RESUMEN
Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr-/- mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3-/-/Ldlr-/- mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.
Asunto(s)
Reprogramación Celular , Dieta Occidental , Epigénesis Genética , Inmunidad Innata , Memoria Inmunológica , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Adulto , Anciano , Animales , Células Cultivadas , Femenino , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Células Mieloides/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Sitios de Carácter Cuantitativo , Receptores de LDL/genéticaRESUMEN
The process of aging is associated with the appearance of low-grade subclinical inflammation, termed inflammaging, that can accelerate age-related diseases. In Western societies the age-related inflammatory response can additionally be aggravated by an inflammatory response related to modern lifestyles and excess calorie consumption, a pathophysiologic inflammatory response that was coined metaflammation. Here, we summarize the current knowledge of mechanisms that drive both of these processes and focus our discussion the emerging concept that a key innate immune pathway, the NLRP3 inflammasome, is centrally involved in the recognition of triggers that appear during physiological aging and during metabolic stress. We further discuss how these processes are involved in the pathogenesis of common age-related pathologies and highlight potential strategies by which the detrimental inflammatory responses could be pharmacologically addressed.
Asunto(s)
Envejecimiento , Inflamasomas/inmunología , Inflamación , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Fisiológico/inmunología , Animales , Citocinas/metabolismo , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Estilo de Vida , Transducción de SeñalRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/análisis , Linfocitos T CD8-positivos/inmunología , Factores Reguladores del Interferón/fisiología , Células Supresoras de Origen Mieloide/inmunología , Neoplasias Pancreáticas/inmunología , Microambiente Tumoral/inmunología , Animales , Apoptosis , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Terapia de Inmunosupresión , Ratones , Ratones Noqueados , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
BACKGROUND: CD16-chimeric antigen receptors (CAR) T cells recognise the Fc-portion of therapeutic antibodies, which can enable the selective targeting of different antigens. Limited evidence exists as to which CD16-CAR design and antibody partner might be most effective. We have hypothesised that the use of high-affinity CD16 variants, with increased Fc-terminus antibody affinity, combined with Fc-engineered antibodies, would provide superior CD16-CAR T cell efficacy. METHODS: CD16-CAR T (wild-type or variants) cells were co-cultured with Panc-1 pancreatic cancer, Raji lymphoma or A375 melanoma cells in the presence or absence of anti-CD20, anti-MCSP, wild-type or the glycoengineered antibody variants. The endpoints were proliferation, activation, and cytotoxicity in vitro. RESULTS: The CD16 158 V variant of CD16-CAR T cells showed increased cytotoxic activity against all the tested cancer cells in the presence of the wild-type antibody directed against MCSP or CD20. Glycoengineered antibodies enhanced CD16-CAR T cell activity irrespective of CD16 polymorphisms as compared with the wild-type antibody. The combination of the glycoengineered antibodies with the CD16-CAR 158 V variant synergised as seen by the increase in all endpoints. CONCLUSION: These results indicate that CD16-CAR with the high-affinity CD16 variant 158 V, combined with Fc-engineered antibodies, have high anti-tumour efficacy.
Asunto(s)
Inmunoterapia Adoptiva , Inmunoterapia , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Polimorfismo Genético , Receptores Quiméricos de Antígenos/uso terapéutico , Receptores de IgG/inmunología , Rituximab/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.
Asunto(s)
Colesterol/inmunología , Inmunidad/inmunología , Inflamasomas/inmunología , Interleucina-18/inmunología , Fosfolípidos/inmunología , Saponinas/inmunología , Adenosina Trifosfato/inmunología , Adenosina Trifosfato/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Western Blotting , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Colesterol/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Combinación de Medicamentos , Humanos , Inmunidad/efectos de los fármacos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Lisosomas/efectos de los fármacos , Lisosomas/inmunología , Lisosomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Fosfolípidos/farmacología , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Saponinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.
Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/patología , Proteínas Portadoras/metabolismo , Colesterol/química , Colesterol/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Aterosclerosis/inducido químicamente , Trasplante de Médula Ósea , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Catepsina B/metabolismo , Catepsina L/metabolismo , Colesterol/farmacología , Cristalización , Proteínas del Citoesqueleto/deficiencia , Dieta Aterogénica , Femenino , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Interleucina-1/deficiencia , Interleucina-18/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/patología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Cavidad Peritoneal/patología , Fagocitos/efectos de los fármacos , Fagocitos/patología , Fagocitos/fisiología , Receptores de LDL/deficiencia , Factores de TiempoRESUMEN
BACKGROUND: Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model. MATERIALS AND METHODS: Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8(+) T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively. RESULTS: Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8(+) T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8(+) T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination. CONCLUSIONS: Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy.
Asunto(s)
Adenocarcinoma/inmunología , Antimetabolitos Antineoplásicos/toxicidad , Linfocitos B/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Desoxicitidina/análogos & derivados , Terapia de Inmunosupresión , Neoplasias Pancreáticas/inmunología , Adenocarcinoma/tratamiento farmacológico , Animales , Especificidad de Anticuerpos , Antimetabolitos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/uso terapéutico , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/farmacología , Línea Celular Tumoral , Terapia Combinada , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Desoxicitidina/toxicidad , Ensayos de Selección de Medicamentos Antitumorales , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Fragmentos de Péptidos/inmunología , Escape del Tumor/efectos de los fármacos , GemcitabinaRESUMEN
Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Colesterol/administración & dosificación , Reactividad Cruzada/inmunología , Pruebas Inmunológicas de Citotoxicidad/métodos , Células Dendríticas/inmunología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Fosfolípidos/administración & dosificación , Saponinas/administración & dosificación , Animales , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Células Cultivadas , Colesterol/inmunología , Células Dendríticas/metabolismo , Combinación de Medicamentos , Femenino , Técnicas de Sustitución del Gen , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/patología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Fosfolípidos/inmunología , Quillaja/inmunología , Saponinas/inmunologíaRESUMEN
Intratumoral cytotoxic CD8+ T cells (CTL) enter a dysfunctional state characterized by expression of coinhibitory receptors, loss of effector function, and changes in the transcriptional landscape. Even though several regulators of T-cell exhaustion have been identified, the molecular mechanisms inducing T-cell exhaustion remain unclear. Here, we show that IL18 receptor (IL18R) signaling induces CD8+ T-cell exhaustion in a murine pancreatic cancer model. Adoptive transfer of Il18r-/- OT-1 CD8+ CTLs resulted in enhanced rejection of subcutaneous tumors expressing ovalbumin (OVA) as a model antigen (PancOVA), compared with wild-type OT-1 CTLs. Transferred intratumoral IL18R-deficient CTLs expressed higher levels of effector cytokines TNF and IFNγ and had reduced expression of coinhibitory receptors (PD-1, TIM-3, 2B4, LAG-3) and the transcription factors Eomes and TOX. Lower expression of coinhibitory receptors and TOX on IL18R-deficient versus IL18R-sufficient CD8+ T cells were confirmed in an orthotopic KPC model. IL18R-induced T-cell exhaustion was regulated by IL2/STAT5 and AKT/mTOR pathways, as demonstrated in an in vitro exhaustion assay. Concordantly, mice deficient in NLRP3, the molecular complex activating IL18, had decreased expression of coinhibitory receptors on intratumoral T cells and similar changes in signaling pathways at the transcriptome level. Thus, molecular pathways promoting T-cell exhaustion indicate an involvement of an NLRP3-expressing tumor microenvironment, which mediates IL18 release. The Cancer Genome Atlas analysis of patients with pancreatic carcinoma showed an association between NLRP3-mediated IL18 signaling and shorter survival. These findings indicate NLRP3-mediated IL18R signaling as a regulator of intratumoral T-cell exhaustion and a possible target for immunotherapy. See related Spotlight by Stromnes, p. 400.
