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1.
Cell Rep Methods ; 3(4): 100460, 2023 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-37159663

RESUMEN

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.


Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Saco Vitelino , Células Madre Hematopoyéticas , Organoides , Actividades Cotidianas
2.
Cell Rep ; 40(5): 111153, 2022 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-35926468

RESUMEN

Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.


Asunto(s)
Neoplasias , Linfocitos T , Presentación de Antígeno , Sistemas CRISPR-Cas/genética , Humanos , Neoplasias/genética , Oncogenes , Análisis de Sistemas
3.
Med ; 3(10): 682-704.e8, 2022 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-36007524

RESUMEN

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.


Asunto(s)
Linfocitos Infiltrantes de Tumor , Linfocitos T , Traslado Adoptivo , Animales , Citocinas/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , Ratones
4.
Curr Opin Immunol ; 74: 39-45, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34710751

RESUMEN

The administration of T cells as cellular therapy against advanced cancers has brought clinical benefit to many patients and has progressed the field of cancer research. However, current cell therapy treatments are not curative in most patients, particularly in those with solid tumors, and it remains to be seen how broadly and efficaciously they may be applied going forward. Recent research has begun to elucidate key factors that regulate the efficacy of cell therapy in cancer patients, including T cell stemness and the ability to effectively target tumor antigens and overcome tumor heterogeneity. In this review, we discuss key properties of clinically effective anti-cancer T cell therapies along with strategies to improve T cell characteristics to augment clinical efficacy in solid tumors.


Asunto(s)
Neoplasias , Linfocitos T , Antígenos de Neoplasias , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Factores Inmunológicos/uso terapéutico , Inmunoterapia , Inmunoterapia Adoptiva , Neoplasias/patología
5.
Cancer Cell ; 37(6): 818-833.e9, 2020 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-32516591

RESUMEN

T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.


Asunto(s)
Neoplasias de la Mama/terapia , Sistemas CRISPR-Cas , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/terapia , Fenotipo , Linfocitos T/trasplante , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Diferenciación Celular , Femenino , Ingeniería Genética , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/genética
6.
J Exp Med ; 216(11): 2619-2634, 2019 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-31405895

RESUMEN

Host conditioning has emerged as an important component of effective adoptive cell transfer-based immunotherapy for cancer. High levels of IL-1ß are induced by host conditioning, but its impact on the antitumor function of T cells remains unclear. We found that the administration of IL-1ß increased the population size and functionality of adoptively transferred T cells within the tumor. Most importantly, IL-1ß enhanced the ability of tumor-specific T cells to trigger the regression of large, established B16 melanoma tumors in mice. Mechanistically, we showed that the increase in T cell numbers was associated with superior tissue homing and survival abilities and was largely mediated by IL-1ß-stimulated host cells. In addition, IL-1ß enhanced T cell functionality indirectly via its actions on radio-resistant host cells in an IL-2- and IL-15-dependent manner. Our findings not only underscore the potential of provoking inflammation to enhance antitumor immunity but also uncover novel host regulations of T cell responses.


Asunto(s)
Inmunoterapia Adoptiva/métodos , Interleucina-1beta/inmunología , Activación de Linfocitos/inmunología , Melanoma Experimental/terapia , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Línea Celular Tumoral , Citocinas/sangre , Interleucina-1beta/metabolismo , Interleucina-6/sangre , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T/metabolismo , Linfocitos T/trasplante , Factor de Necrosis Tumoral alfa/sangre
7.
PLoS Genet ; 15(5): e1008020, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31125342

RESUMEN

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity.


Asunto(s)
Inmunidad Adaptativa , Enfermedades Autoinmunes del Sistema Nervioso/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Malformaciones del Sistema Nervioso/genética , Ribonucleasa H/genética , Animales , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/mortalidad , Enfermedades Autoinmunes del Sistema Nervioso/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Metástasis Linfática , Ratones , Ratones Desnudos , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/mortalidad , Malformaciones del Sistema Nervioso/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Ribonucleasa H/antagonistas & inhibidores , Ribonucleasa H/inmunología , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de Supervivencia , Linfocitos T/inmunología , Linfocitos T/patología
8.
J Clin Invest ; 129(4): 1551-1565, 2019 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-30694219

RESUMEN

Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.


