RESUMEN
In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell-mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood-brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Señalización del Calcio/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Activación de Linfocitos/inmunología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Linfocitos T/inmunología , Animales , Autoantígenos/inmunología , Autoinmunidad/inmunología , Barrera Hematoencefálica/inmunología , Encéfalo/inmunología , Línea Celular , Femenino , Factores de Transcripción NFATC/metabolismo , Ratas , Ratas Endogámicas Lew , Migración Transendotelial y Transepitelial/inmunologíaRESUMEN
The H3K9me3-specific histone methyltransferase Setdb1 impacts on transcriptional regulation by repressing both developmental genes and retrotransposons. How impaired retrotransposon silencing may lead to developmental phenotypes is currently unclear. Here, we show that loss of Setdb1 in pro-B cells completely abrogates B cell development. In pro-B cells, Setdb1 is dispensable for silencing of lineage-inappropriate developmental genes. Instead, we detect strong derepression of endogenous murine leukemia virus (MLV) copies. This activation coincides with an unusual change in chromatin structure, with only partial loss of H3K9me3 and unchanged DNA methylation, but strongly increased H3K4me3. Production of MLV proteins leads to activation of the unfolded protein response pathway and apoptosis. Thus, our data demonstrate that B cell development depends on the proper repression of retrotransposon sequences through Setdb1.
Asunto(s)
Apoptosis/genética , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Retroelementos/genética , Respuesta de Proteína Desplegada/genética , Animales , Perfilación de la Expresión Génica , Silenciador del Gen , Células HEK293 , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Virus de la Leucemia Murina/genética , Lisina/metabolismo , Metilación , Ratones , Secuencias Repetitivas de Ácidos Nucleicos/genética , Transcripción GenéticaRESUMEN
The BAFF-APRIL system is best known for its control of B cell homeostasis, and it is a target of therapeutic intervention in autoimmune diseases and lymphoma. By analyzing the expression of the three receptors of this system, B cell maturation Ag (BCMA), transmembrane activator and CAML interactor, and BAFF receptor, in sorted human immune cell subsets, we found that BCMA was transcribed in plasmacytoid dendritic cells (pDCs) in both blood and lymphoid tissue. Circulating human pDCs contained BCMA protein without displaying it on the cell surface. After engagement of TLR7/8 or TLR9, BCMA was detected also on the cell surface of pDCs. The display of BCMA on the surface of human pDCs was accompanied by release of soluble BCMA (sBCMA); inhibition of γ-secretase enhanced surface expression of BCMA and reduced the release of sBCMA by pDCs. In contrast with human pDCs, murine pDCs did not express BCMA, not even after TLR9 activation. In this study, we extend the spectrum of BCMA expression to human pDCs. sBCMA derived from pDCs might determine local availability of its high-affinity ligand APRIL, because sBCMA has been shown to function as an APRIL-specific decoy. Further, therapeutic trials targeting BCMA in patients with multiple myeloma should consider possible effects on pDCs.
Asunto(s)
Antígeno de Maduración de Linfocitos B/inmunología , Células Dendríticas/inmunología , Transducción de Señal/inmunología , Animales , Receptor del Factor Activador de Células B/inmunología , Antígeno de Maduración de Linfocitos B/biosíntesis , Separación Celular , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
The bloodbrain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.
Asunto(s)
Encéfalo/patología , Movimiento Celular , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Pulmón/patología , Linfocitos T/patología , Traslado Adoptivo , Animales , Autoinmunidad/inmunología , Barrera Hematoencefálica/inmunología , Encéfalo/citología , Encéfalo/inmunología , Moléculas de Adhesión Celular Neuronal/metabolismo , Circulación Cerebrovascular , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Memoria Inmunológica , Pulmón/citología , Pulmón/inmunología , Activación de Linfocitos , Vaina de Mielina/inmunología , Factores de Crecimiento Nervioso/metabolismo , Ratas , Ratas Endogámicas Lew , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
More than 50% of mammalian genomes consist of retrotransposon sequences. Silencing of retrotransposons by heterochromatin is essential to ensure genomic stability and transcriptional integrity. Here, we identified a short sequence element in intracisternal A particle (IAP) retrotransposons that is sufficient to trigger heterochromatin formation. We used this sequence in a genome-wide shRNA screen and identified the chromatin remodeler Atrx as a novel regulator of IAP silencing. Atrx binds to IAP elements and is necessary for efficient heterochromatin formation. In addition, Atrx facilitates a robust and largely inaccessible heterochromatin structure as Atrx knockout cells display increased chromatin accessibility at retrotransposons and non-repetitive heterochromatic loci. In summary, we demonstrate a direct role of Atrx in the establishment and robust maintenance of heterochromatin.
