RESUMEN
Tumor-associated myeloid cells (TAMCs) are key drivers of immunosuppression in the tumor microenvironment, which profoundly impedes the clinical response to immune-dependent and conventional therapeutic modalities. As a hallmark of glioblastoma (GBM), TAMCs are massively recruited to reach up to 50% of the brain tumor mass. Therefore, they have recently been recognized as an appealing therapeutic target to blunt immunosuppression in GBM with the hope of maximizing the clinical outcome of antitumor therapies. Here we report a nano-immunotherapy approach capable of actively targeting TAMCs in vivo. As we found that programmed death-ligand 1 (PD-L1) is highly expressed on glioma-associated TAMCs, we rationally designed a lipid nanoparticle (LNP) formulation surface-functionalized with an anti-PD-L1 therapeutic antibody (αPD-L1). We demonstrated that this system (αPD-L1-LNP) enabled effective and specific delivery of therapeutic payload to TAMCs. Specifically, encapsulation of dinaciclib, a cyclin-dependent kinase inhibitor, into PD-L1-targeted LNPs led to a robust depletion of TAMCs and an attenuation of their immunosuppressive functions. Importantly, the delivery efficiency of PD-L1-targeted LNPs was robustly enhanced in the context of radiation therapy (RT) owing to the RT-induced up-regulation of PD-L1 on glioma-infiltrating TAMCs. Accordingly, RT combined with our nano-immunotherapy led to dramatically extended survival of mice in 2 syngeneic glioma models, GL261 and CT2A. The high targeting efficiency of αPD-L1-LNP to human TAMCs from GBM patients further validated the clinical relevance. Thus, this study establishes a therapeutic approach with immense potential to improve the clinical response in the treatment of GBM and warrants a rapid translation into clinical practice.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Mieloides/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Óxidos N-Cíclicos , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , Humanos , Indolizinas , Ratones , Células Mieloides/efectos de los fármacos , Células Mieloides/efectos de la radiación , Nanopartículas , Compuestos de Piridinio/administración & dosificación , Compuestos de Piridinio/uso terapéutico , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Amino acid deprivation is a strategy that malignancies utilize to blunt anti-tumor T-cell immune responses. It has been proposed that amino acid insufficiency in T-cells is detected by GCN2 kinase, which through phosphorylation of EIF2α, shuts down global protein synthesis leading to T-cell arrest. The role of this amino acid stress sensor in the context of malignant brain tumors has not yet been studied, and may elucidate important insights into the mechanisms of T-cell survival in this harsh environment. Using animal models of glioblastoma and animals with deficiency in GCN2, we explored the importance of this pathway in T-cell function within brain tumors. Our results show that GCN2 deficiency limited CD8+ T-cell activation and expression of cytotoxic markers in two separate murine models of glioblastoma in vivo. Importantly, adoptive transfer of antigen-specific T-cells from GCN2 KO mice did not control tumor burden as well as wild-type CD8+ T-cells. Our in vitro and in vivo data demonstrated that reduction in amino acid availability caused GCN2 deficient CD8+ T-cells to become rapidly necrotic. Mechanistically, reduced CD8+ T-cell activation and necrosis was due to a disruption in TCR signaling, as we observed reductions in PKCθ and phoshpo-PKCθ on CD8+ T-cells from GCN2 KO mice in the absence of tryptophan. Validating these observations, treatment of wild-type CD8+ T-cells with a downstream inhibitor of GCN2 activation also triggered necrosis of CD8+ T-cells in the absence of tryptophan. In conclusion, our data demonstrate the vital importance of intact GCN2 signaling on CD8+ T-cell function and survival in glioblastoma.