Asunto(s)
Interleucina-18 , Neoplasias Pancreáticas , Ratones , Animales , Interleucina-2 , Agotamiento de Células T , Receptores de Interleucina-18 , Factor de Transcripción STAT5 , Proteína con Dominio Pirina 3 de la Familia NLR , Linfocitos T CD8-positivos/inmunología , Neoplasias Pancreáticas/genética , Serina-Treonina Quinasas TOR , Inflamación , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation due to dysregulation of the mucosal immune system. The cytokines IL-1ß and IL-18 appear early in intestinal inflammation and their pro-forms are processed via the caspase-1-activating multiprotein complex, the Nlrp3 inflammasome. Previously, we reported that the uptake of dextran sodium sulfate (DSS) by macrophages activates the Nlrp3 inflammasome and that Nlrp3(-/-) mice are protected in the acute DSS colitis model. Of note, other groups have reported opposing effects in regards to DSS susceptibility in Nlrp3(-/-) mice. Recently, mice lacking inflammasomes were found to develop a distinct intestinal microflora. METHODS: To reconcile the contradicting observations, we investigated the role of Nlrp3 deficiency in two different IBD models: acute DSS colitis and TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis. In addition, we investigated the impact of the intestinal flora on disease severity by performing cohousing experiments of wild-type and Nlrp3(-/-) mice, as well as by antibiotic treatment. RESULTS: Nlrp3(-/-) mice treated with either DSS or TNBS exhibited attenuated colitis and lower mortality. This protective effect correlated with an increased frequency of CD103+ lamina propria dendritic cells expressing a tolerogenic phenotype in Nlrp3(-/-) mice in steady state conditions. Interestingly, after cohousing, Nlrp3(-/-) mice were as susceptible as wild-type mice, indicating that transmission of endogenous bacterial flora between the two mouse strains might increase susceptibility of Nlrp3(-/-) mice towards DSS-induced colitis. Accordingly, treatment with antibiotics almost completely prevented colitis in the DSS model. CONCLUSIONS: The composition of the intestinal microflora significantly influences disease severity in IBD models comparing wild-type and Nlrp3(-/-) mice. This observation may - at least in part - explain contradictory results concerning the role of the inflammasome in different labs. Further studies are required to define the role of the Nlrp3 inflammasome in noninflamed mucosa under steady state conditions and in IBD.
Asunto(s)
Proteínas Portadoras/metabolismo , Ambiente , Predisposición Genética a la Enfermedad , Inflamasomas/metabolismo , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Sustancias Protectoras/metabolismo , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Colitis/inmunología , Colitis/patología , Colitis/fisiopatología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Tolerancia Inmunológica/inmunología , Inflamasomas/deficiencia , Inflamasomas/genética , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/patología , Intestinos/inmunología , Intestinos/microbiología , Intestinos/patología , Metagenoma , Ratones , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Proteína con Dominio Pirina 3 de la Familia NLR , Fenotipo , Ácido TrinitrobencenosulfónicoRESUMEN
Vaccines based on immune stimulatory complexes (ISCOM) induce T-cell responses against tumor antigen (Ag). However, immune responses are impaired in pancreatic cancer patients. We investigated the efficacy of an ISCOM vaccine in a murine pancreatic carcinoma model. Panc02 cells expressing OVA as a model Ag were induced subcutaneously or orthotopically in the pancreas of C57BL/6 mice. Treatment consisted of an OVA containing ISCOM vaccine, either used alone or in combination with the TLR9 agonist CpG. The ISCOM vaccine effectively induced Ag-specific CTL capable of killing tumor cells. However, in mice with established tumors CTL induction by the vaccine was inefficient and did not affect tumor growth. Lack of efficacy correlated with increased numbers of Treg. Depletion of Treg with anti-CD25 mAb restored CTL induction and prolonged survival. Adding low-dose CpG to the ISCOM vaccine reduced Treg numbers, enhanced CTL responses and induced regression of pancreatic tumors in a CD8(+) T cell-dependent manner. Mice cured from the primary tumor mounted a memory T-cell response against wild-type Panc02 tumors, indicative of epitope spreading. Combining ISCOM vaccines with TLR agonists is a promising strategy for breaking tumor immune evasion and deserves further evaluation for the treatment of pancreatic carcinoma.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Modelos Animales de Enfermedad , Inmunoterapia , Oligodesoxirribonucleótidos/uso terapéutico , Neoplasias Pancreáticas/inmunología , Linfocitos T Reguladores/inmunología , Receptor Toll-Like 9/agonistas , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Adenocarcinoma/prevención & control , Animales , Complejo Antígeno-Anticuerpo/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Evasión Inmune , Inmunización , Metástasis Linfática , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/genética , Ovalbúmina/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/prevención & control , Tasa de Supervivencia , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: The proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. Here, the role of the NLRP3 inflammasome in experimental colitis induced by dextran sodium sulfate (DSS) was examined. METHODS: IL-1beta production in response to DSS was studied in macrophages of wild-type, caspase-1(-/-), NLRP3(-/-), ASC(-/-), cathepsin B(-/-) or cathepsin L(-/-) mice. Colitis was induced in C57BL/6 and NLRP3(-/-) mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. RESULTS: Macrophages incubated with DSS in vitro secreted high levels of IL-1beta in a caspase-1-dependent manner. IL-1beta secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1beta secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3(-/-) mice developed a less severe colitis than wild-type mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection comparable with NLRP3 deficiency. CONCLUSIONS: The NLRP3 inflammasome was identified as a critical mechanism of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for patients with IBD.
