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In the version of this article initially published, the name of one author was incorrect (James P. Santo). The correct name is James P. Di Santo. The error has been corrected in the HTML and PDF versions of the article.
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The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases.
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Variación Genética/inmunología , Inmunidad Innata/genética , Inmunidad Adaptativa/genética , Adulto , Anciano , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.
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COVID-19 , Genética de Población , SARS-CoV-2 , Análisis de Expresión Génica de una Sola Célula , Animales , Humanos , Diferenciación Celular , COVID-19/genética , COVID-19/inmunología , COVID-19/virología , Citomegalovirus/fisiología , Pueblos del Este de Asia/genética , Introgresión Genética , Virus de la Influenza A/patogenicidad , Virus de la Influenza A/fisiología , Interferones/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Células Mieloides/inmunología , Hombre de Neandertal/genética , Hombre de Neandertal/inmunología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Selección Genética , Latencia del VirusRESUMEN
Functional molecular characterization of the cochlea has mainly been driven by the deciphering of the genetic architecture of sensorineural deafness. As a result, the search for curative treatments, which are sorely lacking in the hearing field, has become a potentially achievable objective, particularly via cochlear gene and cell therapies. To this end, a complete inventory of cochlear cell types, with an in-depth characterization of their gene expression profiles right up to their final differentiation, is indispensable. We therefore generated a single-cell transcriptomic atlas of the mouse cochlea based on an analysis of more than 120,000 cells on postnatal day 8 (P8), during the prehearing period, P12, corresponding to hearing onset, and P20, when cochlear maturation is almost complete. By combining whole-cell and nuclear transcript analyses with extensive in situ RNA hybridization assays, we characterized the transcriptomic signatures covering nearly all cochlear cell types and developed cell type-specific markers. Three cell types were discovered; two of them contribute to the modiolus which houses the primary auditory neurons and blood vessels, and the third one consists in cells lining the scala vestibuli. The results also shed light on the molecular basis of the tonotopic gradient of the biophysical characteristics of the basilar membrane that critically underlies cochlear passive sound frequency analysis. Finally, overlooked expression of deafness genes in several cochlear cell types was also unveiled. This atlas paves the way for the deciphering of the gene regulatory networks controlling cochlear cell differentiation and maturation, essential for the development of effective targeted treatments.
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Sordera , Transcriptoma , Animales , Ratones , Cóclea/fisiología , Membrana Basilar , Audición/fisiología , Sordera/metabolismoRESUMEN
Standardization of immunophenotyping procedures has become a high priority. We have developed a suite of whole-blood, syringe-based assay systems that can be used to reproducibly assess induced innate or adaptive immune responses. By eliminating preanalytical errors associated with immune monitoring, we have defined the protein signatures induced by (1) medically relevant bacteria, fungi, and viruses; (2) agonists specific for defined host sensors; (3) clinically employed cytokines; and (4) activators of T cell immunity. Our results provide an initial assessment of healthy donor reference values for induced cytokines and chemokines and we report the failure to release interleukin-1α as a common immunological phenotype. The observed naturally occurring variation of the immune response may help to explain differential susceptibility to disease or response to therapeutic intervention. The implementation of a general solution for assessment of functional immune responses will help support harmonization of clinical studies and data sharing.
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Inmunidad Adaptativa/inmunología , Inmunidad Innata/inmunología , Monitorización Inmunológica/métodos , Antígenos/inmunología , Citocinas/sangre , Citocinas/metabolismo , Voluntarios Sanos , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Monitorización Inmunológica/normas , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
MicroRNAs (miRNAs) are critical regulators of gene expression, and their role in a wide variety of biological processes, including host antimicrobial defense, is increasingly well described. Consistent with their diverse functional effects, miRNA expression is highly context dependent and shows marked changes upon cellular activation. However, the genetic control of miRNA expression in response to external stimuli and the impact of such perturbations on miRNA-mediated regulatory networks at the population level remain to be determined. Here we assessed changes in miRNA expression upon Mycobacterium tuberculosis infection and mapped expression quantitative trait loci (eQTL) in dendritic cells from a panel of healthy individuals. Genome-wide expression profiling revealed that â¼40% of miRNAs are differentially expressed upon infection. We find that the expression of 3% of miRNAs is controlled by proximate genetic factors, which are enriched in a promoter-specific histone modification associated with active transcription. Notably, we identify two infection-specific response eQTLs, for miR-326 and miR-1260, providing an initial assessment of the impact of genotype-environment interactions on miRNA molecular phenotypes. Furthermore, we show that infection coincides with a marked remodeling of the genome-wide relationships between miRNA and mRNA expression levels. This observation, supplemented by experimental data using the model of miR-29a, sheds light on the role of a set of miRNAs in cellular responses to infection. Collectively, this study increases our understanding of the genetic architecture of miRNA expression in response to infection, and highlights the wide-reaching impact of altering miRNA expression on the transcriptional landscape of a cell.
