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Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.
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Neoplasias Hematológicas , Hematopoyesis , Inmunoconjugados , Antígenos Comunes de Leucocito , Animales , Femenino , Humanos , Masculino , Ratones , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/inmunología , Hematopoyesis/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Línea Celular Tumoral , Especificidad de AnticuerposRESUMEN
3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.
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MicroARNs , Poliadenilación , Regiones no Traducidas 3' , MicroARNs/genética , MicroARNs/metabolismo , RNA-Seq , ARN Mensajero/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD).
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Subunidad alfa del Receptor de Interleucina-3 , Leucemia Mieloide Aguda , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Epítopos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Inmunoterapia , Células Madre Hematopoyéticas/metabolismo , Inmunoterapia AdoptivaRESUMEN
Owing to Karl Landsteiner's discovery of blood groups, blood transfusions became safe cellular therapies in the early 1900s. Since then, cellular therapy made great advances from transfusions with unmodified cells to today's commercially available chimeric antigen receptor (CAR) T cells requiring complex manufacturing. Modern cellular therapy products can be improved using basic knowledge of cell biology and molecular genetics. Emerging genome engineering tools are becoming ever more versatile and precise and thus catalyze rapid progress towards programmable therapeutic cells that compute input and respond with defined output. Despite a large body of literature describing important functions of non-coding RNAs including microRNAs (miRNAs), the vast majority of cell engineering efforts focuses on proteins. However, miRNAs form an important layer of posttranscriptional regulation of gene expression. Here, we highlight examples of how miRNAs can successfully be incorporated into engineered cellular therapies.
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Tratamiento Basado en Trasplante de Células y Tejidos , MicroARNs , MicroARNs/genéticaRESUMEN
Elucidating the mechanisms by which immune cells become dysfunctional in tumors is critical to developing next-generation immunotherapies. We profiled proteomes of cancer tissue as well as monocyte/macrophages, CD4+ and CD8+ T cells, and NK cells isolated from tumors, liver, and blood of 48 patients with hepatocellular carcinoma. We found that tumor macrophages induce the sphingosine-1-phospate-degrading enzyme SGPL1, which dampened their inflammatory phenotype and anti-tumor function in vivo. We further discovered that the signaling scaffold protein AFAP1L2, typically only found in activated NK cells, is also upregulated in chronically stimulated CD8+ T cells in tumors. Ablation of AFAP1L2 in CD8+ T cells increased their viability upon repeated stimulation and enhanced their anti-tumor activity synergistically with PD-L1 blockade in mouse models. Our data reveal new targets for immunotherapy and provide a resource on immune cell proteomes in liver cancer.
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Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.
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Neoplasias , Agotamiento de Células T , Humanos , Linfocitos T CD8-positivos , Inmunoterapia , Linfocitos Infiltrantes de TumorRESUMEN
CD28 provides the prototypical costimulatory signal required for productive T-cell activation. Known molecular consequences of CD28 costimulation are mostly based on studies of protein signaling molecules. The microRNA cluster miR-17â¼92 is induced by T cell receptor stimulation and further enhanced by combined CD28 costimulation. We demonstrate that transgenic miR-17â¼92 cell-intrinsically largely overcomes defects caused by CD28 deficiency. Combining genetics, transcriptomics, bioinformatics, and biochemical miRNA:mRNA interaction maps we empirically validate miR-17â¼92 target genes that include several negative regulators of T cell activation. CD28-deficient T cells exhibit derepressed miR-17â¼92 target genes during activation. CRISPR/Cas9-mediated ablation of the miR-17â¼92 targets Pten and Nrbp1 in naive CD28-/- CD4+ T cells differentially increases proliferation and expression of the activation markers CD25 and CD44, respectively. Thus, we propose that miR-17â¼92 constitutes a central mediator for T cell activation, integrating signals by the TCR and CD28 costimulation by dampening multiple brakes that prevent T cell activation.