Asunto(s)
Neoplasias Encefálicas/inmunología , Linfocitos T CD8-positivos/inmunología , Glioblastoma/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Escape del Tumor/inmunología , Traslado Adoptivo , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/trasplante , Línea Celular Tumoral/trasplante , Supervivencia Celular/inmunología , Modelos Animales de Enfermedad , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Activación de Linfocitos , Ratones , Ratones Noqueados , Necrosis/genética , Necrosis/inmunología , Fosforilación/inmunología , Biosíntesis de Proteínas/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunologíaRESUMEN
Although radiotherapy has been established as a major therapeutic modality for glioma, radical new avenues are critically needed to prevent inevitable tumor recurrence. Herein, we utilized a magnetic nanoparticle-based platform with cationic polymer modification to promote radiotherapy for glioma treatment. We found that the nanoplatform induced cytotoxicity to glioma cells under radiation as well as promoting significant survival benefits in both immunocompetent and aythmic mice with glioma. Utilizing the magnetic properties of the nanoparticles, we were able to ascertain that myeloid derived suppressor cells (MDSC) were taking up nanoparticles in the brain tumor. The observed efficacy was attributed to destruction of glioma cells as well as MDSCs repolarization from immunosuppressive phenotype to a pro-inflammatory phenotype, which promoted antitumor effects and synergistically promoted radio-therapeutic effects. Our nanoparticles provide a robust dual-targeting platform for glioma radiotherapy by simultaneous eradication of tumor cells and manipulation of myeloid phenotypes in the central nervous system.
Asunto(s)
Glioma/terapia , Células Supresoras de Origen Mieloide/patología , Nanopartículas/química , Radioterapia/métodos , Animales , Compuestos Férricos/química , Ratones , Ratones Endogámicos C57BLRESUMEN
The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher-Thy1.1-yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.
Asunto(s)
Leucodistrofia de Células Globoides/complicaciones , Leucodistrofia de Células Globoides/patología , Enfermedades Neuromusculares/etiología , Enfermedades Neuromusculares/metabolismo , Animales , Animales Recién Nacidos , Axones/metabolismo , Axones/patología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Perros , Galactosilceramidasa/genética , Regulación de la Expresión Génica/genética , Leucodistrofia de Células Globoides/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Contracción Muscular/genética , Músculo Esquelético/crecimiento & desarrollo , Enfermedades Neuromusculares/patología , Psicosina/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Potenciales Sinápticos/efectos de los fármacos , Potenciales Sinápticos/genéticaRESUMEN
The membrane-bound receptor for platelet-derived growth factor A (PDGFRα) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFRα in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFRα function. PDGFRα is less associated with detergent-resistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFRα levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFRα in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFRα levels in OLs and its impact in myelination.
Asunto(s)
Cerebrósido Sulfatasa/genética , Ácidos Grasos/metabolismo , Leucodistrofia Metacromática/patología , Células-Madre Neurales/patología , Oligodendroglía/patología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Animales , Células Cultivadas , Cerebrósido Sulfatasa/metabolismo , Exosomas/genética , Exosomas/metabolismo , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/metabolismo , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis , Oligodendroglía/citología , Oligodendroglía/metabolismo , Proteolisis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Transducción de Señal , Transcripción GenéticaRESUMEN
Demyelination is a major contributor to the general decay of neural functions in children with Krabbe disease. However, recent reports have indicated a significant involvement of neurons and axons in the neuropathology of the disease. In this study, we have investigated the nature of cellular inclusions in the Krabbe brain. Brain samples from the twitcher mouse model for Krabbe disease and from patients affected with the infantile and late-onset forms of the disease were examined for the presence of neuronal inclusions. Our experiments demonstrated the presence of cytoplasmic aggregates of thioflavin-S-reactive material in both human and murine mutant brains. Most of these inclusions were associated with neurons. A few inclusions were detected to be associated with microglia and none were associated with astrocytes or oligodendrocytes. Thioflavin-S-reactive inclusions increased in abundance, paralleling the development of neurological symptoms, and distributed throughout the twitcher brain in areas of major involvement in cognition and motor functions. Electron microscopy confirmed the presence of aggregates of stereotypic ß-sheet folded proteinaceous material. Immunochemical analyses identified the presence of aggregated forms of α-synuclein and ubiquitin, proteins involved in the formation of Lewy bodies in Parkinson's disease and other neurodegenerative conditions. In vitro assays demonstrated that psychosine, the neurotoxic sphingolipid accumulated in Krabbe disease, accelerated the fibrillization of α-synuclein. This study demonstrates the occurrence of neuronal deposits of fibrillized proteins including α-synuclein, identifying Krabbe disease as a new α-synucleinopathy.
