Obesity is an established risk factor for cancer in many tissues. In the mammalian intestine, a pro-obesity high-fat diet (HFD) promotes regeneration and tumorigenesis by enhancing intestinal stem cell (ISC) numbers, proliferation, and function. Although PPAR (peroxisome proliferator-activated receptor) nuclear receptor activity has been proposed to facilitate these effects, their exact role is unclear. Here we find that, in loss-of-function in vivo models, PPARα and PPARδ contribute to the HFD response in ISCs. Mechanistically, both PPARs do so by robustly inducing a downstream fatty acid oxidation (FAO) metabolic program. Pharmacologic and genetic disruption of CPT1A (the rate-controlling enzyme of mitochondrial FAO) blunts the HFD phenotype in ISCs. Furthermore, inhibition of CPT1A dampens the pro-tumorigenic consequences of a HFD on early tumor incidence and progression. These findings demonstrate that inhibition of a HFD-activated FAO program creates a therapeutic opportunity to counter the effects of a HFD on ISCs and intestinal tumorigenesis.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

COVID-19, caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple-organ failure, but little is known about its pathophysiology. Here, we generated single-cell atlases of 23 lung, 16 kidney, 16 liver and 19 heart COVID-19 autopsy donor tissue samples, and spatial atlases of 14 lung donors. Integrated computational analysis uncovered substantial remodeling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in COVID-19 donor heart tissue, and mapped cell types and genes implicated with disease severity based on COVID-19 GWAS. Our foundational dataset elucidates the biological impact of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle income countries. It is thought to be a primary cause of most global growth-stunting cases and a key contributing factor to childhood malnutrition and diminished oral vaccine responses. While EE has been shown to be the byproduct of recurrent enteric infection, to date, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE severity by performing high-throughput single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE from Lusaka, Zambia (8 HIV negative, 3 HIV positive) and 6 adults without EE in Boston, USA. Using the resulting cellular atlas, we scored existing bulk-transcriptomic signatures of reduced villus height and decreased plasma LPS levels in EE, finding that these signatures may be driven by an increased abundance of surface mucosal cells (a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract). In addition, we identified several cell subsets whose fractional abundances associated with histological determined EE severity, small intestinal region, and HIV infection. Furthermore, by comparing distal duodenal EE samples with those from two U.S. control cohorts, we identified broadly decreased epithelial proliferative signaling, lower fractional abundances of goblet cells, and a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells but with increased pro-inflammatory cytokine expression in EE. Altogether, our work illuminates the epithelial and immune correlates of EE severity and provides new molecular targets for intervention.

Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αβ T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of major histocompatibility complex (MHC) class I-related (MR1)-restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis (Mtb) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in HIV-negative individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with MAIT cell tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in HIV-negative people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.

Mast cells (MCs) play a pathobiologic role in type 2 (T2) allergic inflammatory diseases of the airway, including asthma and chronic rhinosinusitis with nasal polyposis (CRSwNP). Distinct MC subsets infiltrate the airway mucosa in T2 disease, including subepithelial MCs expressing the proteases tryptase and chymase (MCTC) and epithelial MCs expressing tryptase without chymase (MCT). However, mechanisms underlying MC expansion and the transcriptional programs underlying their heterogeneity are poorly understood. Here, we use flow cytometry and single-cell RNA-sequencing (scRNA-seq) to conduct a comprehensive analysis of human MC hyperplasia in CRSwNP, a T2 cytokine–mediated inflammatory disease. We link discrete cell surface phenotypes to the distinct transcriptomes of CRSwNP MCT and MCTC, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38highCD117high MCs that is markedly expanded during T2 inflammation. These CD38highCD117high MCs exhibit an intermediate phenotype relative to the expanded MCT and MCTC subsets. CD38highCD117high MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease, a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MCT–like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MCTC in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest that in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.

Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here, we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Hemorrhage Evacuation trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-Seq (scRNA-Seq), this is the first report to our knowledge that characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution. Our analysis revealed shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic rebleed that our transcriptional data indicated occurred prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods such as scRNA-Seq would enable greater understanding of complex intracerebral events.