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Proteínas Portadoras/fisiología , Colitis/fisiopatología , Animales , Caspasa 1/fisiología , Colitis/inducido químicamente , Colitis/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/fisiopatología , Interleucina-1beta/metabolismo , Lisosomas/fisiología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
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2',5'-Oligoadenilato Sintetasa/inmunología , Endorribonucleasas/inmunología , Neoplasias/inmunología , Receptores de Ácido Retinoico/inmunología , 2',5'-Oligoadenilato Sintetasa/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Línea Celular Tumoral , Técnicas de Cocultivo , Proteína 58 DEAD Box/genética , Endorribonucleasas/genética , Humanos , Helicasa Inducida por Interferón IFIH1/genética , Ligandos , Ratones , Receptores Inmunológicos/genéticaRESUMEN
The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.
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Inmunoterapia Adoptiva , Neoplasias Pancreáticas , Receptores CXCR6/metabolismo , Linfocitos T , Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Mesotelina , Ratones , Neoplasias Pancreáticas/terapia , Receptores de Quimiocina/genéticaRESUMEN
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.
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Anticuerpos Neutralizantes/farmacología , Proteína 58 DEAD Box/inmunología , Inmunoterapia/métodos , Leucemia Mieloide Aguda/terapia , ARN Bicatenario/farmacología , Receptores Virales/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proteína 58 DEAD Box/genética , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Xenoinjertos , Humanos , Memoria Inmunológica/efectos de los fármacos , Interferones/genética , Interferones/inmunología , Isoinjertos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/mortalidad , Ratones , Receptores Virales/agonistas , Receptores Virales/genética , Inducción de Remisión , Transducción de Señal , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.
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Ácido Homogentísico/química , Microscopía Intravital/métodos , Activación de Macrófagos , Macrófagos/citología , Técnicas Fotoacústicas/métodos , Pigmentos Biológicos/química , Coloración y Etiquetado/métodos , Animales , Materiales Biocompatibles , Diferenciación Celular , Citocinas/metabolismo , Oro , Células HEK293 , Células HeLa , Humanos , L-Lactato Deshidrogenasa/metabolismo , Macrófagos/metabolismo , Melaninas , Ratones , Nanopartículas , NanotubosRESUMEN
Microphthalmia-associated transcription factor (MITF) is a key transcription factor in melanoma development and progression. MITF amplification and downregulation have been observed in a significant proportion of melanoma patients and correlate with clinical outcomes. Here, we have investigated the effect of MITF on melanoma chemokine expression and immune cell attraction. In B16F10 melanoma cells, MITF knockdown reduced expression of CXCL10, with concomitantly decreased attraction of immune cells and accelerated tumor outgrowth. Conversely, overexpression of MITF in YUMM1.1 melanoma cells also led to an increased immune cell attraction in vitro. Subcutaneous YUMM1.1 melanomas overexpressing MITF however showed a reduced immune infiltration of lymphocytes and an increased tumor growth. In human melanoma cell lines, silencing of MITF enhanced chemokine production and immune cell attraction, while overexpression of MITF led to lower immune cell attraction. In summary, our results show that MITF regulates chemokine expression in murine and in human melanoma cells, and affects in vivo immune cell attraction and tumor growth. These results reveal a functional relationship between MITF and immune cell infiltration, which may be exploited for cancer therapy.
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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.
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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.
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PURPOSE: Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing. EXPERIMENTAL DESIGN: BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell-induced lysis were characterized in three murine and human tumor models in vitro and in vivo. RESULTS: Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo. CONCLUSIONS: We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.