Asunto(s)
Encéfalo/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Cuerpos de Lewy/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Benzotiazoles , Encéfalo/fisiopatología , Encéfalo/ultraestructura , Estudios de Casos y Controles , Cognición , Modelos Animales de Enfermedad , Colorantes Fluorescentes , Humanos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patología , Leucodistrofia de Células Globoides/fisiopatología , Leucodistrofia de Células Globoides/psicología , Cuerpos de Lewy/ultraestructura , Ratones , Actividad Motora , Mutación , Neuronas/ultraestructura , Psicosina/metabolismo , Tiazoles , alfa-Sinucleína/genéticaRESUMEN
Loss of function of galactosylceramidase lysosomal activity causes demyelination and vulnerability of various neuronal populations in Krabbe disease. Psychosine, a lipid-raft-associated sphingolipid that accumulates in this disease, is thought to trigger these abnormalities. Myelin-free in vitro analyses showed that psychosine inhibited fast axonal transport through the activation of axonal PP1 and GSK3ß in the axon. Abnormal levels of activated GSK3ß and abnormally phosphorylated kinesin light chains were found in nerve samples from a mouse model of Krabbe disease. Administration of GSK3ß inhibitors significantly ameliorated transport defects in vitro and in vivo in peripheral axons of the mutant mouse. This study identifies psychosine as a pathogenic sphingolipid able to block fast axonal transport and is the first to provide a molecular mechanism underlying dying-back degeneration in this genetic leukodystrophy.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Leucodistrofia de Células Globoides/patología , Proteínas Motoras Moleculares/metabolismo , Neuronas/patología , Psicosina/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Glucógeno Sintasa Quinasa 3 beta , Leucodistrofia de Células Globoides/tratamiento farmacológico , Leucodistrofia de Células Globoides/genética , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Nervio Ciático/patología , Factores de TiempoRESUMEN
Sulfated galactosylceramides (sulfatides) are glycosphingolipids associated with cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) and are highly expressed in brain tissue. Although it is known that sulfatide species show heterogeneity in their fatty acid acyl group composition throughout brain development, their lipid raft distribution and biological relevance is poorly understood. We validated a fast and sensitive ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to measure developmentally regulated sulfatide species (C16:0, C18:0, C24:1, and C24:0) in central nervous system (CNS) lipid rafts isolated without using detergent. Our UHPLC-MS/MS assay showed good accuracy and precision with a linear range of 5 to 1,000 nM for C18:0 and C24:1 sulfatides and 10 to 1,000 nM for C16:0 and C24:0 sulfatides. We applied this quantitative analysis to detergent-free lipid rafts isolated from wild-type mice and arylsulfatase A-deficient (ASA knockout) mice that accumulate sulfatides. All four sulfatide species were more abundant in raft membranes than in non-raft membranes, with a significant increase in lipid rafts isolated from ASA knockout mice. This is the first description of an analytical method to study these sulfatide species in raft and non-raft membranes and has the potential to be applied to preparations from other tissues.
Asunto(s)
Encéfalo/metabolismo , Cerebrósido Sulfatasa/fisiología , Microdominios de Membrana/metabolismo , Sulfoglicoesfingolípidos/análisis , Animales , Animales Recién Nacidos , Western Blotting , Colesterol/análisis , Cromatografía Líquida de Alta Presión , Ratones , Ratones Noqueados , Espectrometría de Masas en TándemRESUMEN
Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.