Asunto(s)
Traslado Adoptivo , Ingeniería Genética , Neoplasias Experimentales/terapia , Receptores Quiméricos de Antígenos , Transducción de Señal/inmunología , Microambiente Tumoral/inmunología , Animales , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/uso terapéutico , Transducción de Señal/genética , Microambiente Tumoral/genética , Receptor fas/genética , Receptor fas/inmunología
9.
Nat Commun ; 9(1): 2611, 2018 07 04.
Artículo en Inglés | MEDLINE | ID: mdl-29973593

RESUMEN

Tumor-derived soluble factors promote the production of Gr-1+CD11b+ immature myeloid cells, and TGFß signaling is critical in their immune suppressive function. Here, we report that miR-130a and miR-145 directly target TGFß receptor II (TßRII) and are down-regulated in these myeloid cells, leading to increased TßRII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decreased tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNγ-producing cytotoxic CD8 T lymphocytes. miR-130a- and miR-145-targeted molecular networks including TGFß and IGF1R pathways were correlated with higher tumor stages in cancer patients. Lastly, miR-130a and miR-145 mimics, as well as IGF1R inhibitor NT157 improved anti-tumor immunity and inhibited metastasis in preclinical mouse models. These results demonstrated that miR-130a and miR-145 can reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Inmunidad Innata/efectos de los fármacos , Neoplasias Pulmonares/genética , Neoplasias Mamarias Experimentales/genética , MicroARNs/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Animales , Antineoplásicos/farmacología , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inyecciones Intravenosas , Interferón gamma/genética , Interferón gamma/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Transgénicos , MicroARNs/inmunología , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/patología , Oligorribonucleótidos/administración & dosificación , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Pirogalol/análogos & derivados , Pirogalol/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta/inmunología , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Transducción de Señal , Sulfonamidas/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología
10.
Nat Commun ; 9(1): 1944, 2018 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-29789598

RESUMEN

Cancer recurrence after initial diagnosis and treatment is a major cause of breast cancer (BC) mortality, which results from the metastatic outbreak of dormant tumour cells. Alterations in the tumour microenvironment can trigger signalling pathways in dormant cells leading to their proliferation. However, processes involved in the initial and the long-term survival of disseminated dormant BC cells remain largely unknown. Here we show that autophagy is a critical mechanism for the survival of disseminated dormant BC cells. Pharmacologic or genetic inhibition of autophagy in dormant BC cells results in significantly decreased cell survival and metastatic burden in mouse and human 3D in vitro and in vivo preclinical models of dormancy. In vivo experiments identify autophagy gene autophagy-related 7 (ATG7) to be essential for autophagy activation. Mechanistically, inhibition of the autophagic flux in dormant BC cells leads to the accumulation of damaged mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis.


Asunto(s)
Proteína 7 Relacionada con la Autofagia/genética , Autofagia/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/genética , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagia/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/antagonistas & inhibidores , Beclina-1/genética , Beclina-1/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/farmacología , Femenino , Humanos , Hidroxicloroquina/farmacología , Metástasis Linfática , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/metabolismo , Recurrencia , Transducción de Señal
11.
JCI Insight ; 2(23)2017 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-29212954

RESUMEN

Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR-modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.


Asunto(s)
Inmunoterapia Adoptiva/métodos , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores Quiméricos de Antígenos/inmunología , Subgrupos de Linfocitos T/inmunología , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular , Femenino , Proteína Forkhead Box O1/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Memoria Inmunológica , Selectina L/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/inmunología , Transducción Genética/métodos , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Nature ; 548(7669): 537-542, 2017 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-28783722

RESUMEN

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.


