RESUMEN
Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno CTLA-4/antagonistas & inhibidores , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Exoma , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunomodulación/efectos de los fármacos , Inmunomodulación/genética , Ratones , Terapia Molecular Dirigida , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Transcriptoma , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca(2+)-coordinating residues. The data explain the Ca(2+)-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.
Asunto(s)
Activación de Complemento/inmunología , Complemento C1/química , Complemento C1q/química , Complemento C1s/química , Inmunidad Innata/inmunología , Modelos Moleculares , Conformación Proteica , Animales , Células CHO , Cromatografía de Afinidad , Cromatografía en Gel , Activación de Complemento/genética , Complemento C1q/metabolismo , Complemento C1s/metabolismo , Cricetinae , Cricetulus , Cristalización , Escherichia coli , Unión ProteicaAsunto(s)
Linfoma de Células B Grandes Difuso/diagnóstico por imagen , Neoplasias de la Médula Espinal/diagnóstico por imagen , Anciano , Vértebras Cervicales , Humanos , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/cirugía , Masculino , Radiografía , Neoplasias de la Médula Espinal/patología , Neoplasias de la Médula Espinal/cirugíaRESUMEN
Notch1 activation regulates T lineage commitment and early T cell development. Fringe glycosyltransferases alter the sensitivity of Notch receptors to Delta-like versus Jagged Notch ligands, but their functions in T lymphopoiesis have not been defined. Here we show that developmental stage-specific expression of the glycosyltransferase lunatic fringe (Lfng) is required for coordination of the access of T cell progenitors to intrathymic niches that support Notch1-dependent phases of T cell development. Lfng-null progenitors generated few thymocytes in competitive assays, whereas Lfng overexpression converted thymocytes into 'supercompetitors' with enhanced binding of Delta-like ligands and blocked T lymphopoiesis from normal progenitors. We suggest that the ability of Lfng and Notch1 to control progenitor competition for limiting cortical niches is an important mechanism for the homeostatic regulation of thymus size.
Asunto(s)
Glicosiltransferasas/fisiología , Linfopoyesis , Receptor Notch1/fisiología , Linfocitos T/citología , Timo/crecimiento & desarrollo , Animales , Proteínas de Unión al Calcio , Diferenciación Celular/genética , Linaje de la Célula/genética , Glicosiltransferasas/genética , Uniones Intercelulares/inmunología , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Linfopoyesis/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Tamaño de los Órganos , Receptor Notch1/genética , Proteínas Represoras/metabolismo , Células Madre/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citologíaRESUMEN
Recent studies have shown that disruption of Notch1 signaling in lymphocyte progenitors (LP) inhibits T cell development and promotes B cell development in the thymus. Conversely, inappropriate activation of Notch1 in LP inhibits B cell development and causes ectopic T cell development in the bone marrow. These observations imply that Notch1 activation must be spatially regulated to ensure that LP generate B cells in the bone marrow and T cells in the thymus. However, Notch ligands are expressed in both tissues. Studies in flies and worms have revealed that Notch activation is extremely sensitive to small changes in the amount of receptor or ligand expressed, and defined multiple mechanisms that limit Notch activation to discrete cells at specific times during development. Here, we describe how some of these mechanisms might regulate Notch activity in LP during the T/B lineage decision.
Asunto(s)
Linfocitos B/inmunología , Receptores de Superficie Celular/metabolismo , Linfocitos T/inmunología , Factores de Transcripción , Animales , Linaje de la Célula , Proteínas de Drosophila , Endocitosis , Glicosilación , Inmunoglobulinas , Hormonas Juveniles/metabolismo , Ratones , N-Acetilglucosaminiltransferasas/metabolismo , Receptor Notch1 , Receptores de Superficie Celular/fisiología , Receptores de Citocinas/agonistas , Receptores de Citocinas/metabolismo , Transducción de SeñalRESUMEN
Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane. The functions of its homologues, UCP2 and UCP3, in other tissues are debated. UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic. Mild uncoupling would, however, decrease the mitochondrial production of reactive oxygen species, which are important mediators of oxidative damage. Here we show that superoxide increases mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3. Superoxide-induced uncoupling requires fatty acids and is inhibited by purine nucleotides. It correlates with the tissue expression of UCPs, appears in mitochondria from yeast expressing UCP1, and is absent in skeletal muscle mitochondria from UCP3 knockout mice. Our findings indicate that the interaction of superoxide with UCPs may be a mechanism for decreasing the concentrations of reactive oxygen species inside mitochondria.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales , Superóxidos/farmacología , Desacopladores/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Animales , Proteínas Portadoras/genética , Privación de Alimentos , Eliminación de Gen , Gliburida/farmacología , Calor , Membranas Intracelulares/metabolismo , Canales Iónicos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Riñón/citología , Riñón/metabolismo , Hígado/citología , Hígado/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Miocardio/citología , Miocardio/metabolismo , Proteínas/metabolismo , Protones , Ratas , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Bazo/citología , Bazo/metabolismo , Desacopladores/farmacología , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 microg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.
Asunto(s)
Proteínas Portadoras/metabolismo , Mitocondrias Musculares/metabolismo , Mitocondrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Cricetinae , ADN Complementario/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Cuerpos de Inclusión/metabolismo , Canales Iónicos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales , Músculo Esquelético/metabolismo , Protones , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Superóxidos/farmacología , Proteína Desacopladora 1 , Proteína Desacopladora 3RESUMEN
The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.