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The immune system consists of many specialized cell populations that communicate with each other to achieve systemic immune responses. Our analyses of various measured immune cell population frequencies in healthy humans and their responses to diverse stimuli show that human immune variation is continuous in nature, rather than characterized by discrete groups of similar individuals. We show that the same three key combinations of immune cell population frequencies can define an individual's immunotype and predict a diverse set of functional responses to cytokine stimulation. We find that, even though interindividual variations in specific cell population frequencies can be large, unrelated individuals of younger age have more homogeneous immunotypes than older individuals. Across age groups, cytomegalovirus seropositive individuals displayed immunotypes characteristic of older individuals. The conceptual framework for defining immunotypes suggested by our results could guide the development of better therapies that appropriately modulate collective immunotypes, rather than individual immune components. [ABSTRACT FROM AUTHOR]

Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches relying on manual "gating." Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and often the number of subpopulations is unknown a priori. Here we describe ACCENSE, a tool that combines nonlinear dimensionality reduction with density-based partitioning, and displays multivariate cellular phenotypes on a 2D plot. We apply ACCENSE to 35-parameter mass cytometry data from CD8+ T cells derived from specific pathogen-free and germ-free mice, and stratify cells into phenotypic subpopulations. Our results show significant heterogeneity within the known CD8+ T-cell subpopulations, and of particular note is that we find a large novel subpopulation in both specific pathogen-free and germ-free mice that has not been described previously. This subpopulation possesses a phenotypic signature that is distinct from conventional naive and memory subpopulations when analyzed by ACCENSE, but is not distinguishable on a biaxial plot of standard markers. We are able to automatically identify cellular subpopulations based on all proteins analyzed, thus aiding the full utilization of powerful new single-cell technologies such as mass cytometry. [ABSTRACT FROM AUTHOR]

Mutational escape from vaccine-induced immune responses has thwarted the development of a successful vaccine against AIDS, whose causative agent is HIV, a highly mutable virus. Knowing the virus' fitness as a function of its proteomic sequence can enable rational design of potent vaccines, as this information can focus vaccine-induced immune responses to target mutational vulnerabilities of the virus. Spin models have been proposed as a means to infer intrinsic fitness landscapes of HIV proteins from patient-derived viral protein sequences. These sequences are the product of nonequilibrium viral evolution driven by patient-specific immune responses and are subject to phylogenetic constraints. How can such sequence data allow inference of intrinsic fitness landscapes? We combined computer simulations and variational theory á la Feynman to show that, in most circumstances, spin models inferred from patient-derived viral sequences reflect the correct rank order of the fitness of mutant viral strains. Our findings are relevant for diverse viruses. [ABSTRACT FROM AUTHOR]

Cellular immune control of HIV is mediated, in part, by induction of single amino acid mutations that reduce viral fitness, but compensatory mutations limit this effect. Here, we sought to determine if higher order constraints on viral evolution exist, because some coordinately linked combinations of mutations may hurt viability. Immune targeting of multiple sites in such a multidimensionally conserved region might render the virus particularly vulnerable, because viable escape pathways would be greatly restricted. We analyzed available HIV sequences using a method from physics to reveal distinct groups of amino acids whose mutations are collectively coordinated ("HIV sectors"). From the standpoint of mutations at individual sites, one such group in Gag is as conserved as other collectively coevolving groups of sites in Gag. However, it exhibits higher order conservation indicating constraints on the viability of viral strains with multiple mutations. Mapping amino acids from this group onto protein structures shows that combined mutations likely destabilize multiprotein structural interactions critical for viral function. Persons who durably control HIV without medications preferentially target the sector in Gag predicted to be most vulnerable. By sequencing circulating viruses from these individuals, we find that individual mutations occur with similar frequency in this sector as in other targeted Gag sectors. However, multiple mutations within this sector are very rare, indicating previously unrecognized multidimensional constraints on HIV evolution. Targeting such regions with higher order evolutionary constraints provides a novel approach to immunogen design for a vaccine against HIV and other rapidly mutating viruses. [ABSTRACT FROM AUTHOR]