Asunto(s)
Glioblastoma , Ratones , Animales , Humanos , Glioblastoma/metabolismo , Creatina , Hipoxia/metabolismo , Células Mieloides/metabolismo , Células Progenitoras Mieloides , Línea Celular TumoralRESUMEN
As a key component of the standard of care for glioblastoma, radiotherapy induces several immune resistance mechanisms, such as upregulation of CD47 and PD-L1. Here, leveraging these radiotherapy-elicited processes, we generate a bridging-lipid nanoparticle (B-LNP) that engages tumor-associated myeloid cells (TAMCs) to glioblastoma cells via anti-CD47/PD-L1 dual ligation. We show that the engager B-LNPs block CD47 and PD-L1 and promote TAMC phagocytic activity. To enhance subsequent T cell recruitment and antitumor responses after tumor engulfment, the B-LNP was encapsulated with diABZI, a non-nucleotidyl agonist for stimulator of interferon genes. In vivo treatment with diABZI-loaded B-LNPs induced a transcriptomic and metabolic switch in TAMCs, turning these immunosuppressive cells into antitumor effectors, which induced T cell infiltration and activation in brain tumors. In preclinical murine models, B-LNP/diABZI administration synergized with radiotherapy to promote brain tumor regression and induce immunological memory against glioma. In summary, our study describes a nanotechnology-based approach that hijacks irradiation-triggered immune checkpoint molecules to boost potent and long-lasting antitumor immunity against glioblastoma.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Animales , Humanos , Ratones , Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Antígeno CD47/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , InterferonesRESUMEN
Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in other solid tumors suggest a central role of B-cell immunity in driving immune-checkpoint-blockade efficacy. Using single-cell and single-nuclei transcriptomics of human glioblastoma and melanoma brain metastasis, we found that tumor-associated B-cells have high expression of checkpoint molecules, known to block B-cell-receptor downstream effector function such as plasmablast differentiation and antigen-presentation. We also identified TGFß-1/TGFß receptor-2 interaction as a crucial modulator of B-cell suppression. Treatment of glioblastoma patients with pembrolizumab induced expression of B-cell checkpoint molecules and TGFß-receptor-2. Abrogation of TGFß using different conditional knockouts expanded germinal-center-like intratumoral B-cells, enhancing immune-checkpoint-blockade efficacy. Finally, blocking αVß8 integrin (which controls the release of active TGFß) and PD-1 significantly increased B-cell-dependent animal survival and immunological memory. Our study highlights the importance of intratumoral B-cell immunity and a remodeled approach to boost the effects of immunotherapy against brain tumors.
RESUMEN
Understanding the spatial relationship and functional interaction of immune cells in glioblastoma (GBM) is critical for developing new therapeutics that overcome the highly immunosuppressive tumor microenvironment. Our study showed that B and T cells form clusters within the GBM microenvironment within a 15-µm radius, suggesting that B and T cells could form immune synapses within the GBM. However, GBM-infiltrating B cells suppress the activation of CD8+ T cells. To overcome this immunosuppression, we leveraged B-cell functions by activating them with CD40 agonism, IFNγ, and BAFF to generate a potent antigen-presenting B cells named BVax. BVax had improved antigen cross-presentation potential compared to naïve B cells and were primed to use the IL15-IL15Ra mechanism to enhance T cell activation. Compared to naïve B cells, BVax could improve CD8 T cell activation and proliferation. Compared to dendritic cells (DCs), which are the current gold standard professional antigen-presenting cell, BVax promoted highly proliferative T cells in-vitro that had a stem-like memory T cell phenotype characterized by CD62L+CD44- expression, high TCF-1 expression, and low PD-1 and granzyme B expression. Adoptive transfer of BVax-activated CD8+ T cells into tumor-bearing brains led to T cell reactivation with higher TCF-1 expression and elevated granzyme B production compared to DC-activated CD8+ T cells. Adoptive transfer of BVax into an irradiated immunocompetent tumor-bearing host promoted more CD8+ T cell proliferation than adoptive transfer of DCs. Moreover, highly proliferative CD8+ T cells in the BVax group had less PD-1 expression than those highly proliferative CD8+ T cells in the DC group. The findings of this study suggest that BVax and DC could generate distinctive CD8+ T cells, which potentially serve multiple purposes in cellular vaccine development.
