RESUMEN
In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).
Asunto(s)
Monkeypox virus , Mpox , Vacuna contra Viruela , Animales , Humanos , Ratones , Macaca fascicularis , Monkeypox virus/genética , Mpox/inmunología , Mpox/prevención & control , Vacunas Combinadas , Virus Vaccinia/genéticaRESUMEN
Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.
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Embrión de Mamíferos , Células Madre Embrionarias , Animales , Técnicas de Cocultivo , Macaca fascicularis , Células Madre Embrionarias/metabolismo , Diferenciación Celular , Endodermo/metabolismo , Linaje de la CélulaRESUMEN
T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).
Asunto(s)
Vacuna BNT162 , COVID-19 , Animales , Cricetinae , Humanos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Epítopos , SARS-CoV-2/genéticaRESUMEN
Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.
Asunto(s)
Metilación de ADN , Neoplasias de la Próstata , Animales , Humanos , Masculino , Ratones , Carcinogénesis/genética , ADN , Epigénesis Genética , Neoplasias de la Próstata/genética , Células Neoplásicas CirculantesRESUMEN
Medullary thymic epithelial cells (mTECs) generate immunological self-tolerance by ectopically expressing peripheral-tissue antigens (PTAs) within the thymus to preview the peripheral self to maturing T cells. Recent work, drawing inspiration from old histological observations, has shown that subtypes of mTECs, collectively termed mimetic cells, co-opt developmental programs from throughout the organism to express biologically coherent groups of PTAs. Here, we review key aspects of mimetic cells, especially as they relate to the larger contexts of molecular, cellular, developmental, and evolutionary biology. We highlight lineage-defining transcription factors as key regulators of mimetic cells and speculate as to what other factors, including Aire and the chromatin potential of mTECs, permit mimetic cell differentiation and function. Last, we consider what mimetic cells can teach us about not only the thymus but also other tissues.
RESUMEN
Medullary thymic epithelial cells (mTECs) ectopically express thousands of peripheral-tissue antigens (PTAs), which drive deletion or phenotypic diversion of self-reactive immature T cells during thymic differentiation. Failure of PTA expression causes multiorgan autoimmunity. By assaying chromatin accessibility in individual mTECs, we uncovered signatures of lineage-defining transcription factors (TFs) for skin, lung, liver, and intestinal cells-including Grhl, FoxA, FoxJ1, Hnf4, Sox8, and SpiB-in distinct mTEC subtypes. Transcriptomic and histologic analyses showed that these subtypes, which we collectively term mimetic cells, expressed PTAs in a biologically logical fashion, mirroring extra-thymic cell types while maintaining mTEC identity. Lineage-defining TFs bound to mimetic-cell open chromatin regions and were required for mimetic cell accumulation, whereas the tolerogenic factor Aire was partially and variably required. Expression of a model antigen in mimetic cells sufficed to induce cognate T cell tolerance. Thus, mTECs co-opt lineage-defining TFs to drive mimetic cell accumulation, PTA expression, and self-tolerance.
Asunto(s)
Células Epiteliales , Linfocitos T , Animales , Antígenos , Diferenciación Celular , Cromatina/metabolismo , Células Epiteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Linfocitos T/metabolismo , Timo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Recent studies have begun to reveal critical roles for the brain's professional phagocytes, microglia, and their receptors in the control of neurotoxic amyloid beta (Aß) and myelin debris accumulation in neurodegenerative disease. However, the critical intracellular molecules that orchestrate neuroprotective functions of microglia remain poorly understood. In our studies, we find that targeted deletion of SYK in microglia leads to exacerbated Aß deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer's disease (AD). Disruption of SYK signaling in this AD model was further shown to impede the development of disease-associated microglia (DAM), alter AKT/GSK3ß-signaling, and restrict Aß phagocytosis by microglia. Conversely, receptor-mediated activation of SYK limits Aß load. We also found that SYK critically regulates microglial phagocytosis and DAM acquisition in demyelinating disease. Collectively, these results broaden our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material.
