ABSTRACT
The choice of developing thymocytes to become CD8+ cytotoxic or CD4+ helper T cells has been intensely studied, but many of the underlying mechanisms remain to be elucidated. Recent multiomics approaches have provided much higher resolution analysis of gene expression in developing thymocytes than was previously achievable, thereby offering a fresh perspective on this question. Focusing on our recent studies using CITE-seq (cellular indexing of transcriptomes and epitopes) analyses of mouse thymocytes, we present a detailed timeline of RNA and protein expression changes during CD8 versus CD4 T cell differentiation. We also revisit our current understanding of the links between T cell receptor signaling and expression of the lineage-defining transcription factors ThPOK and RUNX3. Finally, we propose a sequential selection model to explain the tight linkage between MHC-I versus MHC-II recognition and T cell lineage choice. This model incorporates key aspects of previously proposed kinetic signaling, instructive, and stochastic/selection models.
Subject(s)
CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Cell Differentiation , Cell Lineage , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Humans , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , Core Binding Factor Alpha 3 Subunit/metabolism , Core Binding Factor Alpha 3 Subunit/genetics , Mice , Transcription Factors/metabolism , Transcriptome , MultiomicsABSTRACT
During development innate lymphoid cells and specialized lymphocyte subsets colonize peripheral tissues, where they contribute to organogenesis and later constitute the first line of protection while maintaining tissue homeostasis. A few of these subsets are produced only during embryonic development and remain in the tissues throughout life. They are generated through a unique developmental program initiated in lympho-myeloid-primed progenitors, which lose myeloid and B cell potential. They either differentiate into innate lymphoid cells or migrate to the thymus to give rise to embryonic T cell receptor-invariant T cells. At later developmental stages, adaptive T lymphocytes are derived from lympho-myeloid progenitors that colonize the thymus, while lymphoid progenitors become specialized in the production of B cells. This sequence of events highlights the requirement for stratification in the establishment of immune functions that determine efficient seeding of peripheral tissues by a limited number of cells.
Subject(s)
B-Lymphocytes/immunology , Lymphocytes/physiology , Lymphoid Progenitor Cells/physiology , Natural Killer T-Cells/immunology , Thymus Gland/immunology , Animals , Cell Differentiation , Cell Lineage , Cellular Microenvironment , Cytokines/metabolism , Humans , Immunity, Innate , Lymphocyte Activation , Paracrine Communication , TranscriptomeABSTRACT
We comprehensively review memory B cells (MBCs), covering the definition of MBCs and their identities and subsets, how MBCs are generated, where they are localized, how they are maintained, and how they are reactivated. Whereas naive B cells adopt multiple fates upon stimulation, MBCs are more restricted in their responses. Evolving work reveals that the MBC compartment in mice and humans consists of distinct subpopulations with differing effector functions. We discuss the various approaches to define subsets and subset-specific roles. A major theme is the need to both deliver faster effector function upon reexposure and readapt to antigenically variant pathogens while avoiding burnout, which would be the result if all MBCs generated only terminal effector function. We discuss cell-intrinsic differences in gene expression and signaling that underlie differences in function between MBCs and naive B cells and among MBC subsets and how this leads to memory responses.
Subject(s)
B-Lymphocyte Subsets/immunology , B-Lymphocytes/immunology , Immunologic Memory , Vaccines/immunology , Animals , Humans , Immunity, Humoral , Lymphocyte Activation , Mice , TranscriptomeABSTRACT
Transcriptomics, the high-throughput characterization of RNAs, has been instrumental in defining pathogenic signatures in human autoimmunity and autoinflammation. It enabled the identification of new therapeutic targets in IFN-, IL-1- and IL-17-mediated diseases. Applied to immunomonitoring, transcriptomics is starting to unravel diagnostic and prognostic signatures that stratify patients, track molecular changes associated with disease activity, define personalized treatment strategies, and generally inform clinical practice. Herein, we review the use of transcriptomics to define mechanistic, diagnostic, and predictive signatures in human autoimmunity and autoinflammation. We discuss some of the analytical approaches applied to extract biological knowledge from high-dimensional data sets. Finally, we touch upon emerging applications of transcriptomics to study eQTLs, B and T cell repertoire diversity, and isoform usage.