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Genoma Humano , MicroARNs/metabolismo , Transcripción Genética , Tuberculosis/genética , Estudios de Casos y Controles , Interacción Gen-Ambiente , Humanos , MicroARNs/genética , Regiones Promotoras Genéticas , Sitios de Carácter Cuantitativo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tuberculosis/metabolismoRESUMEN
BACKGROUND: A successful host immune response to infection is dependent upon both innate and adaptive immune effector mechanisms. Cutaneous leishmaniasis results in an adaptive Th1 CD4(+) T cell response that efficiently clears the parasite, but may also result in scaring. However the role of innate mechanisms during parasite clearance remains less well defined. METHODS: We examined a unique cohort of individuals, living in a Leishmania major endemic region, that were stratified among 3 distinct clinical groups in a cross-sectional study. Specifically, patients were classified either as healed (n = 17), asymptomatic (23), or naïve to infection (18) based upon the classical Leishmanin Skin Test (LST) and the presence or absence of scars. Utilizing a multiplexed immunoassay approach we characterized the induced cytokine and chemokine response to L. major. RESULTS: A subset of innate immune molecules was induced in all groups. By contrast, T cell-associated cytokines were largely induced in exposed groups as compared to L. major-infection naïve individuals. Two exceptions were IL-17A and IL-12p70, induced and not induced, respectively, in naïve individuals. In addition, GM-CSF was more strongly induced in healed patients as compared to the other two groups. Surprisingly an IL-13 response was the best cytokine for classifying previously infected donors. CONCLUSIONS: Exploratory data analysis, utilizing principle component analysis (PCA), revealed distinct patient clusters of the healed and naïve groups based on the most differentially induced proteins. Asymptomatic previously infected individuals were more difficult to assign to a particular cluster based on these induced proteins. Analysis of these proteins may enable the identification of biomarkers associated with disease, leading to a better understanding of the protective mechanisms of immune response against leishmaniasis.
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Biomarcadores/metabolismo , Leishmaniasis Cutánea/diagnóstico , Adolescente , Antígenos de Protozoos/inmunología , Niño , Estudios Transversales , Citocinas/análisis , Femenino , Humanos , Inmunoensayo , Interleucina-13/análisis , Interleucina-17/análisis , Leishmania major/aislamiento & purificación , Leishmania major/patogenicidad , Leishmaniasis Cutánea/parasitología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Análisis de Componente Principal , Sensibilidad y Especificidad , Pruebas CutáneasRESUMEN
Multi-parametric flow cytometry is a key technology for characterization of immune cell phenotypes. However, robust high-dimensional post-analytic strategies for automated data analysis in large numbers of donors are still lacking. Here, we report a computational pipeline, called FlowGM, which minimizes operator input, is insensitive to compensation settings, and can be adapted to different analytic panels. A Gaussian Mixture Model (GMM)-based approach was utilized for initial clustering, with the number of clusters determined using Bayesian Information Criterion. Meta-clustering in a reference donor permitted automated identification of 24 cell types across four panels. Cluster labels were integrated into FCS files, thus permitting comparisons to manual gating. Cell numbers and coefficient of variation (CV) were similar between FlowGM and conventional gating for lymphocyte populations, but notably FlowGM provided improved discrimination of "hard-to-gate" monocyte and dendritic cell (DC) subsets. FlowGM thus provides rapid high-dimensional analysis of cell phenotypes and is amenable to cohort studies.
