ABSTRACT
Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.
Subject(s)
Gene Knock-In Techniques/methods , Genetic Engineering/methods , Immunotherapy/methods , Animals , Blood Cells , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Humans , Mice , Mice, Inbred NOD , Mice, SCID , RNA, Guide, Kinetoplastida/genetics , Single-Cell Analysis/methods , T-Lymphocytes , Transcriptome/geneticsABSTRACT
The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.
Subject(s)
Brain/cytology , CD4-Positive T-Lymphocytes/metabolism , Fetus/cytology , Microglia/cytology , Microglia/metabolism , Synapses/metabolism , Adult , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Behavior Rating Scale , Blood Cells/cytology , Blood Cells/metabolism , Brain/embryology , Brain/metabolism , Child , Female , Fetus/embryology , Humans , Lectins, C-Type/metabolism , Lung/cytology , Lung/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Neurogenesis/genetics , Parabiosis , Pyramidal Cells/metabolism , Pyramidal Cells/physiology , Single-Cell Analysis , Spleen/cytology , Spleen/metabolism , Synapses/immunology , TranscriptomeABSTRACT
Anemia is a major comorbidity in aging, chronic kidney and inflammatory diseases, and hematologic malignancies. However, the transcriptomic networks governing hematopoietic differentiation in blood cell development remain incompletely defined. Here we report that the atypical kinase RIOK2 (right open reading frame kinase 2) is a master transcription factor (TF) that not only drives erythroid differentiation, but also simultaneously suppresses megakaryopoiesis and myelopoiesis in primary human stem and progenitor cells. Our study reveals the previously uncharacterized winged helix-turn-helix DNA-binding domain and two transactivation domains of RIOK2 that are critical to regulate key hematopoietic TFs GATA1, GATA2, SPI1, RUNX3 and KLF1. This establishes RIOK2 as an integral component of the transcriptional regulatory network governing human hematopoietic differentiation. Importantly, RIOK2 mRNA expression significantly correlates with these TFs and other hematopoietic genes in myelodysplastic syndromes, acute myeloid leukemia and chronic kidney disease. Further investigation of RIOK2-mediated transcriptional pathways should yield therapeutic approaches to correct defective hematopoiesis in hematologic disorders.
Subject(s)
Blood Cells/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Cell Differentiation/physiology , Cell Line, Tumor , Cells, Cultured , Erythropoiesis/physiology , Gene Expression Regulation/physiology , HEK293 Cells , Hematopoietic Stem Cells/metabolism , Humans , K562 Cells , Leukemia, Myeloid, Acute/metabolism , Myelodysplastic Syndromes/metabolism , Myelopoiesis/physiology , Transcription Factors/metabolism , Transcription, Genetic/physiologyABSTRACT
Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.
Subject(s)
Blood Cells/metabolism , Bone Marrow Cells/metabolism , Cell Separation , Flow Cytometry , Gene Expression Profiling , Proteome , Proteomics , Single-Cell Analysis , Transcriptome , Age Factors , Blood Cells/immunology , Blood Cells/pathology , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , Cells, Cultured , Databases, Genetic , Healthy Aging/genetics , Healthy Aging/immunology , Healthy Aging/metabolism , Humans , Leukemia/genetics , Leukemia/immunology , Leukemia/metabolism , Leukemia/pathology , RNA-Seq , Systems BiologyABSTRACT
The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.
Subject(s)
Viral Vaccines/administration & dosage , Zika Virus Infection/immunology , Zika Virus Infection/prevention & control , Zika Virus/physiology , Aedes/virology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Blood Cells/virology , Embryo, Mammalian/virology , Female , Fetus/virology , Humans , Lipids/administration & dosage , Male , Mice , Mice, Inbred C57BL , Mutation , RNA, Messenger/genetics , RNA, Messenger/immunology , Specific Pathogen-Free Organisms , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology , Viral Vaccines/immunology , Zika Virus Infection/virologyABSTRACT
A class of cis-regulatory elements, called enhancers, play a central role in orchestrating spatiotemporally precise gene-expression programs during development. Consequently, divergence in enhancer sequence and activity is thought to be an important mediator of inter- and intra-species phenotypic variation. Here, we give an overview of emerging principles of enhancer function, current models of enhancer architecture, genomic substrates from which enhancers emerge during evolution, and the influence of three-dimensional genome organization on long-range gene regulation. We discuss intricate relationships between distinct elements within complex regulatory landscapes and consider their potential impact on specificity and robustness of transcriptional regulation.