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Chronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/genética , Cirrosis Hepática/genética , Neoplasias Hepáticas/genética , Estudios Longitudinales , MicroARNs/genética , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
The CRISPR/Cas technology allows for genome editing in primary T cells. We herein describe the activation of primary murine CD4+ or CD8+ T cells, followed by electroporation with plasmid or ribonucleoproteins (RNP) for gene modification. Gene edited T cells can subsequently be transferred to host mice for in vivo studies or cultured in vitro for further characterization. This protocol enables sophisticated genetic analysis of T cells using commonly available virus-free reagents.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Sistemas CRISPR-Cas , Edición Génica , Plásmidos/genética , Ribonucleoproteínas/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Electroporación , Activación de Linfocitos , Ratones , Fenotipo , Plásmidos/metabolismo , Cultivo Primario de Células , Proyectos de Investigación , Ribonucleoproteínas/metabolismo , Flujo de TrabajoRESUMEN
Myoepithelial neoplasms of soft tissue are rare tumors with clinical, morphological, immunohistochemical, and genetic heterogeneity. The morphological spectrum of these tumors is broad, and the diagnosis often requires immunostaining to confirm myoepithelial differentiation. Rarely, tumors show a morphology that is typical for myoepithelial neoplasms, while the immunophenotype fails to confirm myoepithelial differentiation. For such lesions, the term "myoepithelioma-like" tumor was introduced. Recently, two cases of myoepithelioma-like tumors of the hands and one case of the foot were described with previously never reported OGT-FOXO gene fusions. Here, we report a 50-year-old woman, with a myoepithelial-like tumor localized in the soft tissue of the forearm and carrying a OGT-FOXO1 fusion gene. Our findings extend the spectrum of mesenchymal tumors involving members of the FOXO family of transcription factors and point to the existence of a family of soft tissue tumors that carry the gene fusion of the OGT-FOXO family.
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Biomarcadores de Tumor/genética , Proteína Forkhead Box O1/genética , Fusión Génica , Mioepitelioma/genética , N-Acetilglucosaminiltransferasas/genética , Neoplasias de los Tejidos Blandos/genética , Biomarcadores de Tumor/análisis , Femenino , Antebrazo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Mioepitelioma/química , Mioepitelioma/patología , Mioepitelioma/cirugía , Neoplasias de los Tejidos Blandos/química , Neoplasias de los Tejidos Blandos/patología , Neoplasias de los Tejidos Blandos/cirugía , Resultado del TratamientoRESUMEN
Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.
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Linfocitos T CD4-Positivos/inmunología , Ingeniería Celular/métodos , Factores de Transcripción Forkhead/genética , Edición Génica/métodos , Plásmidos/genética , Animales , Linfocitos T CD4-Positivos/trasplante , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Eliminación de Gen , Inmunoterapia Adoptiva/métodos , Antígenos Comunes de Leucocito/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.
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Aminopiridinas/uso terapéutico , Antineoplásicos/uso terapéutico , Morfolinas/uso terapéutico , Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Administración Oral , Aminopiridinas/administración & dosificación , Aminopiridinas/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proliferación Celular/efectos de los fármacos , Perros , Humanos , Ratones , Modelos Moleculares , Morfolinas/administración & dosificación , Morfolinas/farmacocinética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Desnudas , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The phosphoinositide 3-kinase γ (PI3Kγ) plays a major role in leukocyte recruitment during acute inflammation and has been proposed to inhibit classical macrophage activation by driving immunosuppressive gene expression. PI3Kγ plays an important role in diet-induced obesity and insulin resistance. In seeking to determine the underlying molecular mechanisms, we showed that PI3Kγ action in high-fat diet-induced inflammation and insulin resistance depended largely on its role in the control of adiposity, which was due to PI3Kγ activity in a nonhematopoietic cell type. However, PI3Kγ activity in leukocytes was required for efficient neutrophil recruitment to adipose tissue. Neutrophil recruitment was correlated with proinflammatory gene expression in macrophages in adipose tissue, which triggered insulin resistance early during the development of obesity. Our data challenge the concept that PI3Kγ is a general suppressor of classical macrophage activation and indicate that PI3Kγ controls macrophage gene expression by non-cell-autonomous mechanisms, the outcome of which is context-dependent.
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Tejido Adiposo/patología , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Dieta Alta en Grasa/efectos adversos , Inflamación/prevención & control , Resistencia a la Insulina , Leucocitos/enzimología , Obesidad/complicaciones , Animales , Perfilación de la Expresión Génica , Inflamación/etiología , Leucocitos/patología , Metabolismo de los Lípidos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/fisiopatología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Genomic alterations in GNAS, the gene coding for the Gαs heterotrimeric G protein, are associated with a large number of human diseases. Here, we explored the role of Gαs on stem cell fate decisions by using the mouse epidermis as a model system. Conditional epidermal deletion of Gnas or repression of PKA signalling caused a remarkable expansion of the stem cell compartment, resulting in rapid basal-cell carcinoma formation. In contrast, inducible expression of active Gαs in the epidermis caused hair follicle stem cell exhaustion and hair loss. Mechanistically, we found that Gαs-PKA disruption promotes the cell autonomous Sonic Hedgehog pathway stimulation and Hippo signalling inhibition, resulting in the non-canonical activation of GLI and YAP1. Our study highlights an important tumour suppressive function of Gαs-PKA, limiting the proliferation of epithelial stem cells and maintaining proper hair follicle homeostasis. These findings could have broad implications in multiple pathophysiological conditions, including cancer.