Asunto(s)
Genes Esenciales/genética , Inmunoterapia , Neoplasias/genética , Neoplasias/terapia , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Femenino , Genoma/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interferón gamma/inmunología , Janus Quinasa 1/metabolismo , Bases del Conocimiento , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/terapia , Ratones , Mutación , Neoplasias/inmunología , Neoplasias/metabolismo , Reproducibilidad de los Resultados , Linfocitos T Citotóxicos/metabolismo
13.
Nature ; 537(7621): 539-543, 2016 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-27626381

RESUMEN

Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.


Asunto(s)
Cationes Monovalentes/metabolismo , Melanoma/inmunología , Potasio/metabolismo , Linfocitos T/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Humanos , Tolerancia Inmunológica/inmunología , Inmunoterapia/métodos , Canal de Potasio Kv1.3/metabolismo , Masculino , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Potenciales de la Membrana , Ratones , Necrosis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Análisis de Supervivencia , Linfocitos T/metabolismo , Serina-Treonina Quinasas TOR/metabolismo
14.
PLoS Pathog ; 12(2): e1005442, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26915097

RESUMEN

Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic Trypanosoma brucei parasites were compared in inos(-/-) and wild-type mice. Inos(-/-) mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In inos(-/-) mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Encefalitis/metabolismo , Macrófagos Peritoneales/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Animales , Citocinas/metabolismo , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/metabolismo , Trypanosoma brucei brucei/metabolismo
15.
J Med Chem ; 56(24): 9861-73, 2013 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-24283924

RESUMEN

Novel methods for treatment of African trypanosomiasis, caused by infection with Trypanosoma brucei are needed. Cordycepin (3'-deoxyadenosine, 1a) is a powerful trypanocidal compound in vitro but is ineffective in vivo because of rapid metabolic degradation by adenosine deaminase (ADA). We elucidated the structural moieties of cordycepin required for trypanocidal activity and designed analogues that retained trypanotoxicity while gaining resistance to ADA-mediated metabolism. 2-Fluorocordycepin (2-fluoro-3'-deoxyadenosine, 1b) was identified as a selective, potent, and ADA-resistant trypanocidal compound that cured T. brucei infection in mice. Compound 1b is transported through the high affinity TbAT1/P2 adenosine transporter and is a substrate of T. b. brucei adenosine kinase. 1b has good preclinical properties suitable for an oral drug, albeit a relatively short plasma half-life. We present a rapid and efficient synthesis of 2-halogenated cordycepins, also useful synthons for the development of additional novel C2-substituted 3'-deoxyadenosine analogues to be evaluated in development of experimental therapeutics.


Asunto(s)
Desoxiadenosinas/farmacología , Tripanocidas/farmacología , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Desoxiadenosinas/síntesis química , Desoxiadenosinas/química , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/química , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei rhodesiense/efectos de los fármacos
16.
J Biol Chem ; 288(38): 27456-27468, 2013 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-23900839

RESUMEN

Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis.


Asunto(s)
NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Tiazoles/farmacología , Tripanocidas/farmacología , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Masculino , Ratones , NADH NADPH Oxidorreductasas/metabolismo , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas Protozoarias/metabolismo , Tiazoles/química , Tripanocidas/química , Tripanosomiasis Africana/enzimología
17.
Antimicrob Agents Chemother ; 57(2): 1012-8, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23254423