Asunto(s)
Glioblastoma , Humanos , Granzimas , Linfocitos T CD8-positivos , Receptor de Muerte Celular Programada 1 , Células Presentadoras de Antígenos , Proliferación Celular , Microambiente TumoralRESUMEN
Patients with Krabbe disease, a genetic demyelinating syndrome caused by deficiency of galactosyl-ceramidase and the resulting accumulation of galactosyl-sphingolipids, develop signs of a dying-back axonopathy compounded by a deficiency of large-caliber axons. Here, we show that axonal caliber in Twitcher mice, an animal model for Krabbe disease, is impaired in peripheral axons and is accompanied by a progressive reduction in the abundance and phosphorylation of the three neurofilament (NF) subunits. These changes correlate with an increase in the density of NFs per cross-sectional area in numerous mutant peripheral axons and abnormal increases in the activity of two serine/threonine phosphatases (PP1 and PP2A) in mutant tissue. Similarly, acutely isolated mutant cortical neurons show abnormal phosphorylation of NFs. Psychosine, the neurotoxin accumulated in Krabbe disease, was sufficient to induce abnormal dephosphorylation of NF subunits in a normal motor neuron cell line as well as in acutely isolated normal cortical neurons. This in vitro effect was mediated by PP1 and PP2A, which specifically dephosphorylated NFs. These results demonstrate that the reduced caliber observed in some axons in Krabbe disease involves abnormal dephosphorylation of NFs. We propose that a psychosine-driven pathogenic mechanism through deregulated phosphotransferase activities may be involved in this process.
Asunto(s)
Proteínas de Neurofilamentos/metabolismo , Proteína Fosfatasa 1/fisiología , Proteína Fosfatasa 2/fisiología , Psicosina/farmacología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vaina de Mielina/genética , Vaina de Mielina/patología , Vaina de Mielina/fisiología , Inhibición Neural/genética , Proteínas de Neurofilamentos/antagonistas & inhibidores , Neurotoxinas/farmacología , Fosforilación/fisiología , Regulación hacia Arriba/genéticaRESUMEN
Loss-of-function of the lysosomal enzyme galactosyl-ceramidase causes the accumulation of the lipid raft-associated sphingolipid psychosine, the disruption of postnatal myelination, neurodegeneration and early death in most cases of infantile Krabbe disease. This work presents a first study towards understanding the progression of axonal defects in this disease using the Twitcher mutant mouse. Axonal swellings were detected in axons within the mutant spinal cord as early as 1 week after birth. As the disease progressed, more axonopathic profiles were found in other regions of the nervous system, including peripheral nerves and various brain areas. Isolated mutant neurons recapitulated axonal and neuronal defects in the absence of mutant myelinating glia, suggesting an autonomous neuronal defect. Psychosine was sufficient to induce axonal defects and cell death in cultures of acutely isolated neurons. Interestingly, axonopathy in young Twitcher mice occurred in the absence of demyelination and of neuronal apoptosis. Neuronal damage occurred at later stages, when mutant mice were moribund and demyelinated. Altogether, these findings suggest a progressive dying-back neuronal dysfunction in Twitcher mutants.
Asunto(s)
Axones/patología , Progresión de la Enfermedad , Leucodistrofia de Células Globoides/etiología , Leucodistrofia de Células Globoides/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Axones/efectos de los fármacos , Células Cultivadas , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Leucodistrofia de Células Globoides/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/patología , Psicosina/farmacología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/patología , Médula Espinal/efectos de los fármacos , Médula Espinal/patologíaRESUMEN
Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution.
