RESUMO
How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.
Assuntos
COVID-19 , Sistema Respiratório , SARS-CoV-2 , Humanos , Cílios/fisiologia , Cílios/virologia , COVID-19/virologia , Sistema Respiratório/citologia , Sistema Respiratório/virologia , SARS-CoV-2/fisiologia , Microvilosidades/fisiologia , Microvilosidades/virologia , Internalização do Vírus , Células Epiteliais/fisiologia , Células Epiteliais/virologiaRESUMO
Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.
Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , HumanosRESUMO
Bacille Calmette-Guérin (BCG) vaccination can confer nonspecific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. We show that mice vaccinated intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 B.1.351) or PR8 influenza. Protection was first evident between 14 and 21 d post-vaccination and lasted â¼3 months. Notably, BCG induced a biphasic innate response and robust antigen-specific type 1 helper T cell (TH1 cell) responses in the lungs. MyD88 signaling was essential for innate and TH1 cell responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or interferon (IFN)-γ activity before infection obliterated innate activation and protection. Single-cell and spatial transcriptomics revealed CD4-dependent expression of IFN-stimulated genes in lung myeloid and epithelial cells. Notably, BCG also induced protection against weight loss after mouse-adapted SARS-CoV-2 BA.5, SARS-CoV and SHC014 coronavirus infections. Thus, BCG elicits integrated organ immunity, where CD4+ T cells feed back on tissue myeloid and epithelial cells to imprint prolonged and broad innate antiviral resistance.
Assuntos
Imunidade Adaptativa , Vacina BCG , Animais , Camundongos , Humanos , Retroalimentação , Vacinação , Redução de Peso , Antivirais , Imunidade InataRESUMO
Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.
Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Invasividade Neoplásica/imunologia , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia/métodos , Masculino , Microambiente Tumoral/imunologiaRESUMO
Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.
Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno/genética , Análise de Célula Única , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Efeito Espectador , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Ebolavirus/genética , Chaperona BiP do Retículo Endoplasmático , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/patologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferons/genética , Interferons/metabolismo , Macaca mulatta , Macrófagos/metabolismo , Monócitos/metabolismo , Mielopoese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcriptoma/genéticaRESUMO
To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Genômica/métodos , Neoplasias Cutâneas/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , RNA-Seq , Análise de Célula Única , Pele/metabolismo , Neoplasias Cutâneas/patologia , Transcriptoma , Transplante HeterólogoRESUMO
AI is rapidly becoming part of many aspects of daily life, with an impact that reaches all fields of research. We asked investigators to share their thoughts on how AI is changing immunology research, what is necessary to move forward, the potential and the pitfalls, and what will remain unchanged as the field journeys into a new era.
Assuntos
Alergia e Imunologia , Inteligência Artificial , Humanos , AnimaisRESUMO
A highly multiplexed cytometric imaging approach, termed co-detection by indexing (CODEX), is used here to create multiplexed datasets of normal and lupus (MRL/lpr) murine spleens. CODEX iteratively visualizes antibody binding events using DNA barcodes, fluorescent dNTP analogs, and an in situ polymerization-based indexing procedure. An algorithmic pipeline for single-cell antigen quantification in tightly packed tissues was developed and used to overlay well-known morphological features with de novo characterization of lymphoid tissue architecture at a single-cell and cellular neighborhood levels. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell-interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed spatial cytometry demonstrated here allows for quantitative systemic characterization of tissue architecture in normal and clinically aberrant samples.
Assuntos
Anticorpos/química , Modelos Animais de Doenças , Processamento de Imagem Assistida por Computador/métodos , Lúpus Eritematoso Sistêmico/patologia , Sondas de Oligonucleotídeos/química , Baço/patologia , Animais , Feminino , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos MRL lprRESUMO
Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 T cells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.
Assuntos
Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Subpopulações de Linfócitos T/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Medula Óssea/imunologia , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Tecido Linfoide/imunologia , Masculino , Melanoma/imunologia , Melanoma/terapia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Microambiente TumoralRESUMO
Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.
Assuntos
Infecções por Poxviridae/imunologia , Poxviridae/fisiologia , Domínios Proteicos/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Alelos , Animais , Extinção Biológica , Humanos , Imunidade , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , FosforilaçãoRESUMO
Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.
Assuntos
Infecções por HIV , Ácidos Nucleicos , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD4-Positivos , Vírus de DNA , Terapia de Imunossupressão , Macaca mulatta , Macrófagos , Vírus da Imunodeficiência Símia/fisiologia , Carga ViralRESUMO
Technology development in biological research often aims to either increase the number of cellular features that can be surveyed simultaneously or enhance the resolution at which such observations are possible. For decades, flow cytometry has balanced these goals to fill a critical need by enabling the measurement of multiple features in single cells, commonly to examine complex or hierarchical cellular systems. Recently, a format for flow cytometry has been developed that leverages the precision of mass spectrometry. This fusion of the two technologies, termed mass cytometry, provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. In this Primer, we review the current state of mass cytometry, providing an overview of the instrumentation, its present capabilities, and methods of data analysis, as well as thoughts on future developments and applications.
Assuntos
Citometria de Fluxo/métodos , Espectrometria de Massas/métodos , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Humanos , Análise de Célula ÚnicaRESUMO
Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.
Assuntos
Biologia Computacional/métodos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatologia , Análise de Célula Única/métodos , Medula Óssea/patologia , Criança , Estudos de Coortes , Heterogeneidade Genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , TranscriptomaRESUMO
Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and "corrupted" developmental processes including cancer.
Assuntos
Algoritmos , Linfócitos B/citologia , Linfopoese , Humanos , Interleucina-7/metabolismo , Células Precursoras de Linfócitos B/citologia , Fator de Transcrição STAT5/metabolismo , Recombinação V(D)JRESUMO
The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health1. The intesting has a length of over nine metres, along which there are differences in structure and function2. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.
Assuntos
Intestinos , Análise de Célula Única , Humanos , Diferenciação Celular/genética , Cromatina/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Mucosa Intestinal/citologia , Intestinos/citologia , Intestinos/imunologia , Análise da Expressão Gênica de Célula ÚnicaRESUMO
The growth of omic data presents evolving challenges in data manipulation, analysis and integration. Addressing these challenges, Bioconductor provides an extensive community-driven biological data analysis platform. Meanwhile, tidy R programming offers a revolutionary data organization and manipulation standard. Here we present the tidyomics software ecosystem, bridging Bioconductor to the tidy R paradigm. This ecosystem aims to streamline omic analysis, ease learning and encourage cross-disciplinary collaborations. We demonstrate the effectiveness of tidyomics by analyzing 7.5 million peripheral blood mononuclear cells from the Human Cell Atlas, spanning six data frameworks and ten analysis tools.
Assuntos
Software , Humanos , Biologia Computacional/métodos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/citologia , Genômica/métodos , Análise de DadosRESUMO
The ability to align individual cellular information from multiple experimental sources is fundamental for a systems-level understanding of biological processes. However, currently available tools are mainly designed for single-cell transcriptomics matching and integration, and generally rely on a large number of shared features across datasets for cell matching. This approach underperforms when applied to single-cell proteomic datasets due to the limited number of parameters simultaneously accessed and lack of shared markers across these experiments. Here, we introduce a cell-matching algorithm, matching with partial overlap (MARIO) that accounts for both shared and distinct features, while consisting of vital filtering steps to avoid suboptimal matching. MARIO accurately matches and integrates data from different single-cell proteomic and multimodal methods, including spatial techniques and has cross-species capabilities. MARIO robustly matched tissue macrophages identified from COVID-19 lung autopsies via codetection by indexing imaging to macrophages recovered from COVID-19 bronchoalveolar lavage fluid by cellular indexing of transcriptomes and epitopes by sequencing, revealing unique immune responses within the lung microenvironment of patients with COVID.
Assuntos
COVID-19 , Proteômica , Humanos , Proteômica/métodos , Perfilação da Expressão Gênica/métodos , Transcriptoma , Pulmão , Análise de Célula Única/métodosRESUMO
Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.