Our tissues are complex and dynamic multicellular ecosystems. Studies of tissue physiology have helped establish their basic cellular components, and powerful profiling methods have refined lists of cell types/states and their attributes. Yet, fundamental questions remain as to what influences a cell’s specific role(s) within a tissue and how the collective activity of many cells drives emergent tissue-level functional and dysfunctional behaviors.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial–macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients’ demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Infection with SARS-CoV-2, the virus that causes COVID-19, can lead to severe lower respiratory illness including pneumonia and acute respiratory distress syndrome, which can result in profound morbidity and mortality. However, many infected individuals are either asymptomatic or have isolated upper respiratory symptoms, which suggests that the upper airways represent the initial site of viral infection, and that some individuals are able to largely constrain viral pathology to the nasal and oropharyngeal tissues. Which cell types in the human nasopharynx are the primary targets of SARS-CoV-2 infection, and how infection influences the cellular organization of the respiratory epithelium remains incompletely understood. Here, we present nasopharyngeal samples from a cohort of 35 individuals with COVID-19, representing a wide spectrum of disease states from ambulatory to critically ill, as well as 23 healthy and intubated patients without COVID-19. Using standard nasopharyngeal swabs, we collected viable cells and performed single-cell RNA-sequencing (scRNA-seq), simultaneously profiling both host and viral RNA. We find that following infection with SARS-CoV-2, the upper respiratory epithelium undergoes massive reorganization: secretory cells diversify and expand, and mature epithelial cells are preferentially lost. Further, we observe evidence for deuterosomal cell and immature ciliated cell expansion, potentially representing active repopulation of lost ciliated cells through coupled secretory cell differentiation. Epithelial cells from participants with mild/moderate COVID-19 show extensive induction of genes associated with anti-viral and type I interferon responses. In contrast, cells from participants with severe lower respiratory symptoms appear globally muted in their anti-viral capacity, despite substantially higher local inflammatory myeloid populations and equivalent nasal viral loads. This suggests an essential role for intrinsic, local epithelial immunity in curbing and constraining viral-induced pathology. Using a custom computational pipeline, we characterized cell-associated SARS-CoV-2 RNA and identified rare cells with RNA intermediates strongly suggestive of active replication. Both within and across individuals, we find remarkable diversity and heterogeneity among SARS-CoV-2 RNA+ host cells, including developing/immature and interferon-responsive ciliated cells, KRT13+ “hillock”-like cells, and unique subsets of secretory, goblet, and squamous cells. Finally, SARS-CoV-2 RNA+ cells, as compared to uninfected bystanders, are enriched for genes involved in susceptibility (e.g., CTSL, TMPRSS2) or response (e.g., MX1, IFITM3, EIF2AK2) to infection. Together, this work defines both protective and detrimental host responses to SARS-CoV-2, determines the direct viral targets of infection, and suggests that failed anti-viral epithelial immunity in the nasal mucosa may underlie the progression to severe COVID-19.

Opportunities to interrogate the immune responses in the injured tissue of living patients suffering from acute sterile injuries such as stroke and heart attack are limited. We leveraged a clinical trial of minimally invasive neurosurgery for patients with intracerebral hemorrhage (ICH), a severely disabling subtype of stroke, to investigate the dynamics of inflammation at the site of brain injury over time. Longitudinal transcriptional profiling of CD14+monocytes/macrophages and neutrophils from hematomas of patients with ICH revealed that the myeloid response to ICH within the hematoma is distinct from that in the blood and occurs in stages conserved across the patient cohort. Initially, hematoma myeloid cells expressed a robust anabolic proinflammatory profile characterized by activation of hypoxia-inducible factors (HIFs) and expression of genes encoding immune factors and glycolysis. Subsequently, inflammatory gene expression decreased over time, whereas anti-inflammatory circuits were maintained and phagocytic and antioxidative pathways up-regulated. During this transition to immune resolution, glycolysis gene expression and levels of the potent proresolution lipid mediator prostaglandin E2 remained elevated in the hematoma, and unexpectedly, these elevations correlated with positive patient outcomes. Ex vivo activation of human macrophages by ICH-associated stimuli highlighted an important role for HIFs in production of both inflammatory and anti-inflammatory factors, including PGE2, which, in turn, augmented VEGF production. Our findings define the time course of myeloid activation in the human brain after ICH, revealing a conserved progression of immune responses from proinflammatory to proresolution states in humans after brain injury and identifying transcriptional programs associated with neurological recovery.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRMcells during aGVHD in primate transplant recipients.

