Lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential.

Barrier tissue immune responses are regulated in part by nociceptors. Nociceptor ablation alters local immune responses at peripheral sites and within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Using high-resolution imaging, viral tracing, single-cell transcriptomics, and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is responsive to lymph-borne inflammatory signals. Transcriptomics profiling revealed that multiple sensory neuron subsets, predominantly peptidergic nociceptors, innervate LNs, distinct from those innervating surrounding skin. To uncover LN-resident cells that may interact with LN-innervating sensory neurons, we generated a LN single-cell transcriptomics atlas and nominated nociceptor target populations and interaction modalities. Optogenetic stimulation of LN-innervating sensory fibers triggered rapid transcriptional changes in the predicted interacting cell types, particularly endothelium, stromal cells, and innate leukocytes. Thus, a unique population of sensory neurons monitors peripheral LNs and may locally regulate gene expression.

Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19.

SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA+ host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1high goblet, and KRT13+ "hillock"-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19.

Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Identification of a Master Regulator of Differentiation in Toxoplasma.

Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Acute-stage tachyzoites differentiate into chronic-stage bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. The molecular basis of this differentiation is unknown, despite being efficiently triggered by stresses in culture. Through Cas9-mediated screening and single-cell profiling, we identify a Myb-like transcription factor (BFD1) necessary for differentiation in cell culture and in mice. BFD1 accumulates during stress and its synthetic expression is sufficient to drive differentiation. Consistent with its function as a transcription factor, BFD1 binds the promoters of many stage-specific genes and represents a counterpoint to the ApiAP2 factors that dominate our current view of parasite gene regulation. BFD1 provides a genetic switch to study and control Toxoplasma differentiation and will inform prevention and treatment of chronic infections.

Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.

The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Single-Cell RNA-Seq Reveals AML Hierarchies Relevant to Disease Progression and Immunity.

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies. VIDEO ABSTRACT.

Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

CD4+ T cells re-wire granuloma cellularity and regulatory networks to promote immunomodulation following Mtb reinfection.

Immunological priming-in the context of either prior infection or vaccination-elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrated that prior Mtb infection elicited a long-lasting protective response against subsequent Mtb exposure and was CD4+ T cell dependent. By analyzing data from primary infection, reinfection, and reinfection-CD4+ T cell-depleted granulomas, we found that the presence of CD4+ T cells during reinfection resulted in a less inflammatory lung milieu characterized by reprogrammed CD8+ T cells, reduced neutrophilia, and blunted type 1 immune signaling among myeloid cells. These results open avenues for developing vaccines and therapeutics that not only target lymphocytes but also modulate innate immune cells to limit tuberculosis (TB) disease.

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation.

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.

A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade.

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.

IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

TCR sequencing paired with massively parallel 3' RNA-seq reveals clonotypic T cell signatures.

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.

Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo.

Single-Cell Genomics Unveils Critical Regulators of Th17 Cell Pathogenicity.

Extensive cellular heterogeneity exists within specific immune-cell subtypes classified as a single lineage, but its molecular underpinnings are rarely characterized at a genomic scale. Here, we use single-cell RNA-seq to investigate the molecular mechanisms governing heterogeneity and pathogenicity of Th17 cells isolated from the central nervous system (CNS) and lymph nodes (LN) at the peak of autoimmune encephalomyelitis (EAE) or differentiated in vitro under either pathogenic or non-pathogenic polarization conditions. Computational analysis relates a spectrum of cellular states in vivo to in-vitro-differentiated Th17 cells and unveils genes governing pathogenicity and disease susceptibility. Using knockout mice, we validate four new genes: Gpr65, Plzp, Toso, and Cd5l (in a companion paper). Cellular heterogeneity thus informs Th17 function in autoimmunity and can identify targets for selective suppression of pathogenic Th17 cells while potentially sparing non-pathogenic tissue-protective ones.

Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.

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.