Asunto(s)
ADN Helicasas/genética , ADN Helicasas/metabolismo , Genes de Partícula A Intracisternal , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ensamble y Desensamble de Cromatina , Inestabilidad Genómica , Heterocromatina/genética , Interferencia de ARN , ARN Interferente Pequeño , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Ag recognition via the TCR is necessary for the expansion of specific T cells that then contribute to adaptive immunity as effector and memory cells. Because CD4+ and CD8+ T cells differ in terms of their priming APCs and MHC ligands we compared their requirements of Ag persistence during their expansion phase side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal mouse T cells were thus analyzed after transient and continuous TCR signals. Following equally strong stimulation, CD4+ T cell proliferation depended on prolonged Ag presence, whereas CD8+ T cells were able to divide and differentiate into effector cells despite discontinued Ag presentation. CD4+ T cell proliferation was neither affected by Th lineage or memory differentiation nor blocked by coinhibitory signals or missing inflammatory stimuli. Continued CD8+ T cell proliferation was truly independent of self-peptide/MHC-derived signals. The subset divergence was also illustrated by surprisingly broad transcriptional differences supporting a stronger propensity of CD8+ T cells to programmed expansion. These T cell data indicate an intrinsic difference between CD4+ and CD8+ T cells regarding the processing of TCR signals for proliferation. We also found that the presentation of a MHC class II-restricted peptide is more efficiently prolonged by dendritic cell activation in vivo than a class I bound one. In summary, our data demonstrate that CD4+ T cells require continuous stimulation for clonal expansion, whereas CD8+ T cells can divide following a much shorter TCR signal.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Ciclo Celular/efectos de los fármacos , Diferenciación Celular , Análisis por Conglomerados , Células Dendríticas/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Expresión Génica , Perfilación de la Expresión Génica , Orden Génico , Vectores Genéticos/genética , Antígenos H-2/química , Antígenos H-2/inmunología , Memoria Inmunológica , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.
Asunto(s)
Genes Virales/fisiología , Herpesvirus Humano 8/fisiología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Sistemas de Lectura Abierta/fisiología , Activación Viral/fisiología , Linfocitos B , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genéticaRESUMEN
The tissues of the central nervous system are effectively shielded from the blood circulation by specialized vessels that are impermeable not only to cells, but also to most macromolecules circulating in the blood. Despite this seemingly absolute seclusion, central nervous system tissues are subject to immune surveillance and are vulnerable to autoimmune attacks. Using intravital two-photon imaging in a Lewis rat model of experimental autoimmune encephalomyelitis, here we present in real-time the interactive processes between effector T cells and cerebral structures from their first arrival to manifest autoimmune disease. We observed that incoming effector T cells successively scanned three planes. The T cells got arrested to leptomeningeal vessels and immediately monitored the luminal surface, crawling preferentially against the blood flow. After diapedesis, the cells continued their scan on the abluminal vascular surface and the underlying leptomeningeal (pial) membrane. There, the T cells encountered phagocytes that effectively present antigens, foreign as well as myelin proteins. These contacts stimulated the effector T cells to produce pro-inflammatory mediators, and provided a trigger to tissue invasion and the formation of inflammatory infiltrations.