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Algoritmos , Automatización de Laboratorios/métodos , Citometría de Flujo/métodos , Linfocitos B , Teorema de Bayes , Análisis por Conglomerados , Células Dendríticas , Humanos , Células Asesinas Naturales , Monocitos , Neutrófilos , Estándares de Referencia , Programas Informáticos , Estadística como Asunto , Subgrupos de Linfocitos T , Linfocitos TRESUMEN
Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease. Standardized procedures are essential to allow for inter-individual comparisons in the context of population based or clinical studies. Herein we report the approach taken by the Milieu Intérieur Consortium, highlighting the standardized and automated procedures used for immunophenotyping of human whole blood samples. We optimized eight-color antibody panels and procedures for staining and lysis of whole blood samples, and implemented pre-analytic steps with a semi-automated workflow using a robotic system. We report on four panels that were designed to enumerate and phenotype major immune cell populations (PMN, T, B, NK cells, monocytes and DC). This work establishes a foundation for defining reference values in healthy donors. Our approach provides robust protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.
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Automatización de Laboratorios/métodos , Citometría de Flujo/métodos , Inmunofenotipificación/métodos , Antígenos CD/inmunología , Linfocitos B , Células Dendríticas , Humanos , Células Asesinas Naturales , Monocitos , Neutrófilos , Manejo de Especímenes/métodos , Linfocitos TRESUMEN
Recent advances in the identification of susceptibility genes and environmental exposures (pandemic influenza 2009 vaccination) provide strong support that narcolepsy type 1 is an immune-mediated disease. Considering the limited knowledge regarding the immune mechanisms involved in narcolepsy whether related to flu vaccination or not and the recent progresses in cytokine measurement technology, we assessed 30 cytokines, chemokines and growth factors using the Luminex technology in either peripheral (serum) or central (CSF) compartments in a large population of 90 children and adult patients with narcolepsy type 1 in comparison to 58 non-hypocretin deficient hypersomniacs and 41 healthy controls. Furthermore, we compared their levels in patients with narcolepsy whether exposed to pandemic flu vaccine or not, and analyzed the effect of age, duration of disease and symptom severity. Comparison for sera biomarkers between narcolepsy (n = 84, 54 males, median age: 15.5 years old) and healthy controls (n = 41, 13 males, median age: 20 years old) revealed an increased stimulation of the immune system with high release of several pro- and anti-inflammatory serum cytokines and growth factors with interferon-γ, CCL11, epidermal growth factor, and interleukin-2 receptor being independently associated with narcolepsy. Increased levels of interferon-γ, CCL11, and interleukin-12 were found when close to narcolepsy onset. After several adjustments, only one CSF biomarker differed between narcolepsy (n = 44, 26 males, median age: 15 years old) and non-hypocretin deficient hypersomnias (n = 57, 24 males, median age: 36 years old) with higher CCL 3 levels found in narcolepsy. Comparison for sera biomarkers between patients with narcolepsy who developed the disease post-pandemic flu vaccination (n = 36) to those without vaccination (n = 48) revealed an increased stimulation of the immune system with high release of three cytokines, regulated upon activation normal T-cell expressed and secreted, CXCL10, and CXCL9, being independently and significantly increased in the group exposed to the vaccine. No significant differences were found between narcoleptics whether exposed to flu vaccination or not for CSF biomarkers except for a lower CXCL10 level found in the exposed group. To conclude, we highlighted the role of sera cytokine with pro-inflammatory properties and especially interferon-γ being independently associated with narcolepsy close to disease onset. The activity of the interferon-γ network was also increased in the context of narcolepsy after the pandemic flu vaccination being a potential key player in the immune mechanism that triggers narcolepsy and that coordinates the immune response necessary for resolving vaccination assaults.
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Citocinas/sangre , Citocinas/líquido cefalorraquídeo , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Narcolepsia/inmunología , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Niño , Citocinas/inmunología , Femenino , Humanos , Gripe Humana/inmunología , Gripe Humana/virología , Interferón gamma/sangre , Interferón gamma/líquido cefalorraquídeo , Interferón gamma/inmunología , Masculino , Persona de Mediana Edad , Vacunación , Adulto JovenRESUMEN
The mechanisms regulating memory CD8(+) T cell function and homeostasis during aging are unclear. CD8(+) effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8(+) T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8(+) T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8(+) T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-α that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA(-)CD45RO(+) CMV-specific CD8(+) T cells induced CD45RA expression while Ag activated cells remained CD45RO(+). This raises the possibility that non-specific cytokine-driven accumulation of CMV-specific CD8(+)CD45RA(+) T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging.