Asunto(s)
Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Animales , Ratones , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides , Modelos Animales de Enfermedad , Ratones Transgénicos , Microglía/patología , FagocitosisRESUMEN
Searches for the genetic underpinnings of uniquely human traits have focused on human-specific divergence in conserved genomic regions, which reflects adaptive modifications of existing functional elements. However, the study of conserved regions excludes functional elements that descended from previously neutral regions. Here, we demonstrate that the fastest-evolved regions of the human genome, which we term "human ancestor quickly evolved regions" (HAQERs), rapidly diverged in an episodic burst of directional positive selection prior to the human-Neanderthal split, before transitioning to constraint within hominins. HAQERs are enriched for bivalent chromatin states, particularly in gastrointestinal and neurodevelopmental tissues, and genetic variants linked to neurodevelopmental disease. We developed a multiplex, single-cell in vivo enhancer assay to discover that rapid sequence divergence in HAQERs generated hominin-unique enhancers in the developing cerebral cortex. We propose that a lack of pleiotropic constraints and elevated mutation rates poised HAQERs for rapid adaptation and subsequent susceptibility to disease.
Asunto(s)
Hominidae , Hombre de Neandertal , Animales , Humanos , Hominidae/genética , Secuencias Reguladoras de Ácidos Nucleicos , Hombre de Neandertal/genética , Genoma Humano , GenómicaRESUMEN
To understand the role of T cells in the pathogenesis of ulcerative colitis (UC), we analyzed colonic T cells isolated from patients with UC and controls. Here we identified colonic CD4+ and CD8+ T lymphocyte subsets with gene expression profiles resembling stem-like progenitors, previously reported in several mouse models of autoimmune disease. Stem-like T cells were increased in inflamed areas compared to non-inflamed regions from the same patients. Furthermore, TCR sequence analysis indicated stem-like T cells were clonally related to proinflammatory T cells, suggesting their involvement in sustaining effectors that drive inflammation. Using an adoptive transfer colitis model in mice, we demonstrated that CD4+ T cells deficient in either BCL-6 or TCF1, transcription factors that promote T cell stemness, had decreased colon T cells and diminished pathogenicity. Our results establish a strong association between stem-like T cell populations and UC pathogenesis, highlighting the potential of targeting this population to improve clinical outcomes.
Asunto(s)
Colitis Ulcerosa , Factor Nuclear 1-alfa del Hepatocito , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Humanos , Animales , Ratones , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-alfa del Hepatocito/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Células Madre/inmunología , Células Madre/metabolismo , Femenino , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Ratones Noqueados , Colon/inmunología , Colon/patología , Masculino , Ratones Endogámicos C57BL , Traslado Adoptivo , Modelos Animales de Enfermedad , Adulto , Persona de Mediana EdadRESUMEN
The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved â¼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Teléfono Celular/instrumentación , Imagen Óptica/métodos , ARN Viral/análisis , Carga Viral/métodos , Animales , Prueba de Ácido Nucleico para COVID-19/economía , Prueba de Ácido Nucleico para COVID-19/instrumentación , Sistemas CRISPR-Cas , Línea Celular , Proteínas de la Nucleocápside de Coronavirus/genética , Humanos , Nasofaringe/virología , Imagen Óptica/instrumentación , Fosfoproteínas/genética , Pruebas en el Punto de Atención , Interferencia de ARN , ARN Viral/genética , Sensibilidad y Especificidad , Carga Viral/economía , Carga Viral/instrumentaciónRESUMEN
Following acute injury, stromal cells promote tissue regeneration by a diversity of mechanisms. Time-resolved single-cell RNA sequencing of muscle mesenchymal stromal cells (MmSCs) responding to acute injury identified an 'early-responder' subtype that spiked on day 1 and expressed a notable array of transcripts encoding immunomodulators. IL-1ß, TNF-α and oncostatin M each strongly and rapidly induced MmSCs transcribing this immunomodulatory program. Macrophages amplified the program but were not strictly required for its induction. Transfer of the inflammatory MmSC subtype, tagged with a unique surface marker, into healthy hindlimb muscle induced inflammation primarily driven by neutrophils and macrophages. Among the abundant inflammatory transcripts produced by this subtype, Cxcl5 was stroma-specific and highly upregulated with injury. Depletion of this chemokine early after injury revealed a substantial impact on recruitment of neutrophils, a prolongation of inflammation to later times and an effect on tissue regeneration. Mesenchymal stromal cell subtypes expressing a comparable inflammatory program were found in a mouse model of muscular dystrophy and in several other tissues and pathologies in both mice and humans. These 'early-responder' mesenchymal stromal cells, already in place, permit rapid and coordinated mobilization and amplification of critical cell collaborators in response to injury.
Asunto(s)
Inflamación , Células Madre Mesenquimatosas , Humanos , Ratones , Animales , Inflamación/metabolismo , Macrófagos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neutrófilos/metabolismo , Cicatrización de HeridasRESUMEN
KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8+ T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.
Asunto(s)
Envejecimiento/fisiología , Carcinoma Ductal Pancreático/patología , Remodelación Vascular/fisiología , Animales , Linfocitos T CD8-positivos/inmunología , Carcinoma Ductal Pancreático/microbiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Genes ras/genética , Humanos , Inmunoterapia/métodos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Neoplasias Pancreáticas/patología , Proteína de Retinoblastoma/inmunología , Transducción de Señal/genética , Microambiente Tumoral , Remodelación Vascular/genéticaRESUMEN
Lung group 2 innate lymphoid cells (ILC2s) control the nature of immune responses to airway allergens. Some microbial products, including those that stimulate interferons, block ILC2 activation, but whether this occurs after natural infections or causes durable ILC2 inhibition is unclear. In the present study, we cohoused laboratory and pet store mice as a model of physiological microbial exposure. Laboratory mice cohoused for 2 weeks had impaired ILC2 responses and reduced lung eosinophilia to intranasal allergens, whereas these responses were restored in mice cohoused for ≥2 months. ILC2 inhibition at 2 weeks correlated with increased interferon receptor signaling, which waned by 2 months of cohousing. Reinduction of interferons in 2-month cohoused mice blocked ILC2 activation. These findings suggest that ILC2s respond dynamically to environmental cues and that microbial exposures do not control long-term desensitization of innate type 2 responses to allergens.
Asunto(s)
Alérgenos , Inmunidad Innata , Ratones , Animales , Linfocitos , Citocinas , Pulmón , Interferones , Interleucina-33RESUMEN
Influenza viruses inhabit a wide range of host environments using a limited repertoire of protein components. Unlike viruses with stereotyped shapes, influenza produces virions with significant morphological variability even within clonal populations. Whether this tendency to form pleiomorphic virions is coupled to compositional heterogeneity and whether it affects replicative fitness remains unclear. Here, we address these questions by developing a strain of influenza A virus amenable to rapid compositional characterization through quantitative, site-specific labeling of viral proteins. Using this strain, we find that influenza A produces virions with broad variations in size and composition from even single infected cells. This phenotypic variability contributes to virus survival during environmental challenges, including exposure to antivirals. Complementing genetic adaptations that act over larger populations and longer times, this "low-fidelity" assembly of influenza A virus allows small populations to survive environments that fluctuate over individual replication cycles.
Asunto(s)
Virus de la Influenza A/metabolismo , Ensamble de Virus/fisiología , Línea Celular , Células Cultivadas , Humanos , Virus de la Influenza A/fisiología , Gripe Humana/virología , Proteínas Virales , Virión , Replicación Viral/fisiologíaRESUMEN
Animals must respond to the ingestion of food by generating adaptive behaviors, but the role of gut-brain signaling in behavioral regulation is poorly understood. Here, we identify conserved ion channels in an enteric serotonergic neuron that mediate its responses to food ingestion and decipher how these responses drive changes in foraging behavior. We show that the C. elegans serotonergic neuron NSM acts as an enteric sensory neuron that acutely detects food ingestion. We identify the novel and conserved acid-sensing ion channels (ASICs) DEL-7 and DEL-3 as NSM-enriched channels required for feeding-dependent NSM activity, which in turn drives slow locomotion while animals feed. Point mutations that alter the DEL-7 channel change NSM dynamics and associated behavioral dynamics of the organism. This study provides causal links between food ingestion, molecular and physiological properties of an enteric serotonergic neuron, and adaptive feeding behaviors, yielding a new view of how enteric neurons control behavior.
Asunto(s)
Canales Iónicos Sensibles al Ácido/metabolismo , Sistema Nervioso Entérico/metabolismo , Conducta Alimentaria/fisiología , Canales Iónicos Sensibles al Ácido/fisiología , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Sistema Nervioso Entérico/fisiología , Alimentos , Canales Iónicos/metabolismo , Canales Iónicos/fisiología , Locomoción , Neuronas/metabolismo , Células Receptoras Sensoriales/metabolismo , Neuronas Serotoninérgicas/metabolismo , Neuronas Serotoninérgicas/fisiología , Serotonina , Transducción de SeñalRESUMEN
New neurons arise from quiescent adult neural progenitors throughout life in specific regions of the mammalian brain. Little is known about the embryonic origin and establishment of adult neural progenitors. Here, we show that Hopx+ precursors in the mouse dentate neuroepithelium at embryonic day 11.5 give rise to proliferative Hopx+ neural progenitors in the primitive dentate region, and they, in turn, generate granule neurons, but not other neurons, throughout development and then transition into Hopx+ quiescent radial glial-like neural progenitors during an early postnatal period. RNA-seq and ATAC-seq analyses of Hopx+ embryonic, early postnatal, and adult dentate neural progenitors further reveal common molecular and epigenetic signatures and developmental dynamics. Together, our findings support a "continuous" model wherein a common neural progenitor population exclusively contributes to dentate neurogenesis throughout development and adulthood. Adult dentate neurogenesis may therefore represent a lifelong extension of development that maintains heightened plasticity in the mammalian hippocampus.
Asunto(s)
Células Madre Embrionarias/metabolismo , Neurogénesis , Animales , Diferenciación Celular , Giro Dentado/metabolismo , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Hipocampo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismoRESUMEN
A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.
Asunto(s)
Dipeptidasas/metabolismo , Neutrófilos/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Animales , Cilastatina/farmacología , Cilastatina/uso terapéutico , Dipeptidasas/antagonistas & inhibidores , Dipeptidasas/genética , Modelos Animales de Enfermedad , Endotoxemia/mortalidad , Endotoxemia/patología , Endotoxemia/prevención & control , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Infiltración Neutrófila/efectos de los fármacos , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Tasa de SupervivenciaRESUMEN
Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de los Músculos , Rabdomiosarcoma , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/inmunología , Femenino , Xenoinjertos , Humanos , Células K562 , Masculino , Neoplasias de los Músculos/tratamiento farmacológico , Neoplasias de los Músculos/inmunología , Neoplasias de los Músculos/metabolismo , Neoplasias de los Músculos/patología , Trasplante de Neoplasias , Ftalazinas/farmacología , Piperazinas/farmacología , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/inmunología , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Temozolomida/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , Pez Cebra/inmunologíaRESUMEN
The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.
Asunto(s)
Momento de Replicación del ADN/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Animales , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromatina , ADN/genética , Momento de Replicación del ADN/genética , Células Madre Embrionarias , Elementos de Facilitación Genéticos/genética , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Proteínas Represoras/metabolismo , Análisis Espacio-TemporalRESUMEN
Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.