Asunto(s)
Enfermedades del Sistema Nervioso Central/inmunología , Enfermedades del Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Meninges/irrigación sanguínea , Meninges/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos/inmunología , Movimiento Celular , Células Cultivadas , Meninges/patología , Ratones , Ovalbúmina/inmunología , Fagocitos/inmunología , Ratas , Ratas Endogámicas LewRESUMEN
The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called alpha-secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid-beta peptide (Abeta). alpha-Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel alpha-secretase-cleavage site-specific antibody, we found that RNAi-mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP alpha-secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of alpha-cleavage. This finding was further confirmed by mass-spectrometric detection of APP-cleavage fragments. Surprisingly, in different cell lines, the reduction of alpha-secretase cleavage was not paralleled by a corresponding increase in the Abeta-generating beta-secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with gamma-secretase for the cleavage of a C-terminal APP fragment generated by beta-secretase. We conclude that ADAM10 is the physiologically relevant, constitutive alpha-secretase of APP.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/enzimología , Proteína ADAM10 , Animales , Línea Celular , Humanos , Espectrometría de Masas , Ratones , Neuronas/metabolismoRESUMEN
Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Doxorrubicina/farmacología , Terapia Genética/métodos , Metástasis de la Neoplasia/terapia , Factor de Necrosis Tumoral alfa/farmacología , Animales , Bioensayo , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Lentivirus/genética , Ratones , Plásmidos/genética , Transfección/métodos , TransgenesRESUMEN
The transcriptional regulator FOXP3 is an important determinant of regulatory T (Treg) cell development and function and is frequently used to quantitate Treg cells. However, FOXP3 is also expressed in recently activated conventional human T cells. Here, we investigated the FOXP3 expression patterns in Treg and activated T cells at a cellular level. Upon activation, human CD4(+) CD25(-) T cells expressed FOXP3 mainly in the cytoplasm, in sharp contrast to human CD4(+) CD25(+) Treg cells, where we found FOXP3 to be predominantly expressed in the nucleus. A GFP-FOXP3-fusion protein shuttled from the nucleus to the cytoplasm in transfected primary human T cells. We identified two novel leucine-rich nuclear export signals in FOXP3. Site-directed mutagenesis of both sequences completely abolished nuclear export of FOXP3 in human T cells. Both export sequences localized to exons affected by alternative splicing. The three isoforms FOXP3Δ2, FOXP3Δ7, and FOXP3Δ2Δ7 localized preferentially to the nucleus. Additionally, forced expression of nucleus-directed FOXP3 induced a Treg-cell-associated gene expression pattern and induced regulatory capacity. These findings should aid in the interpretation of future studies utilizing FOXP3 expression as a Treg-cell marker and shed some light on the molecular mechanisms controlling subcellular FOXP3 localization in human T cells.
Asunto(s)
Factores de Transcripción Forkhead/análisis , Linfocitos T Reguladores/química , Linfocitos T/química , Núcleo Celular/química , Células Cultivadas , Citoplasma/química , Factores de Transcripción Forkhead/fisiología , Humanos , Activación de Linfocitos , Señales de Exportación Nuclear , Linfocitos T/ultraestructura , Linfocitos T Reguladores/ultraestructuraRESUMEN
OBJECTIVE: Cord blood-derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell-based therapy, including mesenchymal stem cells (MSCs). METHODS AND RESULTS: ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor(+) cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. CONCLUSIONS: Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell-based therapy represents a promising approach for vascular regeneration of ischemic tissue.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Endotelio Vascular/citología , Miembro Posterior/irrigación sanguínea , Isquemia/terapia , Células Madre Mesenquimatosas/citología , Neovascularización Fisiológica/fisiología , Células Madre/citología , Animales , Proliferación Celular , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Femenino , Ganciclovir/farmacología , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Desnudos , Modelos Animales , Fenotipo , Recuperación de la Función/fisiología , Células Madre/efectos de los fármacos , Células Madre/fisiologíaRESUMEN
In recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of being influenced by DNA methylation is rising steadily and includes common diseases such as schizophrenia, bipolar disorder, Alzheimer's disease, diabetes, atherosclerosis, cancer, major psychosis, lupus and Parkinson's disease. Due to cellular heterogeneity of methylation patterns, epigenetic analyses of single cells become a necessity. One rationale is that DNA methylation profiles are highly variable across individual cells, even in the same organ, dependent on the function of the gene, disease state, exposure to environmental factors (e.g. radiation, drugs or nutrition), stochastic fluctuations and various other causes. Using a polymerase chain reaction (PCR)-slide microreaction system, we present here a methylation-sensitive PCR analysis, the restriction enzyme-based single-cell methylation assay (RSMA), in the analysis of DNA methylation patterns in single cells. This method addresses the problems of cell heterogeneity in epigenetics research; it is comparably affordable, avoids complicated microfluidic systems and offers the opportunity for high-throughput screening, as many single cells can be screened in parallel. In addition to this study, critical principles and caveats of single cell methylation analyses are discussed.