Neuronal types in the central nervous system differ dramatically in their resilience to injury or other insults. Here we studied the selective resilience of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of ∼80% of RGCs within 2 weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 46 RGC types in adult retina. We then tracked their survival after ONC; characterized transcriptomic, physiological, and morphological changes that preceded degeneration; and identified genes selectively expressed by each type. Finally, using loss- and gain-of-function assays in vivo , we showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury and demonstrates that differential gene expression can be used to reveal molecular targets for intervention. • 46 transcriptionally distinct type of retinal ganglion cells (RGCs) in mice • RGC types differ dramatically in survival following axonal transection • Different genes expressed by resilient and vulnerable RGC types • Manipulation of differentially expressed genes promotes RGC survival and regeneration High-throughput single-cell RNA-seq characterizes 46 types of adult mouse retinal ganglion cells and documents dramatic differences among them in their ability to survive axotomy. Manipulation of genes differentially expressed between resilient and vulnerable types enhances survival and axon regeneration. [ABSTRACT FROM AUTHOR]

Summary High-acuity vision in primates, including humans, is mediated by a small central retinal region called the fovea. As more accessible organisms lack a fovea, its specialized function and its dysfunction in ocular diseases remain poorly understood. We used 165,000 single-cell RNA-seq profiles to generate comprehensive cellular taxonomies of macaque fovea and peripheral retina. More than 80% of >60 cell types match between the two regions but exhibit substantial differences in proportions and gene expression, some of which we relate to functional differences. Comparison of macaque retinal types with those of mice reveals that interneuron types are tightly conserved. In contrast, projection neuron types and programs diverge, despite exhibiting conserved transcription factor codes. Key macaque types are conserved in humans, allowing mapping of cell-type and region-specific expression of >190 genes associated with 7 human retinal diseases. Our work provides a framework for comparative single-cell analysis across tissue regions and species. Graphical Abstract Highlights • Macaque fovea and peripheral retina each contain >65 cell types • Most types correspond between regions but differ in proportions and gene expression • Greater conservation of interneuron than ganglion cell types between macaque and mouse • Cell-type- and region-specific expression of genes implicated in human blindness Single-cell-based analysis provides a comprehensive molecular and cellular taxonomy of the primate retina. [ABSTRACT FROM AUTHOR]

Summary The identification of cell types and marker genes is critical for dissecting neural development and function, but the size and complexity of the brain has hindered the comprehensive discovery of cell types. We combined single-cell RNA-seq (scRNA-seq) with anatomical brain registration to create a comprehensive map of the zebrafish habenula, a conserved forebrain hub involved in pain processing and learning. Single-cell transcriptomes of ∼13,000 habenular cells with 4× cellular coverage identified 18 neuronal types and dozens of marker genes. Registration of marker genes onto a reference atlas created a resource for anatomical and functional studies and enabled the mapping of active neurons onto neuronal types following aversive stimuli. Strikingly, despite brain growth and functional maturation, cell types were retained between the larval and adult habenula. This study provides a gene expression atlas to dissect habenular development and function and offers a general framework for the comprehensive characterization of other brain regions. [ABSTRACT FROM AUTHOR]

Summary Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class. [ABSTRACT FROM AUTHOR]

• Temporally regulated gene modules are expressed at distinct phases of postmitotic RGC differentiation in mice. • Transcription factors and recognition molecules are selectively expressed by subsets of 45 developing or adult RGC types. • Three models of visual deprivation showed that vision is dispensable for RGC type specification. • Visual deprivation does, however, affect molecular aspects of RGC maturation and maintenance. The development and connectivity of retinal ganglion cells (RGCs), the retina's sole output neurons, are patterned by activity-independent transcriptional programs and activity-dependent remodeling. To inventory the molecular correlates of these influences, we applied high-throughput single-cell RNA sequencing (scRNA-seq) to mouse RGCs at six embryonic and postnatal ages. We identified temporally regulated modules of genes that correlate with, and likely regulate, multiple phases of RGC development, ranging from differentiation and axon guidance to synaptic recognition and refinement. Some of these genes are expressed broadly while others, including key transcription factors and recognition molecules, are selectively expressed by one or a few of the 45 transcriptomically distinct types defined previously in adult mice. Next, we used these results as a foundation to analyze the transcriptomes of RGCs in mice lacking visual experience due to dark rearing from birth or to mutations that ablate either bipolar or photoreceptor cells. 98.5% of visually deprived (VD) RGCs could be unequivocally assigned to a single RGC type based on their transcriptional profiles, demonstrating that visual activity is dispensable for acquisition and maintenance of RGC type identity. However, visual deprivation significantly reduced the transcriptomic distinctions among RGC types, implying that activity is required for complete RGC maturation or maintenance. Consistent with this notion, transcriptomic alternations in VD RGCs significantly overlapped with gene modules found in developing RGCs. Our results provide a resource for mechanistic analyses of RGC differentiation and maturation, and for investigating the role of activity in these processes. [ABSTRACT FROM AUTHOR]