Subject(s)
Enhancer Elements, Genetic , Evolution, Molecular , Genome-Wide Association Study , Transcription, Genetic , Blood/metabolism , Blood Cells/metabolism , Epigenomics , Hematopoiesis , Humans , Quantitative Trait LociABSTRACT
Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation. The vast majority of interactions are uncharted, constituting a major missing link in understanding genome control. Here, we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types. We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers. Promoter interactomes reflect lineage relationships of the hematopoietic tree, consistent with dynamic remodeling of nuclear architecture during differentiation. Interacting regions are enriched in genetic variants linked with altered expression of genes they contact, highlighting their functional role. We exploit this rich resource to connect non-coding disease variants to putative target promoters, prioritizing thousands of disease-candidate genes and implicating disease pathways. Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases.
Subject(s)
Blood Cells/cytology , Disease/genetics , Promoter Regions, Genetic , Cell Lineage , Cell Separation , Chromatin , Enhancer Elements, Genetic , Epigenomics , Genetic Predisposition to Disease , Genome-Wide Association Study , Hematopoiesis , Humans , Polymorphism, Single Nucleotide , Quantitative Trait LociABSTRACT
The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.
Subject(s)
Blood Cells/physiology , Immunity, Cellular , Protein Interaction Maps , Proteome , Proteomics , Animals , Bodily Secretions , Cell Communication , Computer Simulation , Humans , Mass Spectrometry , Social SupportABSTRACT
Mast cells are evolutionarily ancient sentinel cells. Like basophils, mast cells express the high-affinity receptor for immunoglobulin E (IgE) and have been linked to host defense and diverse immune-system-mediated diseases. To better characterize the function of these cells, we assessed the transcriptional profiles of mast cells isolated from peripheral connective tissues and basophils isolated from spleen and blood. We found that mast cells were transcriptionally distinct, clustering independently from all other profiled cells, and that mast cells demonstrated considerably greater heterogeneity across tissues than previously appreciated. We observed minimal homology between mast cells and basophils, which shared more overlap with other circulating granulocytes than with mast cells. The derivation of mast-cell and basophil transcriptional signatures underscores their differential capacities to detect environmental signals and influence the inflammatory milieu.
Subject(s)
Basophils/physiology , Blood Cells/physiology , Connective Tissue Cells/physiology , Mast Cells/physiology , Spleen/cytology , Animals , Cell Separation , Cells, Cultured , Flow Cytometry , Gene Expression Profiling , Immunoglobulin E/metabolism , Male , Mice , Mice, Inbred C57BL , Tissue Array AnalysisABSTRACT
Somatic stem cells replenish many tissues throughout life to repair damage and to maintain tissue homeostasis. Stem cell function is frequently described as following a hierarchical model in which a single master cell undergoes self-renewal and differentiation into multiple cell types and is responsible for most regenerative activity. However, recent data from studies on blood, skin and intestinal epithelium all point to the concomitant action of multiple types of stem cells with distinct everyday roles. Under stress conditions such as acute injury, the surprising developmental flexibility of these stem cells enables them to adapt to diverse roles and to acquire different regeneration capabilities. This paradigm shift raises many new questions about the developmental origins, inter-relationships and molecular regulation of these multiple stem cell types.
Subject(s)
Adult Stem Cells/cytology , Animals , Blood Cells/cytology , Cell Culture Techniques , Hematopoiesis , Humans , Intestines/cytology , Skin/cytologyABSTRACT
Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.