RESUMEN

New drugs for the treatment of human African trypanosomiasis are urgently needed. A number of 2-aminopyrazines/2-aminopyridines were identified as promising leads following a focused screen of 5,500 compounds for Trypanosoma brucei subsp. brucei viability. Described compounds are trypanotoxic in the submicromolar range and show comparably low cytotoxicity on representative mammalian cell lines. Specifically, 6-([6-fluoro-3,4-dihydro-2H-1-benzopyran-4-yl)]oxy)-N-(piperidin-4-yl)pyrazin-2-amine (CBK201352) is trypanotoxic for T. brucei subsp. brucei, T. brucei subsp. gambiense, and T. brucei subsp. rhodesiense and is nontoxic to mammalian cell lines, and in vitro preclinical assays predict promising pharmacokinetic parameters. Mice inoculated intraperitoneally (i.p.) with 25 mg/kg CBK201352 twice daily for 10 days, starting on the day of infection with T. brucei subsp. brucei, show complete clearance of parasites for more than 90 days. Thus, CBK201352 and related analogs are promising leads for the development of novel treatments for human African trypanosomiasis.


Asunto(s)
Aminopiridinas/farmacología , Benzopiranos/farmacología , Piperidinas/farmacología , Pirazinas/farmacología , Tripanocidas/farmacología , Tripanosomiasis Africana/tratamiento farmacológico , Aminopiridinas/uso terapéutico , Animales , Benzopiranos/uso terapéutico , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Piperidinas/uso terapéutico , Pirazinas/uso terapéutico , Tripanocidas/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei rhodesiense/efectos de los fármacos , Tripanosomiasis Africana/parasitología
18.
J Infect Dis ; 205(2): 320-32, 2012 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22116836

RESUMEN

BACKGROUND: The penetration of T cells and trypanosomes into the brain parenchyma is a major pathogenetic event in African trypanosomiasis. METHODS: The role of innate immune responses in the penetration of T cells and Trypanosoma brucei brucei into the brain was studied in knockout mice by using double immunofluorescent staining and real-time polymerase chain reaction. RESULTS: We demonstrate that Toll-like receptor (TLR)-MyD88-mediated signaling is required for T-cell and parasite penetration into the brain and microglial activation, besides controlling parasitemia and antigen-specific T-cell activation. Among different TLR-deficient mice studied, TLR9 mediated parasitemia control and T-cell penetration into the brain. TLR-MyD88 signals increased levels of interferon (IFN) ß and tumor necrosis factor (TNF) α transcripts in the brains of infected mice and both TNF-α and IFN-α/ß, receptors promoted T-cell and trypanosoma infiltration into the brain parenchyma. Both resident and infiltrating inflammatory cells in the brain controlled parasite densities in a TLR2- and TLR9-MyD88-mediated manner. However, neither IFN-α/ß nor TNF-α contributed to parasite control in the brain. CONCLUSIONS: Our data indicate that innate immune TLR signals stimulate the expression of TNF-α and IFN-α/ß that initiate brain invasion of T cells and trypanosomes, and control T. brucei brucei load in the brain by molecules distinct from these.


Asunto(s)
Encéfalo/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , ARN Mensajero/metabolismo , Trypanosoma brucei brucei , Tripanosomiasis Africana/inmunología , Animales , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/parasitología , Encéfalo/parasitología , Encéfalo/patología , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Inmunidad Innata , Interferón beta/metabolismo , Interferón gamma/sangre , Interferón gamma/inmunología , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Carga de Parásitos , Parasitemia/inmunología , Receptor de Interferón alfa y beta/inmunología , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 9/metabolismo , Tripanosomiasis Africana/metabolismo , Tripanosomiasis Africana/parasitología , Factor de Necrosis Tumoral alfa/metabolismo
19.
PLoS Negl Trop Dis ; 3(8): e495, 2009 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-19652702

RESUMEN

BACKGROUND: There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.). METHODOLOGY/PRINCIPAL FINDINGS: Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug. CONCLUSIONS/SIGNIFICANCE: Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

20.
PLoS Negl Trop Dis ; 3(7): e486, 2009 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-19621071

RESUMEN

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs.


Asunto(s)
Testículo/parasitología , Trypanosoma brucei brucei/aislamiento & purificación , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/veterinaria , Animales , Femenino , Genes Reporteros , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo , Masculino , Ratones , Coloración y Etiquetado/métodos , Tripanocidas/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología
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