Asunto(s)
Glioblastoma , Animales , Linfocitos T CD8-positivos/metabolismo , Supervivencia Celular , Eflornitina/metabolismo , Eflornitina/farmacología , Glioblastoma/metabolismo , Concentración de Iones de Hidrógeno , Terapia de Inmunosupresión , Ratones , Células Mieloides/metabolismo , Poliaminas/metabolismo , Microambiente TumoralRESUMEN
Immunotherapy has revolutionized the treatment of many tumors. However, most glioblastoma (GBM) patients have not, so far, benefited from such successes. With the goal of exploring ways to boost anti-GBM immunity, we developed a B cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNγ stimulation. BVax migrates to key secondary lymphoid organs and is proficient at antigen cross-presentation, which promotes both the survival and the functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. This treatment elicited immunological memory that prevented the growth of new tumors upon subsequent reinjection in cured mice. GBM patient-derived BVax was successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.
Asunto(s)
Ligando 4-1BB/inmunología , Linfocitos B/inmunología , Antígenos CD40/inmunología , Vacunas contra el Cáncer/inmunología , Glioblastoma/terapia , Interferón gamma/inmunología , Neoplasias Experimentales/terapia , Ligando 4-1BB/genética , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Antígenos CD40/genética , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/inmunología , Glioblastoma/patología , Interferón gamma/genética , Ratones , Ratones Noqueados , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunologíaRESUMEN
Lipid rafts (LRs) are membrane realms characterized by high concentrations of cholesterol and sphingolipids. Often, they are portrayed as scaffolds on which many different signaling molecules can assemble their cascades. The idea of rafts as scaffolds is garnering significant attention as the consequences of LR disruption have been shown to be manifest in multiple signaling pathways. In this study, LRs in the brain of the twitcher (TWI) mouse, a bona-fide model for infant variants of human globoid cell leukodystrophy or Krabbe disease, were investigated. This mouse has deficient activity of GALC (beta-galactosylceramidase) that leads to a progressive accumulation of some galactosyl-sphingolipids in the brain. We hypothesized that the accumulation of psychosine (galactosyl-sphingosine) in the TWI CNS may result in the disruption of rafts in different cell populations such as neurons and oligodendrocytes, both cellular targets during disease. In this communication, we demonstrate that psychosine specifically accumulates in LRs in the TWI brain and sciatic nerve and in samples from brains of human Krabbe patients. It is also shown that this accumulation is accompanied by an increase in cholesterol in these domains and changes in the distribution of the LR markers flotillin-2 and caveolin-1. Finally, we show evidence that this phenomenon may provide a mechanism by which psychosine can exert its known inhibitory effect on protein kinase C. This study provides a previously undescribed biophysical aspect for the mechanism of pathogenesis in Krabbe disease.
Asunto(s)
Encéfalo/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patología , Microdominios de Membrana/metabolismo , Psicosina/metabolismo , Animales , Encéfalo/patología , Caveolina 1/metabolismo , Preescolar , Colesterol/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Galactosilceramidasa/deficiencia , Células HeLa , Humanos , Microdominios de Membrana/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Proteína Quinasa C/metabolismo , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatologíaRESUMEN
The potent immunosuppression induced by glioblastoma (GBM) is one of the primary obstacles to finding effective immunotherapies. One hallmark of the GBM-associated immunosuppressive landscape is the massive infiltration of myeloid-derived suppressor cells (MDSC) and, to a lesser extent, regulatory T cells (Treg) within the tumor microenvironment. Here, we showed that regulatory B cells (Breg) are a prominent feature of the GBM microenvironment in both preclinical models and clinical samples. Forty percent of GBM patients (n = 60) scored positive for B-cell tumor infiltration. Human and mouse GBM-associated Bregs were characterized by immunosuppressive activity toward activated CD8+ T cells, the overexpression of inhibitory molecules PD-L1 and CD155, and production of immunosuppressive cytokines TGFß and IL10. Local delivery of B cell-depleting anti-CD20 immunotherapy improved overall survival of animals (IgG vs. anti-CD20 mean survival: 18.5 vs. 33 days, P = 0.0001), suggesting a potential role of Bregs in GBM progression. We unveiled that GBM-associated MDSCs promoted regulatory B-cell function by delivering microvesicles transporting membrane-bound PD-L1, able to be up-taken by tumoral B cells. The transfer of functional PD-L1 via microvesicles conferred Bregs the potential to suppress CD8+ T-cell activation and acquisition of an effector phenotype. This work uncovered the role of B cells in GBM physiopathology and provides a mechanism by which the GBM microenvironment controls B cell-mediated immunosuppression.See related Spotlight on p. 1902.