Asunto(s)
Metilación de ADN , Análisis de la Célula Individual , Línea Celular , Línea Celular Tumoral , Islas de CpG , Enzimas de Restricción del ADN , Ensayos Analíticos de Alto Rendimiento , Humanos , Linfocitos/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF-7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2(high) and HER2(low) cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2(low) cell fraction. Accordingly, HER2(low) cells also showed reduced proliferation, ductal-like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2(low) sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2(high) fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti-HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Receptor ErbB-2/metabolismo , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Secuencia de Bases , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cartilla de ADN , Femenino , Humanos , Piranos/administración & dosificación , Piranos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , TrastuzumabRESUMEN
The identification of novel approaches to specifically target the DNA-damage checkpoint response in chemotherapy-resistant cancer stem cells (CSC) of solid tumors has recently attracted great interest. We show here in colon cancer cell lines and primary colon cancer cells that inhibition of checkpoint-modulating phosphoinositide 3-kinase-related (PIK) kinases preferentially depletes the chemoresistant and exclusively tumorigenic CD133(+) cell fraction. We observed a time- and dose-dependent disproportionally pronounced loss of CD133(+) cells and the consecutive lack of in vitro and in vivo tumorigenicity of the remaining cells. Depletion of CD133(+) cells was initiated through apoptosis of cycling CD133(+) cells and further substantiated through subsequent recruitment of quiescent CD133(+) cells into the cell cycle followed by their elimination. Models using specific PIK kinase inhibitors, somatic cell gene targeting, and RNA interference demonstrated that the observed detrimental effects of caffeine on CSC were attributable specifically to the inhibition of the PIK kinase ataxia telangiectasia- and Rad3-related (ATR). Mechanistically, phosphorylation of CHK1 checkpoint homolog (S. pombe; CHK1) was significantly enhanced in CD133(+) as compared with CD133(-) cells on treatment with DNA interstrand-crosslinking (ICL) agents, indicating a preferential activation of the ATR/CHK1-dependent DNA-damage response in tumorigenic CD133(+) cells. Consistently, the chemoresistance of CD133(+) cells toward DNA ICL agents was overcome through inhibition of ATR/CHK1-signaling. In conclusion, our study illustrates a novel target to eliminate the tumorigenic CD133(+) cell population in colon cancer and provides another rationale for the development of specific ATR-inhibitors.
Asunto(s)
Carcinoma/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Transformación Celular Neoplásica/genética , Neoplasias del Colon/patología , Células Madre Neoplásicas/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/terapia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Separación Celular/métodos , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/terapia , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Terapia Genética , Glicoproteínas/metabolismo , Humanos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida/métodos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , ARN Interferente Pequeño/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2(369)) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCR alpha-chain of this TCR specifically recognized HER2(369) not only in combination with the original beta-chain but also with four other beta-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one beta-chain derived from another HER2(369)-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells.
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Presentación de Antígeno/inmunología , Epítopos de Linfocito T/inmunología , Epítopos Inmunodominantes/metabolismo , Isoantígenos/inmunología , Fragmentos de Péptidos/inmunología , Receptor ErbB-2/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Subgrupos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Línea Celular Transformada , Línea Celular Tumoral , Células Clonales , Epítopos de Linfocito T/metabolismo , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Humanos , Células Híbridas , Isoantígenos/metabolismo , Células Jurkat , Células K562 , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Receptor ErbB-2/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.
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Regulación Bacteriana de la Expresión Génica , Inositol/metabolismo , Salmonella typhimurium/metabolismo , Fusión Artificial Génica , Carbono/metabolismo , Medios de Cultivo/química , Citometría de Flujo , Eliminación de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Fenotipo , Regiones Promotoras Genéticas , Salmonella typhimurium/crecimiento & desarrollo , Activación TranscripcionalRESUMEN
Regulatory T cells (Treg) mediate tolerance towards self-antigens by suppression of innate and adaptive immunity. In cancer patients, tumor-infiltrating FoxP3+ Treg suppress local anti-tumor immune responses and are often associated with poor prognosis. Markers that are selectively expressed on tumor-infiltrating Treg may serve as targets for immunotherapy of cancer. Here we show that CD103, an integrin mediating lymphocyte retention in epithelial tissues, is expressed at high levels on tumor-infiltrating FoxP3+ Treg in several types of murine cancer. In the CT26 model of colon cancer up to 90% of the intratumoral FoxP3+ cells expressed CD103 compared to less than 20% in lymphoid organs. CD103+ Treg suppressed T effector cell activation more strongly than CD103(neg) Treg. Expression of CD103 on Treg closely correlated with intratumoral levels of transforming growth factor ß (TGF-ß) and could be induced in a TGF-ß-dependent manner by tumor cell lines. In vivo, gene silencing of TGF-ß reduced the frequency of CD103+ Treg, demonstrating that CD103 expression on tumor-infiltrating Treg is driven by intratumoral TGF-ß. Functional blockade of CD103 using a monoclonal antibody did however not reduce the number of intratumoral Treg, indicating that CD103 is not involved in homing or retention of FoxP3+ cells in the tumor tissue. In conclusion, expression of CD103 is a hallmark of Treg that infiltrate TGF-ß-secreting tumors. CD103 thus represents an interesting target for selective depletion of tumor-infiltrating Treg, a strategy that may help to improve anti-cancer therapy.
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Factores de Transcripción Forkhead/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Animales , Antígenos CD , Línea Celular Tumoral , Femenino , Cadenas alfa de Integrinas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T Reguladores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The clinical picture of experimental autoimmune encephalomyelitis (EAE) is critically dependent on the nature of the target autoantigen and the genetic background of the experimental animals. Potentially lethal EAE is mediated by myelin basic protein (MBP)-specific T cells in Lewis rats, whereas transfer of S100beta- or myelin oligodendrocyte glycoprotein (MOG)-specific T cells causes intense inflammatory response in the central nervous system (CNS) with minimal disease. However, in Dark Agouti rats, the pathogenicity of MOG-specific T cells resembles the one of MBP-specific T cells in the Lewis rat. Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue. Irrespective of their pathogenicity, the migratory activity, gene expression patterns, and immigration of green fluorescent protein(+) T cells into the CNS were similar. However, exclusively highly pathogenic T cells were significantly reactivated within the CNS. Without local effector T cell activation, production of monocyte chemoattractants was insufficient to initiate and propagate a full inflammatory response. Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.
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Encefalomielitis Autoinmune Experimental/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Autoantígenos/metabolismo , Línea Celular , Sistema Nervioso Central/inmunología , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/etiología , Proteínas Fluorescentes Verdes , Mediadores de Inflamación/metabolismo , Proteínas Luminiscentes/genética , Activación de Linfocitos , Especificidad de Órganos , Fenotipo , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND & AIMS: Pancreatic cancers contain exclusively tumorigenic cancer stem cells (CSCs), which are highly resistant to chemotherapy, resulting in a relative increase in CSC numbers during gemcitabine treatment. Signaling through sonic hedgehog and mammalian target of rapamycin (mTOR), respectively, may be essential for CSC self-renewal and could represent putative targets for novel treatment modalities. METHODS: We used in vitro and in vivo models of pancreatic cancer to examine the effects of sonic hedgehog inhibition (cyclopamine/CUR199691) and mTOR blockade (rapamycin) on the tumorigenic CSC population. RESULTS: Surprisingly, neither cyclopamine nor rapamycin alone or as supplements to chemotherapy were capable of effectively diminishing the CSC pool. Only the combined inhibition of both pathways together with chemotherapy reduced the number of CSCs to virtually undetectable levels in vitro and in vivo. Most importantly, in vivo administration of this triple combination in mice with established patient-derived pancreatic tumors was reasonably tolerated and translated into significantly prolonged long-term survival. CONCLUSIONS: The combined blockade of sonic hedgehog and mTOR signaling together with standard chemotherapy is capable of eliminating pancreatic CSCs. Further preclinical investigation of this promising approach may lead to the development of a novel therapeutic strategy to improve the devastating prognosis of patients with pancreatic cancer.