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Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Antígenos Comunes de Leucocito/metabolismo , Factores de Edad , Afinidad de Anticuerpos/inmunología , Antígenos/inmunología , Antígenos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Humanos , Inmunofenotipificación , Interferón-alfa/biosíntesis , Interleucina-15/inmunología , Interleucina-15/metabolismo , Activación de Linfocitos/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Fosfoproteínas/inmunología , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Individual microRNAs (miRNAs) are rapidly down-regulated during conditions of cellular activation and infection, but factors mediating miRNA turnover are poorly understood. Infection of mouse cells with murine cytomegalovirus (MCMV) induces the rapid down-regulation of an antiviral cellular miRNA, miR-27. Here, we identify a transcript produced by MCMV that binds to miR-27 and mediates its degradation. UV-crosslinking and high-throughput sequencing [CRAC (UV-crosslinking and analysis of cDNA)] identified MCMV RNA segments associated with the miRNA-binding protein Argonaute 2 (Ago2). A cluster of hits mapped to a predicted miR-27-binding site in the 3'UTR of the previously uncharacterized ORF, m169. The expression kinetics of the m169 transcript correlated with degradation of miR-27 during infection, and m169 expression inhibited miR-27 functional activity in a reporter assay. siRNA knockdown of m169 demonstrated its requirement for miR-27 degradation following infection and did not affect other host miRNAs. Substitution of the miR-27-binding site in m169 to create complementarity to a different cellular miRNA, miR-24, resulted in down-regulation of only miR-24 following infection. The m169 transcript is cytoplasmic, capped, polyadenylated, and interacts with miRNA-27 through seed pairing: characteristic features of the normal messenger RNA (mRNA) targets of miRNAs. This virus-host interaction reveals a mode of miRNA regulation in which a mRNA directs the degradation of a miRNA. We speculate that RNA-mediated miRNA degradation could be a more general viral strategy for manipulating host cells.
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MicroARNs/antagonistas & inhibidores , Muromegalovirus/genética , Regiones no Traducidas 3'/genética , Animales , Proteínas Argonautas/metabolismo , Secuencia de Bases , Sitios de Unión , Reactivos de Enlaces Cruzados/metabolismo , Citoplasma/metabolismo , ADN Complementario/genética , Regulación de la Expresión Génica/efectos de la radiación , Ensayos Analíticos de Alto Rendimiento , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Datos de Secuencia Molecular , Muromegalovirus/efectos de la radiación , Células 3T3 NIH , Nucleótidos/genética , Estabilidad del ARN/genética , Estabilidad del ARN/efectos de la radiación , Transporte de ARN/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Transcriptoma/genética , Rayos UltravioletaRESUMEN
MicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes.
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MicroARNs/biosíntesis , MicroARNs/genética , Virus/genética , Animales , Expresión Génica , Humanos , MicroARNs/metabolismo , Virus/metabolismoRESUMEN
Persistent viral infections and inflammatory syndromes induce the accumulation of T cells with characteristics of terminal differentiation or senescence. However, the mechanism that regulates the end-stage differentiation of these cells is unclear. Human CD4(+) effector memory (EM) T cells (CD27(-)CD45RA(-)) and also EM T cells that re-express CD45RA (CD27(-)CD45RA(+); EMRA) have many characteristics of end-stage differentiation. These include the expression of surface KLRG1 and CD57, reduced replicative capacity, decreased survival, and high expression of nuclear γH2AX after TCR activation. A paradoxical observation was that although CD4(+) EMRA T cells exhibit defective telomerase activity after activation, they have significantly longer telomeres than central memory (CM)-like (CD27(+)CD45RA(-)) and EM (CD27(-)CD45RA(-)) CD4(+) T cells. This suggested that telomerase activity was actively inhibited in this population. Because proinflammatory cytokines such as TNF-α inhibited telomerase activity in T cells via a p38 MAPK pathway, we investigated the involvement of p38 signaling in CD4(+) EMRA T cells. We found that the expression of both total and phosphorylated p38 was highest in the EM and EMRA compared with that of other CD4(+) T cell subsets. Furthermore, the inhibition of p38 signaling, especially in CD4(+) EMRA T cells, significantly enhanced their telomerase activity and survival after TCR activation. Thus, activation of the p38 MAPK pathway is directly involved in certain senescence characteristics of highly differentiated CD4(+) T cells. In particular, CD4(+) EMRA T cells have features of telomere-independent senescence that are regulated by active cell signaling pathways that are reversible.