Subject(s)
Blood Cells/metabolism , Cell Differentiation/genetics , DNA Mutational Analysis/methods , Muscle, Smooth/metabolism , Mutation , Neurons/metabolism , Single Molecule Imaging/methods , Stem Cells/metabolism , Alzheimer Disease/genetics , Blood Cells/cytology , Cell Division , Cohort Studies , Colon/cytology , Epithelium/metabolism , Granulocytes/cytology , Granulocytes/metabolism , Healthy Volunteers , Humans , Male , Middle Aged , Muscle, Smooth/cytology , Mutagenesis , Mutation Rate , Neurons/cytology , Stem Cells/cytologyABSTRACT
The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.
Subject(s)
Cell Lineage/genetics , Embryonic Development/genetics , Hematopoietic System/embryology , Hematopoietic System/metabolism , Mutation , Blood Cells/cytology , Blood Cells/metabolism , Clone Cells/cytology , Clone Cells/metabolism , DNA Mutational Analysis , Fetus/cytology , Fetus/embryology , Fetus/metabolism , Germ Layers/cytology , Germ Layers/metabolism , Health , Hematopoietic System/cytology , Humans , Karyotyping , Male , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Mutation Rate , Organ Specificity/genetics , Time Factors , Whole Genome Sequencing , WorkflowABSTRACT
I was attracted to hematology because by combining clinical findings with the use of a microscope and simple laboratory tests, one could often make a diagnosis. I was attracted to genetics when I learned about inherited blood disorders, at a time when we had only hints that somatic mutations were also important. It seemed clear that if we understood not only what genetic changes caused what diseases but also the mechanisms through which those genetic changes contribute to cause disease, we could improve management. Thus, I investigated many aspects of the glucose-6-phosphate dehydrogenase system, including cloning of the gene, and in the study of paroxysmal nocturnal hemoglobinuria (PNH), I found that it is a clonal disorder; subsequently, we were able to explain how a nonmalignant clone can expand, and I was involved in the first trial of PNH treatment by complement inhibition. I was fortunate to do clinical and research hematology in five countries; in all of them, I learned from mentors, from colleagues, and from patients.
Subject(s)
Hemoglobinuria, Paroxysmal , Humans , Hemoglobinuria, Paroxysmal/genetics , Hemoglobinuria, Paroxysmal/pathology , Blood Cells/pathology , Clone Cells/pathologyABSTRACT
The vasculature consists of vessels of different sizes that are arranged in a hierarchical pattern. Two cell populations work in concert to establish this pattern during embryonic development and adopt it to changes in blood flow demand later in life: endothelial cells that line the inner surface of blood vessels, and adjacent vascular mural cells, including smooth muscle cells and pericytes. Despite recent progress in elucidating the signalling pathways controlling their crosstalk, much debate remains with regard to how mural cells influence endothelial cell biology and thereby contribute to the regulation of blood vessel formation and diameters. In this Review, I discuss mural cell functions and their interactions with endothelial cells, focusing on how these interactions ensure optimal blood flow patterns. Subsequently, I introduce the signalling pathways controlling mural cell development followed by an overview of mural cell ontogeny with an emphasis on the distinguishing features of mural cells located on different types of blood vessels. Ultimately, I explore therapeutic strategies involving mural cells to alleviate tissue ischemia and improve vascular efficiency in a variety of diseases.