In late 2019 and through 2020, the COVID-19 pandemic swept the world, presenting both scientific and medical challenges associated with understanding and treating a previously unknown disease. To help address the need for great understanding of COVID-19, the scientific community mobilized and banded together rapidly to characterize SARS-CoV-2 infection, pathogenesis and its distinct disease trajectories. The urgency of COVID-19 provided a pressing use-case for leveraging relatively new tools, technologies, and nascent collaborative networks. Single-cell biology is one such example that has emerged over the last decade as a powerful approach that provides unprecedented resolution to the cellular and molecular underpinnings of biological processes. Early foundational work within the single-cell community, including the Human Cell Atlas, utilized published and unpublished data to characterize the putative target cells of SARS-CoV-2 sampled from diverse organs based on expression of the viral receptor ACE2 and associated entry factors TMPRSS2 and CTSL (Muus et al., 2020; Sungnak et al., 2020; Ziegler et al., 2020). This initial characterization of reference data provided an important foundation for framing infection and pathology in the airway as well as other organs. However, initial community analysis was limited to samples derived from uninfected donors and other previously-sampled disease indications. This report provides an overview of a single-cell data resource derived from samples from COVID-19 patients along with initial observations and guidance on data reuse and exploration.

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.

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma–associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation

Recent political and social events, mainly those originating in the USA, have triggered an intense desire for equity in all facets of the human experience. More specifically, actions engendered by the Black Lives Matter movement and others have led to the scrutinizing of equity across a wide range of fields, from politics and business to academia and scientific research. In science, in particular, several major journals have published opinion pieces and editorials seeking greater equity or relating to the ‘non-white’ experience. Many of their readers have been stunned by the revelations. Indeed, the scientific community is only now coming to terms with an unsettling and uncomfortable truth: structural exclusion of non-white people permeates all levels of the scientific enterprise. That being said, with awareness comes opportunity. New frameworks for describing and addressing these issues have recently emerged, creating a structure with which groups can each consider how to best internalize and embody the lessons in their own scientific initiatives.

In the Human Cell Atlas (HCA) consortium, equity has been a point of emphasis from inception in 2016 for one simple reason: the HCA’s success depends upon it. Fundamentally, the HCA is meant to be a foundational resource, inclusive of the many cell types and states found in healthy people across the globe. That resource can then be used to address a wide range of scientific questions and, in the future, to facilitate a better understanding of disease. This mission demands, explicitly, the inclusion of representation along axes of sex, age, ethnicity, environment, socioeconomic status and, in some cases, disease susceptibility in its biospecimens. Moreover, it requires broad participation to ensure comprehensive coverage and identify barriers to success and support continuity, and necessitates reciprocal, balanced benefit from the methods, data and results to ensure global engagement.

To this end, the HCA has set ambitious and dynamic equity goals for itself. Below, we describe key lessons learned through equity activities thus far, as well as our future plans.

Granulomas are complex cellular structures comprised predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated 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 (RR), a dynamic process in which some patients with disseminated lepromatous leprosy (L-lep) transition towards 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 IFN-γ and IL-1β. 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 contribute to the antimicrobial response.

The liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating cellular proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single-cell RNA-seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage-derived WNT/β-catenin signaling. Interestingly, transcriptional compensation is most prominent in non-proliferating cells, clearly delineating two temporally distinct phases of liver recovery. Overall, our work describes a mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage-derived WNT signals required for this compensation.

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively-selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, to clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.

Central to anti-tumor immunity are dendritic cells (DCs), which stimulate long-lived protective T cell responses. Recent studies have demonstrated that DCs can achieve a state of hyperactivation, which is associated with inflammasome activities within living cells. Herein, we report that hyperactive DCs have an enhanced ability to migrate to draining lymph nodes and stimulate potent cytotoxic T lymphocyte (CTL) responses. This enhanced migratory activity is dependent on the chemokine receptor CCR7 and is associated with a unique transcriptional program that is not observed in conventionally activated or pyroptotic DCs. We show that hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor immunity against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses are intrinsic to the cDC1 subset of DCs, depend on the inflammasome-dependent cytokine IL-1β, and enable tumor lysates to serve as immunogens. If these activities are verified in humans, hyperactive DCs may impact immunotherapy.

Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury, particularly over time, in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Haemorrhage Evacuation (MISTIEIII) trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-sequencing, we characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution for the first time. Our analysis revealed rapid shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic re-bleed (second local exposure to blood) that our transcriptional data indicated occurred more than 30 hours prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods like scRNA-seq can inform our understanding of complex intracerebral events.

Prostate cancer is the second most common malignancy in men worldwide and consists of a mixture of tumor and non-tumor cell types. To characterize the prostate cancer tumor microenvironment, we performed single-cell RNA-sequencing on prostate biopsies, prostatectomy specimens, and patient-derived organoids from localized prostate cancer patients. We identify a population of tumor-associated club cells that may act as progenitor cells and uncover heterogeneous cellular states in prostate epithelial cells marked by high androgen signaling states that are enriched in prostate cancer. ERG- tumor cells, compared to ERG+ cells, demonstrate shared heterogeneity with surrounding luminal epithelial cells and appear to give rise to common tumor microenvironment responses. Finally, we show that prostate epithelial organoids recapitulate tumor-associated epithelial cell states and are enriched with distinct cell types and states from their parent tissues. Our results provide diagnostically relevant insights and advance our understanding of the cellular states associated with prostate carcinogenesis.

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.

In humans and nonhuman primates, Mycobacterium tuberculosis lung infection yields a complex multicellular structure: the tuberculosis granuloma. All granulomas are not equivalent, however, even within the same host: in some, local immune activity promotes bacterial clearance, while in others, it allows persistence or outgrowth. Here, we used single-cell RNA-sequencing to define holistically cellular responses associated with control in cynomolgus macaques. Granulomas that facilitated bacterial killing contained significantly higher proportions of CD4+ and CD8+ T cells expressing hybrid Type1-Type17 immune responses or stem-like features and CD8-enriched T cells with specific cytotoxic functions; failure to control correlated with mast cell, plasma cell and fibroblast abundance. Co-registering these data with serial PET-CT imaging suggests that a degree of early immune control can be achieved through cytotoxic activity, but that more robust restriction only arises after the priming of specific adaptive immune responses, defining new targets for vaccination and treatment.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 (“Second-Strand Synthesis”), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well Sincreased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Innate lymphoid cells (ILCs) are important for response to infection and for immune development in early life. HIV infection in adults depletes circulating ILCs, but the impact on children infected from birth remains unknown. We study vertically HIV-infected children from birth to adulthood and find severe and persistent depletion of all circulating ILCs that, unlike CD4+ T cells, are not restored by long-term antiretroviral therapy unless initiated at birth. Remaining ILCs upregulate genes associated with cellular activation and metabolic perturbation. Unlike HIV-infected adults, ILCs are also profoundly depleted in tonsils of vertically infected children. Transcriptional profiling of remaining ILCs reveals ongoing cell-type-specific activity despite anti-retroviral therapy. Collectively, these data suggest an important and ongoing role for ILCs in lymphoid tissue of HIV-infected children from birth, where persistent depletion and sustained transcriptional activity are likely to have long-term immune consequences that merit further investigation.

Our nasal epithelial COVID-19 dataset, along with COVID-19 datasets from other genomics groups, can now be found at covid19cellatlas.org. This work was sponsored by the Chan-Zuckerberg Initiative.

Bulk transcriptomic studies have defined classical and basal-like gene expression subtypes in pancreatic ductal adenocarcinoma (PDAC) that correlate with survival and response to chemotherapy; however, the underlying mechanisms that govern these subtypes and their heterogeneity remain elusive. Here, we performed single-cell RNA-sequencing of 23 metastatic PDAC needle biopsies and matched organoid models to understand how tumor cell-intrinsic features and extrinsic factors in the tumor microenvironment (TME) shape PDAC cancer cell phenotypes. We identify a novel cancer cell state that co-expresses basal-like and classical signatures, demonstrates upregulation of developmental and KRAS-driven gene expression programs, and represents a transitional intermediate between the basal-like and classical poles. Further, we observe structure to the metastatic TME supporting a model whereby reciprocal intercellular signaling shapes the local microenvironment and influences cancer cell transcriptional subtypes. In organoid culture, we find that transcriptional phenotypes are plastic and strongly skew toward the classical expression state, irrespective of genotype. Moreover, we show that patient-relevant transcriptional heterogeneity can be rescued by supplementing organoid media with factors found in the TME in a subtype-specific manner. Collectively, our study demonstrates that distinct microenvironmental signals are critical regulators of clinically relevant PDAC transcriptional states and their plasticity, identifies the necessity for considering the TME in cancer modeling efforts, and provides a generalizable approach for delineating the cell-intrinsic versus -extrinsic factors that govern tumor cell phenotypes.

B cell receptors (BCRs) display a combination of variable (V)-gene-encoded complementarity determining regions (CDRs) and adaptive/hypervariable CDR3 loops to engage antigens. It has long been proposed that the former tune for recognition of pathogens or groups of pathogens. To experimentally evaluate this within the human antibody repertoire, we perform immune challenges in transgenic mice that bear diverse human CDR3 and light chains but are constrained to different human VHgenes. We find that, of six commonly deployed VHsequences, only those CDRs encoded by IGHV1-202 enable polyclonal antibody responses against bacterial lipopolysaccharide (LPS) when introduced to the bloodstream. The LPS is from diverse strains of gram-negative bacteria, and the VH-gene-dependent responses are directed against the non-variable and universal saccrolipid substructure of this antigen. This reveals a broad-spectrum anti-LPS response in which germline-encoded CDRs naturally hardwire the human antibody repertoire for recognition of a conserved microbial target.

Studies are needed to determine the mechanisms of mucosal dysregulation in patients with inflammatory bowel diseases (IBDs) and differences in inflammatory responses of patients with ulcerative colitis (UC) vs Crohn’s disease (CD). We used mass cytometry (CyTOF) to characterize and compare immune cell populations in the mucosa and blood from patients with IBD and without IBD (controls) at single-cell resolution. We performed CyTOF analysis of colonic mucosa samples (n = 87) and peripheral blood mononuclear cells (n = 85) from patients with active or inactive UC or CD and controls. We also performed single-cell RNA sequencing, flow cytometry, and RNA in situ hybridization analyses to validate key findings. We used random forest modeling to identify differences in signatures across subject groups. Compared with controls, colonic mucosa samples from patients with IBD had increased abundances of HLA-DR+CD38+ T cells, including T-regulatory cells that produce inflammatory cytokines; CXCR3+ plasmablasts; and IL1B+ macrophages and monocytes. Colonic mucosa samples from patients with UC were characterized by expansion of IL17A+ CD161+ effector memory T cells and IL17A+ T-regulatory cells; expansion of HLA-DR+CD56+ granulocytes; and reductions in type 3 innate lymphoid cells. Mucosal samples from patients with active CD were characterized by IL1B+HLA-DR+CD38+ T cells, IL1B+TNF+IFNG+ naïve B cells, IL1B+ dendritic cells (DCs), and IL1B+ plasmacytoid DCs. Peripheral blood mononuclear cells from patients with active CD differed from those of active UC in that the peripheral blood mononuclear cells from patients with CD had increased IL1B+ T-regulatory cells, IL1B+ DCs and IL1B+ plasmacytoid DCs, IL1B+ monocytes, and fewer group 1 innate lymphoid cells. Random forest modeling differentiated active UC from active CD in colonic mucosa and blood samples; top discriminating features included many of the cellular populations identified above. We used single-cell technologies to identify immune cell populations specific to mucosa and blood samples from patients with active or inactive CD and UC and controls. This information might be used to develop therapies that target specific cell populations in patients with different types of IBD.

