Insights Gained from Single-Cell Analysis of Immune Cells in the Tumor Microenvironment.

Understanding tumor immune microenvironments is critical for identifying immune modifiers of cancer progression and developing cancer immunotherapies. Recent applications of single-cell RNA sequencing (scRNA-seq) in dissecting tumor microenvironments have brought important insights into the biology of tumor-infiltrating immune cells, including their heterogeneity, dynamics, and potential roles in both disease progression and response to immune checkpoint inhibitors and other immunotherapies. This review focuses on the advances in knowledge of tumor immune microenvironments acquired from scRNA-seq studies across multiple types of human tumors, with a particular emphasis on the study of phenotypic plasticity and lineage dynamics of immune cells in the tumor environment. We also discuss several imminent questions emerging from scRNA-seq observations and their potential solutions on the horizon.

The Myeloid Cell Compartment-Cell by Cell.

Myeloid cells are a major cellular compartment of the immune system comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. Models of cellular ontogeny, activation, differentiation, and tissue-specific functions of myeloid cells have been revisited during the last years with surprising results; for example, most tissue macrophages are yolk sac derived, monocytes and macrophages follow a multidimensional model of activation, and tissue signals have a significant impact on the functionality of all these cells. While these exciting results have brought these cells back to center stage, their enormous plasticity and heterogeneity, during both homeostasis and disease, are far from understood. At the same time, the ongoing revolution in single-cell genomics, with single-cell RNA sequencing (scRNA-seq) leading the way, promises to change this. Prevailing models of hematopoiesis with distinct intermediates are challenged by scRNA-seq data suggesting more continuous developmental trajectories in the myeloid cell compartment. Cell subset structures previously defined by protein marker expression need to be revised based on unbiased analyses of scRNA-seq data. Particularly in inflammatory conditions, myeloid cells exhibit substantially vaster heterogeneity than previously anticipated, and work performed within large international projects, such as the Human Cell Atlas, has already revealed novel tissue macrophage subsets. Based on these exciting developments, we propose the next steps to a full understanding of the myeloid cell compartment in health and diseases.

Systems Immunology: Learning the Rules of the Immune System.

Given the many cell types and molecular components of the human immune system, along with vast variations across individuals, how should we go about developing causal and predictive explanations of immunity? A central strategy in human studies is to leverage natural variation to find relationships among variables, including DNA variants, epigenetic states, immune phenotypes, clinical descriptors, and others. Here, we focus on how natural variation is used to find patterns, infer principles, and develop predictive models for two areas: (a) immune cell activation-how single-cell profiling boosts our ability to discover immune cell types and states-and (b) antigen presentation and recognition-how models can be generated to predict presentation of antigens on MHC molecules and their detection by T cell receptors. These are two examples of a shift in how we find the drivers and targets of immunity, especially in the human system in the context of health and disease.

Pan-cancer single-cell dissection reveals phenotypically distinct B cell subtypes.

Characterizing the compositional and phenotypic characteristics of tumor-infiltrating B cells (TIBs) is important for advancing our understanding of their role in cancer development. Here, we establish a comprehensive resource of human B cells by integrating single-cell RNA sequencing data of B cells from 649 patients across 19 major cancer types. We demonstrate substantial heterogeneity in their total abundance and subtype composition and observe immunoglobulin G (IgG)-skewness of antibody-secreting cell isotypes. Moreover, we identify stress-response memory B cells and tumor-associated atypical B cells (TAABs), two tumor-enriched subpopulations with prognostic potential, shared in a pan-cancer manner. In particular, TAABs, characterized by a high clonal expansion level and proliferative capacity as well as by close interactions with activated CD4 T cells in tumors, are predictive of immunotherapy response. Our integrative resource depicts distinct clinically relevant TIB subsets, laying a foundation for further exploration of functional commonality and diversity of B cells in cancer.

Neutrophil profiling illuminates anti-tumor antigen-presenting potency.

Neutrophils, the most abundant and efficient defenders against pathogens, exert opposing functions across cancer types. However, given their short half-life, it remains challenging to explore how neutrophils adopt specific fates in cancer. Here, we generated and integrated single-cell neutrophil transcriptomes from 17 cancer types (225 samples from 143 patients). Neutrophils exhibited extraordinary complexity, with 10 distinct states including inflammation, angiogenesis, and antigen presentation. Notably, the antigen-presenting program was associated with favorable survival in most cancers and could be evoked by leucine metabolism and subsequent histone H3K27ac modification. These neutrophils could further invoke both (neo)antigen-specific and antigen-independent T cell responses. Neutrophil delivery or a leucine diet fine-tuned the immune balance to enhance anti-PD-1 therapy in various murine cancer models. In summary, these data not only indicate the neutrophil divergence across cancers but also suggest therapeutic opportunities such as antigen-presenting neutrophil delivery.

Proteostasis governs differential temperature sensitivity across embryonic cell types.

Embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. Phenotypes associated with environmental stress suggest that some cell types are more sensitive to stress than others, but the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single-cell RNA sequencing (RNA-seq) to identify cell types and molecular programs driving phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in developmental timing. The notochord is particularly sensitive to temperature, which we map to a specialized cell type: sheath cells. These cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and anatomic defects. Our study demonstrates that whole-animal single-cell RNA-seq can identify mechanisms for developmental robustness and pinpoint cell types that constitute key failure points.