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Linfocitos T CD4-Positivos/inmunología , Senescencia Celular/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Telomerasa/inmunología , Adulto , Western Blotting , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Separación Celular , Citometría de Flujo , Humanos , Memoria Inmunológica/inmunología , Hibridación Fluorescente in Situ , Antígenos Comunes de Leucocito/biosíntesis , Antígenos Comunes de Leucocito/inmunología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citología , Telomerasa/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/biosíntesis , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions. This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.
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Médula Ósea , Encéfalo , Núcleo Celular , ARN Nuclear Pequeño , BioensayoRESUMEN
Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.
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COVID-19 , Citocinas , Endorribonucleasas , SARS-CoV-2 , Síndrome de Respuesta Inflamatoria Sistémica , Niño , Humanos , COVID-19/inmunología , Citocinas/genética , Citocinas/inmunología , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , ARN Bicatenario , SARS-CoV-2/genética , Síndrome de Respuesta Inflamatoria Sistémica/genéticaRESUMEN
DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.
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Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Histona Desacetilasas/metabolismo , Proteínas Represoras , Proteína de Retinoblastoma/fisiología , Células 3T3 , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , División Celular , Línea Celular , Secuencia Conservada , Factores de Transcripción E2F , Glutatión/metabolismo , Histona Desacetilasa 1 , Histona Desacetilasas/genética , Humanos , Ratones , Modelos Biológicos , Mutación , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Factores de Transcripción/metabolismoRESUMEN
HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of ß-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control.
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Linfocitos T CD4-Positivos/inmunología , Controladores de Élite , Infecciones por VIH/inmunología , VIH-1/inmunología , Receptores CCR5/metabolismo , Internalización del Virus , Quimiocinas , Regulación hacia Abajo , Regulación de la Expresión Génica , Productos del Gen gag/metabolismo , Infecciones por VIH/virología , Antígenos de Histocompatibilidad Clase II , Humanos , Mutación , Receptores CCR5/genética , Receptores CXCR3RESUMEN
PURPOSE: CD70 is a costimulatory molecule known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). Clear-cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors; however, the clinical consequences of CD70 expression remain unclear. EXPERIMENTAL DESIGN: Tumor tissue from 25 patients with ccRCC was assessed for the expression of CD27 and CD70 in situ using multiplex immunofluorescence. CD27+ T-cell phenotypes in tumors were analyzed by flow cytometry and their gene expression profile were analyzed by single-cell RNA sequencing then confirmed with public data. Baseline sCD27 was measured in 81 patients with renal cell carcinoma (RCC) treated with immunotherapy (35 for training cohort and 46 for validation cohort). RESULTS: In the tumor microenvironment, CD27+ T cells interacted with CD70-expressing tumor cells. Compared with CD27- T cells, CD27+ T cells exhibited an apoptotic and dysfunctional signature. In patients with RCC, the intratumoral CD27-CD70 interaction was significantly correlated with the plasma sCD27 concentration. High sCD27 levels predicted poor overall survival in patients with RCC treated with anti-programmed cell death protein 1 in both the training and validation cohorts but not in patients treated with antiangiogenic therapy. CONCLUSIONS: In conclusion, we demonstrated that sCD27, a surrogate marker of T-cell dysfunction, is a predictive biomarker of resistance to immunotherapy in RCC. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be extended to other tumors.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Ligando CD27/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Inmunoterapia , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Microambiente TumoralRESUMEN
The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4+ T-cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA+ CD27+ CD4+ T cells over time. In contrast, the increase in CD45RAâ» CD27â» and CD45RA+ CD27â» CD4+ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RAâ» CD27â» and CD45RA+ CD27â» CD4+ T cells in CMV-seropositive donors are specific for this virus. CD45RA+ CD27â» CD4+ T cells have significantly reduced CD28, interleukin-7 receptor α (IL-7Rα) and Bcl-2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T-cell receptor activation compared with the other T-cell subsets in the same donors. Despite this, the CD45RA+ CD27â» subset is as multifunctional as the CD45RAâ» D27+ and CD45RAâ» CD27â» CD4+ T-cell subsets, indicating that they are not an exhausted population. In addition, CD45RA+ CD27â» CD4+ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4+ memory T cells re-expressing CD45RA can be generated from the CD45RAâ» CD27+ population by the addition of IL-7 and during this process these cells down-regulated expression of IL-7R and Bcl-2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA+ CD27â» CD4+ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated.