imunologia , Terapia de Imunossupressão , Interferon gama/metabolismo , Ovalbumina/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Receptor de Morte Celular Programada 1/imunologia , Animais , Linhagem Celular Tumoral , Epitopos , Contagem de Linfócitos , Camundongos , Camundongos Transgênicos , Transdução de Sinais/imunologia , Transdução Genética , Resultado do Tratamento
19.
Immunotherapy ; 7(4): 363-76, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25917628

RESUMO

Despite continuous progress in the understanding of deregulated pathways in pancreatic cancer cells and development of targeted therapies, therapeutic advances with clinical benefit have been scarce over the last decades. The recent success of immunotherapy for some solid cancers has fueled optimism that this approach might also work for pancreatic cancer. However, a highly immunosuppressive microenvironment mediated by tumor, stromal and immune cells creates a major hurdle for immunotherapy. Mouse models have helped to unravel critical immunosuppressive mechanisms that could serve as novel therapeutic targets. Here we review new promising strategies that alone or in combination with other modalities, such as chemotherapy or irradiation, have the potential to lead to tumor immune control and finally better clinical outcome.


Assuntos
Quimiorradioterapia , Imunoterapia/métodos , Neoplasias Pancreáticas/terapia , Animais , Terapia Combinada , Modelos Animais de Doenças , Humanos , Terapia de Imunossupressão , Camundongos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/imunologia , Resultado do Tratamento , Microambiente Tumoral
20.
J Immunother ; 38(3): 116-26, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25751501

RESUMO

Sensitivity of carcinoma cells towards gemcitabine (Gem) has been linked to mitochondrial apoptotic proteins. Recently, we described synergistic efficacy of Gem-based chemoimmunotherapy and a dendritic cell (DC) tumor vaccine in a murine pancreatic carcinoma model. Here, we investigated the role of the mitochondrial proteins Bcl-2, Bcl-xL, and Bax for sensitization of pancreatic carcinoma cells toward T-cell-mediated cytotoxicity alone and in combination with Gem. Bcl-2 expression was silenced by siRNA in Panc02 pancreatic cancer cells expressing the model antigen ovalbumin (PancOVA). Tumor cells were treated with Gem and/or siRNA, and cytotoxicity induced by OVA-specific cytotoxic T lymphocytes (CTL) from OT-1 mice was assessed. Gem-induced and T-cell-induced cytotoxicity was also studied in human Colo357 pancreatic cancer cell lines overexpressing Bax or Bcl-xL. Apoptosis induction by Fas-activating antibody was measured by Annexin V staining. The in vivo capacity of Bcl-2 siRNA to augment CTL efficacy induced by DC vaccinations was assessed in C57BL/6 mice bearing PancOVA tumors. PancOVA cells treated with Bcl-2 siRNA were sensitized towards both Gem and T-cell-mediated killing; combination therapy exhibited an additive effect. Bax overexpression sensitized Colo357 cells to both Gem-mediated and T-cell-mediated cytotoxicity, whereas Bcl-xL overexpression was inhibitory. Combining Bcl-2 silencing with DC therapy improved tumor control in the PancOVA model in vivo without affecting the number of tumor-reactive CTL. In conclusion, expression of Bcl-2, Bcl-xL, and Bax in pancreatic tumor cells determines sensitivity towards both Gem-mediated and CTL-mediated toxicity. Bcl-2 silencing could be exploited therapeutically in tumor vaccine approaches.


Assuntos
Citotoxicidade Imunológica/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Vacinas Anticâncer , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Expressão Gênica , Inativação Gênica , Humanos , Imunoterapia , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Carga Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Receptor fas/genética , Receptor fas/metabolismo , Gencitabina
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