Subject(s)
Blood Cells , Endothelial Cells , Female , Pregnancy , Humans , Cell Differentiation , Embryonic Development , BiologyABSTRACT
Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. Here, we combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, we identified a highly migratory and immune-responsive pupal cell population expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, we present the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/metabolism , Drosophila melanogaster/metabolism , Transcriptome/genetics , Cell Differentiation , Blood Cells/metabolism , Drosophila Proteins/metabolism , Hemocytes , Larva/metabolismABSTRACT
BloodSpot is a specialised database integrating gene expression data from acute myeloid leukaemia (AML) patients related to blood cell development and maturation. The database and interface has helped numerous researchers and clinicians to quickly get an overview of gene expression patterns in healthy and malignant haematopoiesis. Here, we present an update to our framework that includes protein expression data of sorted single cells. With this update we also introduce datasets broadly spanning age groups, which many users have requested, with particular interest for researchers studying paediatric leukaemias. The backend of the database has been rewritten and migrated to a cloud-based environment to accommodate the growth, and provide a better user-experience for our many international users. Users can now enjoy faster transfer speeds and a more responsive interface. In conclusion, the continuing popularity of the database and emergence of new data modalities has prompted us to rewrite and futureproof the back-end, including paediatric centric views, as well as single cell protein data, allowing us to keep the database updated and relevant for the years to come. The database is freely available at www.bloodspot.eu.
Subject(s)
Hematopoiesis , Leukemia, Myeloid, Acute , Child , Humans , Blood Cells , Cell Differentiation , Databases, Genetic , Hematopoiesis/genetics , Leukemia, Myeloid, Acute/genetics , Proteins/geneticsABSTRACT
In animals, microRNAs are amongst the primary non-coding RNAs involved in regulating the gene expression of a cell. Most mRNAs in a cell are targeted by one or many miRNAs. Although several mechanisms can be attributed to the degradation of miRNA and mRNA within a cell, but the involvement of autophagy in the clearance of miRNA and its target mRNA is not known. We discover a leucine-responsive axis in blood cell progenitors that can mediate an autophagy-directed degradation of miRNA-bound mRNA in Drosophila melanogaster and Homo sapiens. This previously unknown miRNA clearance axis is activated upon amino acid deprivation that can traffic miRNA-mRNA-loaded Argonaute for autophagic degradation in a p62-dependent manner. Thus, our research not only reports a novel axis that can address the turnover of a catalytically active miRISC but also elucidates a slicer-independent mechanism through which autophagy can selectively initiate the clearance of target mRNA.
Subject(s)
MicroRNAs , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Autophagy/genetics , Blood CellsABSTRACT
Hematopoietic stem and progenitor cells maintain blood cell homeostasis by integrating various cues provided by specialized microenvironments or niches. Biomechanical forces are emerging as key regulators of hematopoiesis. Here, we report that mechanical stimuli provided by blood flow in the vascular niche control Drosophila hematopoiesis. In vascular niche cells, the mechanosensitive channel Piezo transduces mechanical forces through intracellular calcium upregulation, leading to Notch activation and repression of FGF ligand transcription, known to regulate hematopoietic progenitor maintenance. Our results provide insight into how the vascular niche integrates mechanical stimuli to regulate hematopoiesis.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/metabolism , Drosophila Proteins/metabolism , Hematopoiesis/physiology , Blood Cells , Stem Cells/metabolism , Stem Cell Niche , Ion ChannelsABSTRACT
Variable levels of gene expression between tissues complicates the use of RNA sequencing of patient biosamples to delineate the impact of genomic variants. Here, we describe a gene- and tissue-specific metric to inform the feasibility of RNA sequencing. This overcomes limitations of using expression values alone as a metric to predict RNA-sequencing utility. We have derived a metric, minimum required sequencing depth (MRSD), that estimates the depth of sequencing required from RNA sequencing to achieve user-specified sequencing coverage of a gene, transcript, or group of genes. We applied MRSD across four human biosamples: whole blood, lymphoblastoid cell lines (LCLs), skeletal muscle, and cultured fibroblasts. MRSD has high precision (90.1%-98.2%) and overcomes transcript region-specific sequencing biases. Applying MRSD scoring to established disease gene panels shows that fibroblasts, of these four biosamples, are the optimum source of RNA for 63.1% of gene panels. Using this approach, up to 67.8% of the variants of uncertain significance in ClinVar that are predicted to impact splicing could be assayed by RNA sequencing in at least one of the biosamples. We demonstrate the utility and benefits of MRSD as a metric to inform functional assessment of splicing aberrations, in particular in the context of Mendelian genetic disorders to improve diagnostic yield.