One of the central challenges in the field of allo-immunity is deciphering the mechanisms driving T cells to infiltrate and subsequently occupy target organs to cause disease. The act of CD8-dominated T cell infiltration is critical to acute graft-versus-host disease (aGVHD), wherein donor T cells become activated, tissue-infiltrating and highly cytotoxic, causing wide-spread tissue damage after allogeneic hematopoietic stem cell transplant (allo-HCT). However, in human and non-human primate studies, deconvolving the transcriptional programs of newly recruited relative to resident memory T cells in the gastrointestinal (GI) tract has remained a challenge. In this study, we combined the novel technique of Serial Intravascular Staining (SIVS) with single-cell RNA-Seq (scRNA-seq) to enable detailed dissection of the tightly connected processes by which T cells first infiltrate tissues and then establish a pathogenic tissue residency program after allo-HCT in non-human primates. Our results have enabled the creation of a spatiotemporal map of the transcriptional drivers of CD8 T cell infiltration into the primary aGVHD target-organ, the GI tract. We identify the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphdepletion-driven T cell infiltration. The donor CD8 T cells that infiltrate the GI tract demonstrate a highly activated, cytotoxic phenotype while simultaneously rapidly developing canonical tissue-resident memory (TRM) protein expression and transcriptional signatures, driven by IL-15/IL-21 signaling. Moreover, by combining SIVS and transcriptomic analysis, we have been able to work backwards from this pathogenic TRM programing, and, for the first time, identify a cluster of genes directly associated with tissue invasiveness, prominently including specific chemokines and adhesion molecules and their receptors, as well as a central cytoskeletal transcriptional node. The clinical relevance of this new tissue invasion signature was validated by its ability to discriminate the CD8 T cell transcriptome of patients with GI aGVHD. These results provide new insights into the mechanisms controlling tissue infiltration and pathogenic CD8 TRM transcriptional programing, uncovering critical transitions in allo-immune tissue invasion and destruction.

Ebola virus (EBOV) causes epidemics with high case fatality rates, yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. To better understand EBOV infection in vivo, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cell activity during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, providing insight into pathogenesis. We find that immature, proliferative monocyte-lineage cells with reduced antigen presentation capacity replace conventional circulating monocyte subsets within days of infection, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying viral RNA abundance in individual cells, we identify molecular determinants of tropism and examine temporal dynamics in viral and host gene expression. Within infected cells, we observe that EBOV down-regulates STAT1 mRNA and interferon signaling, and up-regulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating cellular pathways the virus manipulates for its replication. Overall, this study sheds light on EBOV tropism, replication dynamics, and elicited immune response, and provides a framework for characterizing interactions between hosts and emerging viruses in a maximum containment setting.

Despite the epidemics of chronic obstructive pulmonary disease (COPD), the cellular and molecular mechanisms of this disease are far from being understood. Here, we characterize and classify the cellular composition within the alveolar space and peripheral blood of COPD patients and control donors using a clinically applicable single-cell RNA-seq technology corroborated by advanced computational approaches for: machine learning-based cell-type classification, identification of differentially expressed genes, prediction of metabolic changes, and modeling of cellular trajectories within a patient cohort. These high-resolution approaches revealed: massive transcriptional plasticity of macrophages in the alveolar space with increased levels of invading and proliferating cells, loss of MHC expression, reduced cellular motility, altered lipid metabolism, and a metabolic shift reminiscent of mitochondrial dysfunction in COPD patients. Collectively, single-cell omics of multi-tissue samples was used to build the first cellular and molecular framework for COPD pathophysiology as a prerequisite to develop molecular biomarkers and causal therapies against this deadly disease.

Single-cell RNA sequencing (scRNA-seq) has provided a high-dimensional catalog of millions of cells across species and diseases. These data have spurred the development of hundreds of computational tools to derive novel biological insights. Here, we outline the components of scRNA-seq analytical pipelines and the computational methods that underlie these steps. We describe available methods, highlight well-executed benchmarking studies, and identify opportunities for additional benchmarking studies and computational methods. As the biochemical approaches for single-cell omics advance, we propose coupled development of robust analytical pipelines suited for the challenges that new data present and principled selection of analytical methods that are suited for the biological questions to be addressed.