Subject(s)
Autoimmune Diseases/diagnosis , Inflammation/diagnosis , Transcriptome , Autoimmune Diseases/immunology , Datasets as Topic , High-Throughput Nucleotide Sequencing , Humans , Inflammation/immunology , Information Storage and Retrieval , Molecular Targeted Therapy , Monitoring, Immunologic , PrognosisABSTRACT
CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
Subject(s)
Metabolic Engineering , T-Lymphocytes , Humans , Gene Expression Profiling , Metabolic Engineering/methods , RNA , TranscriptomeABSTRACT
In aging, physiologic networks decline in function at rates that differ between individuals, producing a wide distribution of lifespan. Though 70% of human lifespan variance remains unexplained by heritable factors, little is known about the intrinsic sources of physiologic heterogeneity in aging. To understand how complex physiologic networks generate lifespan variation, new methods are needed. Here, we present Asynch-seq, an approach that uses gene-expression heterogeneity within isogenic populations to study the processes generating lifespan variation. By collecting thousands of single-individual transcriptomes, we capture the Caenorhabditis elegans "pan-transcriptome"-a highly resolved atlas of non-genetic variation. We use our atlas to guide a large-scale perturbation screen that identifies the decoupling of total mRNA content between germline and soma as the largest source of physiologic heterogeneity in aging, driven by pleiotropic genes whose knockdown dramatically reduces lifespan variance. Our work demonstrates how systematic mapping of physiologic heterogeneity can be applied to reduce inter-individual disparities in aging.
Subject(s)
Aging , Caenorhabditis elegans , Gene Regulatory Networks , Longevity , Transcriptome , Caenorhabditis elegans/genetics , Caenorhabditis elegans/physiology , Animals , Aging/genetics , Transcriptome/genetics , Longevity/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , RNA, Messenger/metabolism , RNA, Messenger/geneticsABSTRACT
How evolution at the cellular level potentiates macroevolutionary change is central to understanding biological diversification. The >66,000 rove beetle species (Staphylinidae) form the largest metazoan family. Combining genomic and cell type transcriptomic insights spanning the largest clade, Aleocharinae, we retrace evolution of two cell types comprising a defensive gland-a putative catalyst behind staphylinid megadiversity. We identify molecular evolutionary steps leading to benzoquinone production by one cell type via a mechanism convergent with plant toxin release systems, and synthesis by the second cell type of a solvent that weaponizes the total secretion. This cooperative system has been conserved since the Early Cretaceous as Aleocharinae radiated into tens of thousands of lineages. Reprogramming each cell type yielded biochemical novelties enabling ecological specialization-most dramatically in symbionts that infiltrate social insect colonies via host-manipulating secretions. Our findings uncover cell type evolutionary processes underlying the origin and evolvability of a beetle chemical innovation.
Subject(s)
Coleoptera , Animals , Coleoptera/genetics , Coleoptera/metabolism , Evolution, Molecular , Benzoquinones/metabolism , Phylogeny , Genomics , Symbiosis/genetics , Transcriptome , Genome, InsectABSTRACT
Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
Subject(s)
Imaging, Three-Dimensional , Transcriptome , Animals , Mice , Humans , Transcriptome/genetics , Imaging, Three-Dimensional/methods , Software , Gene Expression Profiling/methods , Lymph Nodes/pathology , Lymph Nodes/metabolism , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Brain/metabolism , Mice, Inbred C57BL , Lymphatic Metastasis , FemaleABSTRACT
Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.
Subject(s)
Prosencephalon , Animals , Prosencephalon/metabolism , Prosencephalon/embryology , Mice , Rats , Blastocyst/metabolism , Female , CRISPR-Cas Systems/genetics , Transcriptome , Organogenesis , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Male , Mice, Inbred C57BLABSTRACT
Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.