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Transformación Celular Neoplásica/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Folículo Piloso/citología , Células Madre/citología , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/genética , Cromograninas , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Proteínas Hedgehog/metabolismo , Vía de Señalización Hippo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Noqueados , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAP , Proteína con Dedos de Zinc GLI1RESUMEN
microRNAs (miRNA) are small regulatory RNAs exerting pleiotropic functions in virtually any immune cell-type. Dozens of miRNAs with a known function in the immune system constitute interesting drug targets for immunomodulation. Chemical modifications of nucleic acid-based miRNA mimics and inhibitors largely solved instability issues but delivery to immune cells remains a major challenge. However, recent success targeting the acidic tumor microenvironment is very promising for inflammatory diseases. Moreover, small molecules are being explored as an interesting alternative. Although RNA is often considered 'undruggable' by small molecules recent progress modulating miRNA function through small molecules is encouraging. Computational approaches even allow predictions about specific small molecule/RNA interactions. Finally, recent clinical success demonstrates that drugs targeting RNAs work in humans.
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Sistemas de Liberación de Medicamentos/tendencias , Marcación de Gen/tendencias , Inmunomodulación/fisiología , MicroARNs/genética , MicroARNs/inmunología , Animales , Humanos , MicroARNs/metabolismoRESUMEN
BACKGROUND: Tissue mast cell numbers are dynamically regulated by recruitment of progenitors from the vasculature. It is unclear whether progenitors are recruited during allergic sensitization and whether recruitment promotes allergic responses. OBJECTIVE: We sought to (1) determine the effect of mast cell recruitment on acute allergic responses and (2) to define the role of phosphoinositide 3-kinase (PI3K) isoforms in sequential steps to allergic responses. METHODS: Gene-targeted mice for PI3Kγ or PI3Kδ or mice treated with isoform-specific PI3K inhibitors (a novel PI3Kγ-specific inhibitor [NVS-PI3-4] and the PI3Kδ inhibitor IC87114) were used to monitor IgE-mediated mast cell recruitment, migration, adhesion by means of intravital microscopy, degranulation, TNF-α release, and subsequent endothelial cell activation in vivo or in bone marrow-derived mast cells. RESULTS: Functional PI3Kγ, but not PI3Kδ, was crucial for mast cell accumulation in IgE-challenged skin, TNF-α release from IgE/antigen-stimulated mast cells, and mast cell/endothelial interactions and chemotaxis. PI3Kγ-deficient bone marrow-derived mast cells did not adhere to the endothelium in TNF-α-treated cremaster muscle, whereas PI3Kδ was not required. Depletion of TNF-α blocked IgE-induced mast cell recruitment, which links tissue mast cell-derived cytokine release to endothelial activation and mast cell recruitment. Interference with mast cell recruitment protected against anaphylaxis and was superior to blockage of tissue mast cell degranulation. CONCLUSIONS: Interference with mast cell recruitment to exacerbated tissues provides a novel strategy to alleviate allergic reactions and surpassed attenuation of tissue mast cell degranulation. This results in prolonged drug action and allows for reduction of drug doses required to block anaphylaxis, an important feature for drugs targeting inflammatory disease in general.
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Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Hipersensibilidad/inmunología , Mastocitos/inmunología , Anafilaxia/tratamiento farmacológico , Anafilaxia/inmunología , Anafilaxia/metabolismo , Animales , Antialérgicos/uso terapéutico , Degranulación de la Célula/inmunología , Movimiento Celular , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Células Endoteliales/inmunología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Hipersensibilidad/tratamiento farmacológico , Hipersensibilidad/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.