Multi-omics analysis of mucosal and systemic immunity to SARS-CoV-2 after birth.

The dynamics of immunity to infection in infants remain obscure. Here, we used a multi-omics approach to perform a longitudinal analysis of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infants and young children by analyzing blood samples and weekly nasal swabs collected before, during, and after infection with Omicron and non-Omicron variants. Infection stimulated robust antibody titers that, unlike in adults, showed no sign of decay for up to 300 days. Infants mounted a robust mucosal immune response characterized by inflammatory cytokines, interferon (IFN) α, and T helper (Th) 17 and neutrophil markers (interleukin [IL]-17, IL-8, and CXCL1). The immune response in blood was characterized by upregulation of activation markers on innate cells, no inflammatory cytokines, but several chemokines and IFNα. The latter correlated with viral load and expression of interferon-stimulated genes (ISGs) in myeloid cells measured by single-cell multi-omics. Together, these data provide a snapshot of immunity to infection during the initial weeks and months of life.

SPLASH: A statistical, reference-free genomic algorithm unifies biological discovery.

Today's genomics workflows typically require alignment to a reference sequence, which limits discovery. We introduce a unifying paradigm, SPLASH (Statistically Primary aLignment Agnostic Sequence Homing), which directly analyzes raw sequencing data, using a statistical test to detect a signature of regulation: sample-specific sequence variation. SPLASH detects many types of variation and can be efficiently run at scale. We show that SPLASH identifies complex mutation patterns in SARS-CoV-2, discovers regulated RNA isoforms at the single-cell level, detects the vast sequence diversity of adaptive immune receptors, and uncovers biology in non-model organisms undocumented in their reference genomes: geographic and seasonal variation and diatom association in eelgrass, an oceanic plant impacted by climate change, and tissue-specific transcripts in octopus. SPLASH is a unifying approach to genomic analysis that enables expansive discovery without metadata or references.

Molecular and spatial signatures of mouse brain aging at single-cell resolution.

The diversity and complex organization of cells in the brain have hindered systematic characterization of age-related changes in its cellular and molecular architecture, limiting our ability to understand the mechanisms underlying its functional decline during aging. Here, we generated a high-resolution cell atlas of brain aging within the frontal cortex and striatum using spatially resolved single-cell transcriptomics and quantified changes in gene expression and spatial organization of major cell types in these regions over the mouse lifespan. We observed substantially more pronounced changes in cell state, gene expression, and spatial organization of non-neuronal cells over neurons. Our data revealed molecular and spatial signatures of glial and immune cell activation during aging, particularly enriched in the subcortical white matter, and identified both similarities and notable differences in cell-activation patterns induced by aging and systemic inflammatory challenge. These results provide critical insights into age-related decline and inflammation in the brain.

A neutrophil response linked to tumor control in immunotherapy.

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sellhi state rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12, and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy.

Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states.

We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands to millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogeneous, we found within-population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome.

Human prefrontal cortex gene regulatory dynamics from gestation to adulthood at single-cell resolution.

Human brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation, we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference, we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development.

Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

Single-cell approaches in human microbiome research.

Microbial culturing and meta-omic profiling technologies have significantly advanced our understanding of the taxonomic and functional variation of the human microbiome and its impact on host processes. The next increase in resolution will come by understanding the role of low-abundant and less-prevalent bacteria and the study of individual cell behaviors that underlie the complexity of microbial ecosystems. To this aim, single-cell techniques are being rapidly developed to isolate, culture, and characterize the genomes and transcriptomes of individual microbes in complex communities. Here, we discuss how these single-cell technologies are providing unique insights into the biology and behavior of human microbiomes.

Tissue-resident FOLR2+ macrophages associate with CD8+ T cell infiltration in human breast cancer.

Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2+ tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2+ macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8+ T cells. FOLR2+ macrophages efficiently prime effector CD8+ T cells ex vivo. The density of FOLR2+ macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies.

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq.

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.

Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche.

Immunotherapy is a promising treatment for triple-negative breast cancer (TNBC), but patients relapse, highlighting the need to understand the mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. Quiescent cancer cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC.

GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

Multiple early factors anticipate post-acute COVID-19 sequelae.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex.

Realizing the full utility of brain organoids to study human development requires understanding whether organoids precisely replicate endogenous cellular and molecular events, particularly since acquisition of cell identity in organoids can be impaired by abnormal metabolic states. We present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, from generation of neural progenitors through production of differentiated neuronal and glial subtypes. We show that processes of cellular diversification correlate closely to endogenous ones, irrespective of metabolic state, empowering the use of this atlas to study human fate specification. We define longitudinal molecular trajectories of cortical cell types during organoid development, identify genes with predicted human-specific roles in lineage establishment, and uncover early transcriptional diversity of human callosal neurons. The findings validate this comprehensive atlas of human corticogenesis in vitro as a resource to prime investigation into the mechanisms of human cortical development.