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1.
Cancer Immunol Res ; 11(4): 421-434, 2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36758176

RESUMEN

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 Pancreáticas
2.
Sci Immunol ; 6(61)2021 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-34272227

RESUMEN

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.


Asunto(s)
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/genética
3.
Nat Biomed Eng ; 5(11): 1246-1260, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34083764

RESUMEN

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.


Asunto(s)
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/genética
4.
Cancer Immunol Immunother ; 69(10): 2101-2112, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32448983

RESUMEN

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 Cultivadas
5.
Leukemia ; 34(4): 1017-1026, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31740809

RESUMEN

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.


Asunto(s)
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 Tratamiento
6.
J Immunother Cancer ; 7(1): 349, 2019 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-31843014

RESUMEN

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.
Nat Commun ; 10(1): 5056, 2019 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-31699983

RESUMEN

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.


Asunto(s)
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 , Nanotubos
8.
J Immunother Cancer ; 7(1): 288, 2019 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-31694706

RESUMEN

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.

9.
Clin Cancer Res ; 25(19): 5890-5900, 2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-31285373

RESUMEN

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.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Receptores ErbB/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias Pancreáticas/terapia , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/genética , Molécula de Adhesión Celular Epitelial/inmunología , Molécula de Adhesión Celular Epitelial/metabolismo , Humanos , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Mesotelina , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Neoplasias Pancreáticas/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Br J Cancer ; 120(1): 79-87, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30429531

RESUMEN

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/inmunología
11.
Transl Oncol ; 12(2): 350-360, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30502589

RESUMEN

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.

12.
Semin Immunol ; 40: 61-73, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30268598

RESUMEN

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 Señal
13.
Front Immunol ; 9: 1955, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30214445

RESUMEN

Background: Interaction of the programmed death receptor 1 (PD-1) and its ligand, PD-L1, suppresses T cell activity and permits tumors to evade T cell-mediated immune surveillance. We have recently demonstrated that antigen-specific CD8+ T cells transduced with a PD1-CD28 fusion protein are protected from PD-1-mediated inhibition. We have now investigated the potential of PD1-CD28 fusion protein-transduced CD4+ T cells alone or in combination with CD8+ T cells for immunotherapy of pancreatic cancer and non-Hodgkin lymphoma. Methods: OVA-specific CD4+ and CD8+ were retrovirally transduced with the PD1-CD28 fusion protein. Cytokine release, proliferation, cytotoxic activity, and phenotype of transduced T cells were assessed in the context of Panc02-OVA (murine pancreatic cancer model) and E.G7-PD-L1 (murine T cell lymphoma model) cells. Results: Stimulation of PD1-CD28 fusion protein-transduced CD4+ T cells with anti-CD3 and recombinant PD-L1 induced specific T cell activation, as measured by IFN-y release and T cell proliferation. Coculture with Panc02-OVA or E.G7-PD-L1 tumor cells also led to specific activation of CD4+ T cells. Cytokine release and T cell proliferation was most effective when tumor cells simultaneously encountered genetically engineered CD4+ and CD8+ T cells. Synergy between both cell populations was also observed for specific tumor cell lysis. T cell cytotoxicity was mediated via granzyme B release and mediated enhanced tumor control in vivo. Transduced CD4+ and CD8+ T cells in co-culture with tumor cells developed a predominant central memory phenotype over time. Different ratios of CD4+ and CD8+ transduced T cells led to a significant increase of IFN-y and IL-2 secretion positively correlating with CD4+ T cell numbers used. Mechanistically, IL-2 and MHC-I were central to the synergistic activity of CD4+ and CD8+ T cells, since neutralization of IL-2 prevented the crosstalk between these cell populations. Conclusion: PD1-CD28 fusion protein-transduced CD4+ T cells significantly improved anti-tumoral effect of fusion protein-transduced CD8+ T cells. Thus, our results indicate that PD1-CD28 fusion protein-transduced CD4+ T cells have the potential to overcome the PD-1-PD-L1 immunosuppressive axis in pancreatic cancer and non-Hodgkin lymphoma.


Asunto(s)
Traslado Adoptivo , Antígenos CD28/inmunología , Linfocitos T CD8-positivos , Linfoma no Hodgkin/terapia , Neoplasias Experimentales/terapia , Neoplasias Pancreáticas/terapia , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Colaboradores-Inductores , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Antígenos CD28/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Receptor de Muerte Celular Programada 1/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/trasplante , Transducción Genética
14.
Sci Rep ; 8(1): 8810, 2018 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-29892077

RESUMEN

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.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos Nucleares/inmunología , Reacciones Cruzadas , Receptor de Muerte Celular Programada 1/inmunología , Animales , Línea Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones Endogámicos C57BL , Microscopía Confocal
15.
JCI Insight ; 3(5)2018 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-29515025

RESUMEN

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.


Asunto(s)
Diferenciación Celular/inmunología , Colitis/inmunología , Células Dendríticas/inmunología , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Colon/citología , Colon/inmunología , Colon/patología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Cadenas alfa de Integrinas/inmunología , Cadenas alfa de Integrinas/metabolismo , Interleucina-1beta/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Células Th17/inmunología , Células Th17/metabolismo , Células Th17/trasplante
16.
Cell ; 172(1-2): 162-175.e14, 2018 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-29328911

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/genética
17.
Front Immunol ; 8: 2000, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29403480

RESUMEN

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.

18.
Oncoimmunology ; 5(7): e1189051, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27622045

RESUMEN

Toll-like receptor 7 (TLR7) agonists are potent immune stimulants able to overcome cancer-associated immune suppression. Due to dose-limiting systemic toxicities, only the topically applied TLR7 agonist (imiquimod) has been approved for therapy of skin tumors. There is a need for TLR7-activating compounds with equivalent efficacy but less toxicity. SC1, a novel small molecule agonist for TLR7, is a potent type-1 interferon inducer, comparable to the reference TLR7 agonist resiquimod, yet with lower induction of proinflammatory cytokines. In vivo, SC1 activates NK cells in a TLR7-dependent manner. Mice bearing the NK cell-sensitive lymphoma RMA-S are cured by repeated s. c. administrations of SC1 as efficiently as by the administration of resiquimod. No relevant toxicities were observed. Mechanistically, SC1 reverses NK cell anergy and restores NK cell-mediated tumor cell killing in an IFN-α-dependent manner. TLR7 targeting by SC1-based compounds may form an attractive strategy to activate NK cell responses for cancer therapy.

19.
Cancer Lett ; 381(1): 259-68, 2016 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-26968250

RESUMEN

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.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Escape del Tumor/efectos de los fármacos , Animales , Vacunas contra el Cáncer/uso terapéutico , Humanos , Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fenotipo , Transducción de Señal/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Microambiente Tumoral
20.
Dtsch Arztebl Int ; 112(48): 809-15, 2015 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-26667979

RESUMEN

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.


Asunto(s)
Inmunoterapia/métodos , Terapia Molecular Dirigida/métodos , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Medicina Basada en la Evidencia , Humanos , Ipilimumab , Neoplasias/patología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Resultado del Tratamiento
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