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Linfocitos T CD4-Positivos/inmunología , Hepatitis Viral Animal/inmunología , Tejido Linfoide/inmunología , Bazo/inmunología , Traslado Adoptivo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Quimiocinas/genética , Quimiocinas/inmunología , Quimiocinas/metabolismo , Proteína Ligando Fas/deficiencia , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Citometría de Flujo , Hepatitis Viral Animal/metabolismo , Hepatitis Viral Animal/virología , Interferón gamma/deficiencia , Interferón gamma/genética , Interferón gamma/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , Tejido Linfoide/metabolismo , Tejido Linfoide/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Perforina/deficiencia , Perforina/genética , Perforina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Bazo/virología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Obesity is associated with a chronic low-grade inflammation, and specific antiinflammatory interventions may be beneficial for the treatment of type 2 diabetes and other obesity-related diseases. The lipid kinase PI3Kγ is a central proinflammatory signal transducer that plays a major role in leukocyte chemotaxis, mast cell degranulation, and endothelial cell activation. It was also reported that PI3Kγ activity within hematopoietic cells plays an important role in obesity-induced inflammation and insulin resistance. Here, we show that protection from insulin resistance, metabolic inflammation, and fatty liver in mice lacking functional PI3Kγ is largely consequent to their leaner phenotype. We also show that this phenotype is largely based on decreased fat gain, despite normal caloric intake, consequent to increased energy expenditure. Furthermore, our data show that PI3Kγ action on diet-induced obesity depends on PI3Kγ activity within a nonhematopoietic compartment, where it promotes energetic efficiency for fat mass gain. We also show that metabolic modulation by PI3Kγ depends on its lipid kinase activity and might involve kinase-independent signaling. Thus, PI3Kγ is an unexpected but promising drug target for the treatment of obesity and its complications.
Asunto(s)
Tejido Adiposo Blanco/enzimología , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Resistencia a la Insulina/fisiología , Obesidad/enzimología , Termogénesis/fisiología , Animales , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Dieta Alta en Grasa/efectos adversos , Hígado Graso/enzimología , Hígado Graso/etiología , Hígado Graso/prevención & control , Inflamación/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Obesidad/etiología , Obesidad/prevención & control , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Esterol Esterasa/metabolismo , Delgadez/enzimologíaRESUMEN
PURPOSE: This work employs pharmacological targeting of phosphoinositide 3-kinases (PI3K) in selected neuroblastoma (NB) tumors with the inhibitor AS605240, which has been shown to express low toxicity and relative specificity for the PI3K species γ. METHODS: The expression pattern of PI3K isoforms in 7 NB cell lines and 14 tumor patient samples was determined by Western blotting and immunocytochemistry. The effect of AS605240 on the growth of four selected tumor cell lines was assessed. Two cell lines exhibiting (SK-N-LO) or lacking (SK-N-AS) PI3Kγ expression were chosen for further in vitro analysis, which involved propidium iodide (PI)-based cell cycle staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL-staining) of apoptotic cells and analysis of PI3K/Akt-related signaling pathways via Western blotting and translocation experiments. The action of AS605240 in vivo was addressed by xenograft experiments in severe combined immunodeficiency (SCID) mice, thereby comparing SK-N-LO and SK-N-AS derived tumors. Apoptosis induced in SK-N-LO tumors was shown by immunohistochemical TUNEL-staining. RESULTS: Significant expression of PI3Kγ in neuroblastoma patient biopsies and tumor cell lines was detected. AS605240 induced apoptosis in NB cell lines proportional to this expression and suppressed growth of PI3Kγ positive, but not negative, tumors in a xenograft mouse model. No adverse effects of the inhibitor treatment were observed. CONCLUSIONS: Our observations hint to an oncogenic function of PI3Kγ in distinct neuroblastoma entities and reveal PI3K targeting by AS605240 as a promising molecular therapy of these tumors.
Asunto(s)
Neuroblastoma/prevención & control , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinoxalinas/farmacología , Tiazolidinedionas/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones SCID , Neuroblastoma/enzimología , Neuroblastoma/patología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Phosphoinositide 3-kinases (PI3K) are key molecular players in male fertility. However, the specific roles of different p110 PI3K catalytic subunits within the spermatogenic lineage have not been characterized so far. Herein, we report that male mice expressing a catalytically inactive p110beta develop testicular hypotrophy and impaired spermatogenesis, leading to a phenotype of oligo-azoospermia and defective fertility. The examination of testes from p110beta-defective tubules demonstrates a widespread loss in spermatogenic cells, due to defective proliferation and survival of pre- and postmeiotic cells. In particular, p110beta is crucially needed in c-Kit-mediated spermatogonial expansion, as c-Kit-positive cells are lost in the adult testis and activation of Akt by SCF is blocked by a p110beta inhibitor. These data establish that activation of the p110beta PI3K isoform by c-Kit is required during spermatogenesis, thus opening the way to new treatments for c-Kit positive testicular cancers.