Chronic hepatitis C virus (HCV) infection is one of the leading causes of liver failure and liver cancer, affecting around 3% of the world's population. The extreme sequence variability of the virus resulting from error-prone replication has thwarted the discovery of a universal prophylactic vaccine. It is known that vigorous and multispecific cellular immune responses, involving both helper CD4+ and cytotoxic CD8+ T cells, are associated with the spontaneous clearance of acute HCV infection. Escape mutations in viral epitopes can, however, abrogate protective T-cell responses, leading to viral persistence and associated pathologies. Despite the propensity of the virus to mutate, there might still exist substitutions that incur a fitness cost. In this paper, we identify groups of coevolving residues within HCV nonstructural protein 3 (NS3) by analyzing diverse sequences of this protein using ideas from random matrix theory and associated methods. Our analyses indicate that one of these groups comprises a large percentage of residues for which HCV appears to resist multiple simultaneous substitutions. Targeting multiple residues in this group through vaccine-induced immune responses should either lead to viral recognition or elicit escape substitutions that compromise viral fitness. Our predictions are supported by published clinical data, which suggested that immune genotypes associated with spontaneous clearance of HCV preferentially recognized and targeted this vulnerable group of residues. Moreover, mapping the sites of this group onto the available protein structure provided insight into its functional significance. An epitope-based immunogen is proposed as an alternative to the NS3 epitopes in the peptide-based vaccine IC41. [ABSTRACT FROM AUTHOR]

Across neurodegenerative diseases, common mechanisms may reveal novel therapeutic targets based on neuronal protection, repair, or regeneration, independent of etiology or site of disease pathology. To address these mechanisms and discuss emerging treatments, in April, 2021, Glaucoma Research Foundation, BrightFocus Foundation, and the Melza M. and Frank Theodore Barr Foundation collaborated to bring together key opinion leaders and experts in the field of neurodegenerative disease for a virtual meeting titled "Solving Neurodegeneration". This "think-tank" style meeting focused on uncovering common mechanistic roots of neurodegenerative disease and promising targets for new treatments, catalyzed by the goal of finding new treatments for glaucoma, the world's leading cause of irreversible blindness and the common interest of the three hosting foundations. Glaucoma, which causes vision loss through degeneration of the optic nerve, likely shares early cellular and molecular events with other neurodegenerative diseases of the central nervous system. Here we discuss major areas of mechanistic overlap between neurodegenerative diseases of the central nervous system: neuroinflammation, bioenergetics and metabolism, genetic contributions, and neurovascular interactions. We summarize important discussion points with emphasis on the research areas that are most innovative and promising in the treatment of neurodegeneration yet require further development. The research that is highlighted provides unique opportunities for collaboration that will lead to efforts in preventing neurodegeneration and ultimately vision loss. [ABSTRACT FROM AUTHOR]

Most irreversible blindness results from retinal disease. To advance our understanding of the etiology of blinding diseases, we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptomes of ~85,000 cells from the fovea and peripheral retina of seven adult human donors. Utilizing computational methods, we identified 58 cell types within 6 classes: photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and non-neuronal cells. Nearly all types are shared between the two retinal regions, but there are notable differences in gene expression and proportions between foveal and peripheral cohorts of shared types. We then used the human retinal atlas to map expression of 636 genes implicated as causes of or risk factors for blinding diseases. Many are expressed in striking cell class-, type-, or region-specific patterns. Finally, we compared gene expression signatures of cell types between human and the cynomolgus macaque monkey, Macaca fascicularis. We show that over 90% of human types correspond transcriptomically to those previously identified in macaque, and that expression of disease-related genes is largely conserved between the two species. These results validate the use of the macaque for modeling blinding disease, and provide a foundation for investigating molecular mechanisms underlying visual processing. [ABSTRACT FROM AUTHOR]

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From ∼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa), and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation. [ABSTRACT FROM AUTHOR]

Summary CD8 + T cells contribute to the control of HIV, but it is not clear whether initial immune responses modulate the viral set point. We screened high-risk uninfected women twice a week for plasma HIV RNA and identified 12 hyperacute infections. Onset of viremia elicited a massive HIV-specific CD8 + T cell response, with limited bystander activation of non-HIV memory CD8 + T cells. HIV-specific CD8 + T cells secreted little interferon-γ, underwent rapid apoptosis, and failed to upregulate the interleukin-7 receptor, known to be important for T cell survival. The rapidity to peak CD8 + T cell activation and the absolute magnitude of activation induced by the exponential rise in viremia were inversely correlated with set point viremia. These data indicate that rapid, high magnitude HIV-induced CD8 + T cell responses are crucial for subsequent immune control of acute infection, which has important implications for HIV vaccine design. [ABSTRACT FROM AUTHOR]