Asunto(s)
Linfocitos B/inmunología , Antígeno B7-H1/inmunología , Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Células Supresoras de Origen Mieloide/inmunología , Animales , Antígenos CD20/genética , Antígenos CD20/inmunología , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Glioblastoma/genética , Humanos , Tolerancia Inmunológica , Ratones Endogámicos C57BL , Ratones Noqueados , Microambiente Tumoral/inmunologíaRESUMEN
The immunosuppressive microenvironment is one of the major factors promoting the growth of glioblastoma multiforme (GBM). Infiltration of CD4+CD25+Foxp3+ regulatory T cells (Tregs) into the tumor microenvironment plays a significant role in the suppression of the anti-tumor immunity and portends a dismal prognosis for patients. Glioma-mediated secretion of chemo-attractant C-C motif ligand 2 and 22 (CCL2/22) has previously been shown by our group to promote Treg migration in vitro. In this study, we show that a local implantation of platelet-rich fibrin patch (PRF-P) into the brain of GL261 glioma-bearing mice prolonged the survival of affected animals by 42.85% (p = 0.0011). Analysis performed on brain tumor tissue harvested from PRF-P-treated mice revealed a specific decrease in intra-tumoral lymphocytes with a preferential depletion of immunosuppressive Tregs. Importantly, co-culture of GL261 or chemo-attractants (CCL2/22) with PRF-P abrogated Treg migration. Pharmacological blockade of the CCL2/22 interaction with their receptors potentiated the inhibitory effect of PRF-P on Tregs recruitment in culture. Moreover, our findings revealed the soluble CD40 ligand (sCD40L) as a major Treg inhibitory player produced by activated platelets entrapped within the fibrin matrix of the PRF-P. Blockade of sCD40L restored the migratory capacity of Tregs, emphasizing the role of PRF-P in preventing the Treg migration to glioma tissue. Our findings highlight autologous PRF-P as a personalized, Treg-selective suppression platform that can potentially supplement and enhance the efficacy of glioma therapies.
Asunto(s)
Autoinjertos , Neoplasias Encefálicas/terapia , Glioma/terapia , Fibrina Rica en Plaquetas/fisiología , Linfocitos T Reguladores/inmunología , Animales , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Células Cultivadas , Craneotomía/métodos , Glioma/inmunología , Glioma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T Reguladores/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
The mechanisms by which regulatory T cells (Tregs) migrate to and function within the hypoxic tumor microenvironment are unclear. Our studies indicate that specific ablation of hypoxia-inducible factor 1α (HIF-1α) in Tregs results in enhanced CD8+ T cell suppression versus wild-type Tregs under hypoxia, due to increased pyruvate import into the mitochondria. Importantly, HIF-1α-deficient Tregs are minimally affected by the inhibition of lipid oxidation, a fuel that is critical for Treg metabolism in tumors. Under hypoxia, HIF-1α directs glucose away from mitochondria, leaving Tregs dependent on fatty acids for mitochondrial metabolism within the hypoxic tumor. Indeed, inhibition of lipid oxidation enhances the survival of mice with glioma. Interestingly, HIF-1α-deficient-Treg mice exhibit significantly enhanced animal survival in a murine model of glioma, due to their stymied migratory capacity, explaining their reduced abundance in tumor-bearing mice. Thus HIF-1α acts as a metabolic switch for Tregs between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression.