RESUMEN
Early expansion and long-term persistence predict efficacy of chimeric antigen receptor T cells (CARTs)1-7, but mechanisms governing effector versus memory CART differentiation and whether asymmetric cell division induces differential fates in human CARTs remain unclear. Here we show that target-induced proximity labelling enables isolation of first-division proximal-daughter and distal-daughter CD8 CARTs that asymmetrically distribute their surface proteome and transcriptome, resulting in divergent fates. Target-engaged CARs remain on proximal daughters, which inherit a surface proteome resembling activated-undivided CARTs, whereas the endogenous T cell receptor and CD8 enrich on distal daughters, whose surface proteome resembles resting CARTs, correlating with glycolytic and oxidative metabolism, respectively. Despite memory-precursor phenotype and in vivo longevity, distal daughters demonstrate transient potent cytolytic activity similar to proximal daughters, uncovering an effector-like state in distal daughters destined to become memory CARTs. Both partitioning of pre-existing transcripts and changes in RNA velocity contribute to asymmetry of fate-determining factors, resulting in diametrically opposed transcriptional trajectories. Independent of naive, memory or effector surface immunophenotype, proximal-daughter CARTs use core sets of transcription factors known to support proliferation and effector function. Conversely, transcription factors enriched in distal daughters restrain differentiation and promote longevity, evidenced by diminished long-term in vivo persistence and function of distal-daughter CARTs after IKZF1 disruption. These studies establish asymmetric cell division as a framework for understanding mechanisms of CART differentiation and improving therapeutic outcomes.
Asunto(s)
División Celular Asimétrica , Linfocitos T CD8-positivos , Receptores Quiméricos de Antígenos , Humanos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Transcriptoma , Memoria Inmunológica , Proteoma/metabolismo , Diferenciación Celular , Linaje de la Célula , Animales , Ratones , Femenino , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND & AIMS: Dyskeratosis congenita (DC) is a telomere biology disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction experience multiorgan failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear. METHODS: We used isogenic human induced pluripotent stem cells (iPSCs) harboring a causal DC mutation in DKC1 or a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-corrected control allele to model DC liver pathologies. We differentiated these iPSCs into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single-cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships. RESULTS: Directed differentiation of iPSCs into HEPs and stellate cells and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, with DC HEPs becoming hyperplastic and also eliciting a pathogenic hyperplastic, proinflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant HEPs and hepatostellate organoids could be rescued via suppression of serine/threonine kinase AKT (protein kinase B) activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. CONCLUSIONS: Isogenic iPSC-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.
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Células Madre Pluripotentes Inducidas , Humanos , Hiperplasia/patología , Hígado/patología , Diferenciación Celular/genética , Organoides/patología , Proteínas Nucleares , Proteínas de Ciclo Celular/genéticaRESUMEN
Morphea is a rare multifactorial autoimmune disorder characterized by a complex and dynamic interplay between Th1 and Th2 signaling. Active clinical trials are currently investigating the safety and efficacy of dupilumab for the treatment of primary morphea. Here, we present two cases of morphea that developed in pediatric atopic dermatitis patients treated with dupilumab. These findings may support a causal relationship between IL-4 receptor blockade and the development of the early inflammatory phase of morphea.
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Dermatitis Atópica , Esclerodermia Localizada , Humanos , Niño , Dermatitis Atópica/tratamiento farmacológico , Anticuerpos Monoclonales/efectos adversos , Esclerodermia Localizada/inducido químicamente , Esclerodermia Localizada/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/efectos adversos , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Non-melanoma skin cancers are cutaneous malignancies representing the most common form of cancer in the United States. They are comprised predominantly of basal cell carcinomas and squamous cell carcinomas (cSCC). The incidence of cSCC is increasing, resulting in substantial morbidity and ever higher treatment costs; currently in excess of one billion dollars, per annum. Here, we review research defining the molecular basis and development of cSCC that aims to provide new insights into pathogenesis and drive the development of novel, cost and morbidity saving therapies.
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Carcinoma Basocelular , Carcinoma de Células Escamosas , Neoplasias Cutáneas , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/genética , Humanos , Incidencia , Neoplasias Cutáneas/patología , Estados UnidosRESUMEN
Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating in vivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.
Asunto(s)
Señalización del Calcio , Transición Epitelial-Mesenquimal , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Plasticidad de la CélulaRESUMEN
The central role of calcium (Ca2+) signaling in lymphocyte development and acquisition of functional immunity and tolerance is well established. Ca2+ signals are initiated upon antigen binding to cognate receptors on lymphocytes that trigger store operated Ca2+ entry (SOCE). The underlying mechanism of SOCE in lymphocytes involves TCR and BCR mediated activation of Stromal Interaction Molecule 1 and 2 (STIM1/2) embedded in the ER membrane. Once activated, STIM proteins oligomerize and re-localize to ER domains juxtaposed to the plasma membrane where they activate Orai channels to allow Ca2+ to enter the cell across the plasma membrane. Importantly, STIM/Orai-dependent Ca2+ signals guide antigen induced lymphocyte development and function principally by regulating the activity of transcription factors.The most widely studied of these transcription factors is the Nuclear Factor of Activated T cells (NFAT). NFAT is expressed ubiquitously and the mechanism by which Ca2+ regulates NFAT activation and signaling is well known. By contrast, a mechanistic understanding of how Ca2+ signals also shape the activation and specificity of NF-κB to control the expression of pro-inflammatory genes has lagged. Here we discuss the methodology used to investigate Ca2+ dependent mechanisms of NF-κB activation in lymphocytes. Our approach focuses on three main areas of signal transduction and signaling: (1) antigen receptor engagement and Ca2+ dependent initiation of NF-kB signaling, (2) Ca2+ dependent induction of NF-κB heterodimer activation and nuclear localization, and (3) and how Ca2+ regulates NF-κB dependent expression of target genes and proteins.
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Linfocitos B , Linfocitos B/metabolismo , Calcio/metabolismo , Señalización del Calcio , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2RESUMEN
As access to high-throughput sequencing technology has increased, the bottleneck in biomedical research has shifted from data generation to data analysis. Here, we describe a modular and extensible framework for didactic instruction in bioinformatics using publicly available RNA sequencing data sets from infectious disease studies, with a focus on host-parasite interactions. We highlight lessons learned from adapting this course for virtual learners during the coronavirus disease 2019 (COVID-19) pandemic.
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Biología Computacional/educación , Biología Computacional/métodos , Interacciones Huésped-Parásitos/fisiología , Animales , COVID-19/patología , Análisis de Datos , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/genética , Schistosoma mansoni/fisiología , Toxoplasma/efectos de los fármacos , Toxoplasma/genética , Toxoplasma/fisiologíaRESUMEN
The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.
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Ebolavirus/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Secuencias de Aminoácidos , Angiomotinas , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/transmisión , Fiebre Hemorrágica Ebola/virología , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Microscopía Confocal , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Virión/fisiología , Liberación del VirusRESUMEN
B cell receptor (BCR) engagement induces naive B cells to differentiate and perform critical immune-regulatory functions. Acquisition of functional specificity requires that a cell survive, enter the cell cycle, and proliferate. We establish that quantitatively distinct Ca2+ signals triggered by variations in the extent of BCR engagement dynamically regulate these transitions by controlling nuclear factor κB (NF-κB), NFAT, and mTORC1 activity. Weak BCR engagement induces apoptosis by failing to activate NF-κB-driven anti-apoptotic gene expression. Stronger signals that trigger more robust Ca2+ signals promote NF-κB-dependent survival and NFAT-, mTORC1-, and c-Myc-dependent cell-cycle entry and proliferation. Finally, we establish that CD40 or TLR9 costimulation circumvents these Ca2+-regulated checkpoints of B cell activation and proliferation. As altered BCR signaling is linked to autoimmunity and B cell malignancies, these results have important implications for understanding the pathogenesis of aberrant B cell activation and differentiation and therapeutic approaches to target these responses.
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Calcio/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Apoptosis/inmunología , Linfocitos B/inmunología , Ciclo Celular/inmunología , Diferenciación Celular/inmunología , Proliferación Celular/fisiología , Supervivencia Celular/inmunología , Activación de Linfocitos/inmunología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/inmunología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/inmunología , FN-kappa B/metabolismo , Factores de Transcripción NFATC/inmunología , Factores de Transcripción NFATC/metabolismo , Células Precursoras de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.
Asunto(s)
Aedes/inmunología , Aedes/parasitología , Dirofilaria immitis/crecimiento & desarrollo , Mosquitos Vectores/inmunología , Mosquitos Vectores/parasitología , Aedes/genética , Animales , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Larva/crecimiento & desarrollo , Mosquitos Vectores/genéticaRESUMEN
Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.
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Filoviridae/fisiología , Marburgvirus/fisiología , Imitación Molecular , Proteínas Proto-Oncogénicas c-yes/metabolismo , Proteínas de la Matriz Viral/fisiología , Liberación del Virus , Angiomotinas , Sitios de Unión , Membrana Celular/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Dominios PDZ , Dominios Proteicos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The ability of Toxoplasma gondii to inject the rhoptry kinase ROP16 into host cells results in the activation of the transcription factors STAT3 and STAT6, but it is unclear how these events impact infection. Here, parasites that inject Cre-recombinase with rhoptry proteins were used to distinguish infected macrophages from those only injected with parasite proteins. Transcriptional profiling revealed that injection of rhoptry proteins alone was sufficient to induce an M2 phenotype that is dependent on STAT3 and STAT6, but only infected cells displayed reduced expression of genes associated with antimicrobial activity and protective immunity. In vivo, the absence of STAT3 or STAT6 improved parasite control, while the loss of ROP16 resulted in a marked reduction in parasite numbers and heightened parasite-specific T cell responses. Thus, ROP16 is a virulence factor that can act in cis and trans to promote M2 programs and which limits the magnitude of parasite-specific T cell responses.
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Proteínas Tirosina Quinasas/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T/inmunología , Toxoplasma/inmunología , Factores de Virulencia/inmunología , Animales , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT6/inmunologíaRESUMEN
Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata, an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C. fasciculata is that adherent parasites can be generated both in vitro, allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C. fasciculata. As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C. fasciculata is a powerful model system for probing kinetoplastid-insect interactions.
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Aedes/parasitología , Crithidia fasciculata/genética , Genes Protozoarios , Aedes/anatomía & histología , Animales , Adhesión Celular/genética , Adhesión Celular/fisiología , Crithidia fasciculata/crecimiento & desarrollo , Crithidia fasciculata/fisiología , Femenino , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos , Masculino , ARN Protozoario , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
Caspase-8 is a key integrator of cell survival and cell death decisions during infection and inflammation. Following engagement of tumor necrosis factor superfamily receptors or certain Toll-like receptors (TLRs), caspase-8 initiates cell-extrinsic apoptosis while inhibiting RIPK3-dependent programmed necrosis. In addition, caspase-8 has an important, albeit less well understood, role in cell-intrinsic inflammatory gene expression. Macrophages lacking caspase-8 or the adaptor FADD have defective inflammatory cytokine expression and inflammasome priming in response to bacterial infection or TLR stimulation. How caspase-8 regulates cytokine gene expression, and whether caspase-8-mediated gene regulation has a physiological role during infection, remain poorly defined. Here we demonstrate that both caspase-8 enzymatic activity and scaffolding functions contribute to inflammatory cytokine gene expression. Caspase-8 enzymatic activity was necessary for maximal expression of Il1b and Il12b, but caspase-8 deficient cells exhibited a further decrease in expression of these genes. Furthermore, the ability of TLR stimuli to induce optimal IκB kinase phosphorylation and nuclear translocation of the nuclear factor kappa light chain enhancer of activated B cells family member c-Rel required caspase activity. Interestingly, overexpression of c-Rel was sufficient to restore expression of IL-12 and IL-1ß in caspase-8-deficient cells. Moreover, Ripk3-/-Casp8-/- mice were unable to control infection by the intracellular parasite Toxoplasma gondii, which corresponded to defects in monocyte recruitment to the peritoneal cavity, and exogenous IL-12 restored monocyte recruitment and protection of caspase-8-deficient mice during acute toxoplasmosis. These findings provide insight into how caspase-8 controls inflammatory gene expression and identify a critical role for caspase-8 in host defense against eukaryotic pathogens.