Summary Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. Video Abstract [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1)-specific monoclonal antibodies with extraordinary potency and breadth have recently been described. In humanized mice, combinations of monoclonal antibodies have been shown to suppress viraemia, but the therapeutic potential of these monoclonal antibodies has not yet been evaluated in primates with an intact immune system. Here we show that administration of a cocktail of HIV-1-specific monoclonal antibodies, as well as the single glycan-dependent monoclonal antibody PGT121, resulted in a rapid and precipitous decline of plasma viraemia to undetectable levels in rhesus monkeys chronically infected with the pathogenic simian-human immunodeficiency virus SHIV-SF162P3. A single monoclonal antibody infusion afforded up to a 3.1 log decline of plasma viral RNA in 7 days and also reduced proviral DNA in peripheral blood, gastrointestinal mucosa and lymph nodes without the development of viral resistance. Moreover, after monoclonal antibody administration, host Gag-specific T-lymphocyte responses showed improved functionality. Virus rebounded in most animals after a median of 56 days when serum monoclonal antibody titres had declined to undetectable levels, although, notably, a subset of animals maintained long-term virological control in the absence of further monoclonal antibody infusions. These data demonstrate a profound therapeutic effect of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys as well as an impact on host immune responses. Our findings strongly encourage the investigation of monoclonal antibody therapy for HIV-1 in humans. [ABSTRACT FROM AUTHOR]

The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b , a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs. [Display omitted] • Three glutamatergic L2/3 cell types are specified after eye opening • L2/3 cell types are spatially segregated by graded transcriptomic distinctions • Vision is required for the graded expression of L2/3 cell type markers • Binocular function in L2/3 requires Igsf9b , a graded vision-dependent gene Transcriptomic, genetic, and functional analyses of the developing mouse primary visual cortex (V1) reveal that early sensory experience selectively influences the development of layer 2/3 glutamatergic neurons by regulating spatial and temporal gene expression programs. [ABSTRACT FROM AUTHOR]

Retinal ganglion cells (RGCs) form an array of feature detectors, which convey visual information to central brain regions. Characterizing RGC diversity is required to understand the logic of the underlying functional segregation. Using single-cell transcriptomics, we systematically classified RGCs in adult and larval zebrafish, thereby identifying marker genes for >30 mature types and several developmental intermediates. We used this dataset to engineer transgenic driver lines, enabling specific experimental access to a subset of RGC types. Expression of one or few transcription factors often predicts dendrite morphologies and axonal projections to specific tectal layers and extratectal targets. In vivo calcium imaging revealed that molecularly defined RGCs exhibit specific functional tuning. Finally, chemogenetic ablation of eomesa + RGCs, which comprise melanopsin-expressing types with projections to a small subset of central targets, selectively impaired phototaxis. Together, our study establishes a framework for systematically studying the functional architecture of the visual system. • Transcriptional profiling classifies >30 distinct retinal ganglion cell types • Molecular profiles of RGCs correlate with morphological and physiological features • Genome-engineered driver lines provide selective access to RGC types • Perturbation of a genetically defined visual pathway disrupts phototaxis A molecular catalog of retinal ganglion cell (RGC) types is essential for understanding the organization and development of the visual system. Kölsch et al. used single-cell RNA-seq to identify >30 larval and adult zebrafish RGC types. Using genetic methods, they linked transcriptomically defined types to morphology, physiology, and a visual behavior. [ABSTRACT FROM AUTHOR]

Summary In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals. Graphical Abstract Highlights • Intestinal stem cells (ISCs) are non-conventional antigen-presenting cells by MHCII • Interactions between ISCs and T helper subsets modulate distinct ISC fates • Epithelial MHCII is important for epithelial-cell remodeling following infection • Regulatory T cells and their key cytokine IL-10 support ISC renewal Intestinal stem cells act as non-conventional antigen presenting cells, and these interactions with T helper cells modulate ISC renewal and differentiation to shape the intestine. [ABSTRACT FROM AUTHOR]

The article focuses on research into gene expression trajectories of stem cells of zebrafish embryos during embryogenesis. It states that single-cell RNA sequencing (scRNA-seq) data was analyzed using a simulated diffusion-based computational method with modules of co-expressed genes identified and connected over development time. It comments that a gene expression analysis found intermediate cells could switch specification from one cellular rate to another.