Subject(s)
Poly A , Polyadenylation , Single-Cell Analysis , Single-Cell Analysis/methods , Humans , Poly A/metabolism , Animals , Mice , Introns/genetics , Transcriptome/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , Gene Expression RegulationABSTRACT
Cancer is a disease that stems from a fundamental liability inherent to multicellular life forms in which an individual cell is capable of reneging on the interests of the collective organism. Although cancer is commonly described as an evolutionary process, a less appreciated aspect of tumorigenesis may be the constraints imposed by the organism's developmental programs. Recent work from single-cell transcriptomic analyses across a range of cancer types has revealed the recurrence, plasticity, and co-option of distinct cellular states among cancer cell populations. Here, we note that across diverse cancer types, the observed cell states are proximate within the developmental hierarchy of the cell of origin. We thus posit a model by which cancer cell states are directly constrained by the organism's "developmental map." According to this model, a population of cancer cells traverses the developmental map, thereby generating a heterogeneous set of states whose interactions underpin emergent tumor behavior.
Subject(s)
Models, Biological , Neoplasms , Animals , Humans , Carcinogenesis/pathology , Carcinogenesis/genetics , Neoplasms/pathology , Neoplasms/genetics , Neoplasms/metabolism , Single-Cell Analysis , Transcriptome/genetics , Neoplastic Stem Cells/pathologyABSTRACT
Progress in understanding early human development has been impeded by the scarcity of reference datasets from natural embryos, particularly those with spatial information during crucial stages like gastrulation. We conducted high-resolution spatial transcriptomics profiling on 38,562 spots from 62 transverse sections of an intact Carnegie stage (CS) 8 human embryo. From this spatial transcriptomic dataset, we constructed a 3D model of the CS8 embryo, in which a range of cell subtypes are identified, based on gene expression patterns and positional register, along the anterior-posterior, medial-lateral, and dorsal-ventral axis in the embryo. We further characterized the lineage trajectories of embryonic and extra-embryonic tissues and associated regulons and the regionalization of signaling centers and signaling activities that underpin lineage progression and tissue patterning during gastrulation. Collectively, the findings of this study provide insights into gastrulation and post-gastrulation development of the human embryo.
Subject(s)
Embryo, Mammalian , Gastrulation , Gene Expression Regulation, Developmental , Imaging, Three-Dimensional , Humans , Embryo, Mammalian/metabolism , Transcriptome/genetics , Gastrula/metabolism , Gastrula/embryology , Signal Transduction , Cell Lineage , Gene Expression Profiling , Body Patterning/geneticsABSTRACT
Tumors are not simply a chaotic mass of mutated cells but can follow complex organizational principles, including in space. In this issue of Cell, Mathur and colleagues reconstruct a 3D genomic, epigenomic, and transcriptomic spatial cartograph of glioblastoma, offering a "whole-tumor" perspective with patterns of clonal expansion that are embedded in neurodevelopmental hierarchy.
Subject(s)
Brain Neoplasms , Glioblastoma , Humans , Glioblastoma/genetics , Glioblastoma/pathology , Transcriptome , Gene Expression Profiling , Brain Neoplasms/genetics , Brain Neoplasms/pathologyABSTRACT
Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.
Subject(s)
Gene Regulatory Networks , Single-Cell Analysis , Animals , Female , Humans , Mice , Cerebral Cortex/metabolism , Cerebral Cortex/cytology , CRISPR-Cas Systems/genetics , Dependovirus/genetics , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Genetic Vectors/metabolism , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Neurons/metabolism , Neurons/cytology , Single-Cell Analysis/methods , Transcriptome/genetics , Cell Line , Transcription, GeneticABSTRACT
Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.
Subject(s)
Brain Neoplasms , Glioblastoma , Glioblastoma/pathology , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Brain Neoplasms/pathology , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Spatial Analysis , Transcriptome/genetics , Tumor Microenvironment , Proteomics , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Gene Expression Regulation, NeoplasticABSTRACT
Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.
Subject(s)
Bone Marrow , Hematopoietic Stem Cells , Mesenchymal Stem Cells , Proteomics , Single-Cell Analysis , Transcriptome , Humans , Single-Cell Analysis/methods , Bone Marrow/metabolism , Hematopoietic Stem Cells/metabolism , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Proteomics/methods , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Hematopoiesis , Stem Cell Niche , Bone Marrow Cells/metabolism , Bone Marrow Cells/cytologyABSTRACT
The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. Whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche is unknown. To address this, we compared perinatal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited enrichment in genes mediating m6A mRNA methylation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs but not osteoblasts led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was significantly rescued by deletion of Klf2, an m6A target. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.
Subject(s)
Hematopoietic Stem Cells , Mesenchymal Stem Cells , Methyltransferases , Mice, Inbred C57BL , Stem Cell Niche , Animals , Mice , Adenosine/metabolism , Adenosine/analogs & derivatives , Cell Differentiation , Epigenesis, Genetic , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Kruppel-Like Transcription Factors , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Methyltransferases/metabolism , Methyltransferases/genetics , Osteoblasts/metabolism , Osteoblasts/cytology , Osteogenesis , RNA, Messenger/metabolism , RNA, Messenger/genetics , Transcriptome/genetics , HumansABSTRACT
Dexamethasone is a life-saving treatment for severe COVID-19, yet its mechanism of action is unknown, and many patients deteriorate or die despite timely treatment initiation. Here, we identify dexamethasone treatment-induced cellular and molecular changes associated with improved survival in COVID-19 patients. We observed a reversal of transcriptional hallmark signatures in monocytes associated with severe COVID-19 and the induction of a monocyte substate characterized by the expression of glucocorticoid-response genes. These molecular responses to dexamethasone were detected in circulating and pulmonary monocytes, and they were directly linked to survival. Monocyte single-cell RNA sequencing (scRNA-seq)-derived signatures were enriched in whole blood transcriptomes of patients with fatal outcome in two independent cohorts, highlighting the potential for identifying non-responders refractory to dexamethasone. Our findings link the effects of dexamethasone to specific immunomodulation and reversal of monocyte dysregulation, and they highlight the potential of single-cell omics for monitoring in vivo target engagement of immunomodulatory drugs and for patient stratification for precision medicine approaches.
Subject(s)
COVID-19 Drug Treatment , COVID-19 , Dexamethasone , Monocytes , SARS-CoV-2 , Single-Cell Analysis , Humans , Dexamethasone/pharmacology , Dexamethasone/therapeutic use , Monocytes/metabolism , Monocytes/drug effects , SARS-CoV-2/drug effects , Male , Female , Transcriptome , Middle Aged , Aged , Glucocorticoids/therapeutic use , Glucocorticoids/pharmacology , Lung/pathology , AdultABSTRACT
Over the past decade, mRNA modifications have emerged as important regulators of gene expression control in cells. Fueled in large part by the development of tools for detecting RNA modifications transcriptome wide, researchers have uncovered a diverse epitranscriptome that serves as an additional layer of gene regulation beyond simple RNA sequence. Here, we review the proteins that write, read, and erase these marks, with a particular focus on the most abundant internal modification, N6-methyladenosine (m6A). We first describe the discovery of the key enzymes that deposit and remove m6A and other modifications and discuss how our understanding of these proteins has shaped our views of modification dynamics. We then review current models for the function of m6A reader proteins and how our knowledge of these proteins has evolved. Finally, we highlight important future directions for the field and discuss key questions that remain unanswered.
Subject(s)
Adenosine , Gene Expression Regulation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Adenosine/genetics , Adenosine/metabolism , Proteins/genetics , Proteins/metabolism , TranscriptomeABSTRACT
Hematopoietic stem cells (HSCs) and downstream progenitors have long been studied based on phenotype, cell purification, proliferation, and transplantation into myeloablated recipients. These experiments, complemented by data on expression profiles, mouse mutants, and humans with hematopoietic defects, are the foundation for the current hematopoietic differentiation tree. However, there are fundamental gaps in our knowledge of the quantitative and qualitative operation of the HSC/progenitor system under physiological and pathological conditions in vivo. The hallmarks of HSCs, self-renewal and multipotency, are observed in in vitro assays and cell transplantation experiments; however, the extent to which these features occur naturally in HSCs and progenitors remains uncertain. We focus here on work that strives to address these unresolved questions, with emphasis on fate mapping and modeling of the hematopoietic flow from stem cells toward myeloid and lymphoid lineages during development and adult life.