Barrier tissue epithelia play an essential role in maintaining organismal homeostasis, and changes in their cellular composition have been observed in multiple human diseases. Within the small intestinal epithelium, adult stem cells integrate diverse signals to regulate regeneration and differentiation, thereby establishing overall cellularity. Accordingly, directing stem cell differentiation could provide a tractable approach to alter the abundance or quality of specialized cells of the small intestinal epithelium, including the secretory Paneth, goblet, and enteroendocrine populations. Yet, to date, there has been a lack of suitable tools and rigorous approaches to identify biological targets and pharmacological agents that can modify epithelial composition to enable causal testing of disease-associated changes with novel therapeutic candidates. To empower the search for epithelia-modifying agents, we establish a first-of-its-kind high-throughput phenotypic organoid screen. We demonstrate the ability to screen thousands of samples and uncover biological targets and associated small molecule inhibitors which translate to in vivo. This approach is enabled by employing a functional, cell-type specific, scalable assay on an organoid model designed to represent the physiological cues of in vivo Paneth cell differentiation from adult intestinal stem cells. Further, we miniaturize and adapt the organoid culture system to enable automated plating and screening, thereby providing the ability to test thousands of samples. Strikingly, in our screen we identify inhibitors of the nuclear exporter Xpo1 modulate stem cell fate commitment by inducing a pan-epithelial stress response combined with an interruption of mitogen signaling in cycling intestinal progenitors, thereby significantly increasing the abundance of Paneth cells independent of known WNT and Notch differentiation cues. We extend our observation in vivo, demonstrating that oral administration of Xpo1 inhibitor KPT-330 at doses 1,000-fold lower than conventionally used in hematologic malignancies increases Paneth cell abundance. In total, we provide a framework to identify novel biological cues and therapeutic leads to rebalance intestinal stem cell differentiation and modulate epithelial tissue composition via high-throughput phenotypic screening in rationally-designed organoid model of differentiation.

We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-associated genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells’ potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clinical samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) which causes the disease COVID-19. SARS-CoV- 2 spike (S)-protein binds ACE2, and in concert with host proteases, principally TMPRSS2, promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 amongst tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discover that ACE2 is a human interferon- stimulated gene (ISG) in vitro using airway epithelial cells, and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta- analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross- talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.

Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.

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.

The scale and capabilities of single-cell RNA-sequencing methods have expanded rapidly in recent years, enabling major dis- coveries and large-scale cell mapping efforts. However, these methods have not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single-cell and/or single-nucleus profiling—selecting representa- tive methods based on their usage and our expertise and resources to prepare libraries—including two low-throughput and five high-throughput methods. We tested the methods on three types of samples: cell lines, peripheral blood mononuclear cells and brain tissue, generating 36 libraries in six separate experiments in a single center. To directly compare the methods and avoid processing differences introduced by the existing pipelines, we developed scumi, a flexible computational pipeline that can be used with any single-cell RNA-sequencing method. We evaluated the methods for both basic performance, such as the structure and alignment of reads, sensitivity and extent of multiplets, and for their ability to recover known biological information in the samples.

Cellular immunity is critical for controlling intracellular pathogens, but individual cellular dynamics and cell–cell cooperativity in evolving human immune responses remain poorly understood. Single-cell RNA-sequencing (scRNA-seq) represents a powerful tool for dissecting complex multicellular behaviors in health and disease and nominating testable therapeutic targets. Its application to longitudinal samples could afford an opportunity to uncover cellular factors associated with the evolution of disease progression without potentially confounding inter-individual variability. Here, we present an experimental and computational methodology that uses scRNA-seq to characterize dynamic cellular programs and their molecular drivers, and apply it to HIV infection. By performing scRNA-seq on peripheral blood mononuclear cells from four untreated individuals before and longitudinally during acute infection, we were powered within each to discover gene response modules that vary by time and cell subset. Beyond previously unappreciated individual- and cell-type-specific interferon-stimulated gene upregulation, we describe temporally aligned gene expression responses obscured in bulk analyses, including those involved in proinflammatory T cell differentiation, prolonged monocyte major histocompatibility complex II upregulation and persistent natural killer (NK) cell cytolytic killing. We further identify response features arising in the first weeks of infection, for example proliferating natural killer cells, which potentially may associate with future viral control. Overall, our approach provides a unified framework for characterizing multiple dynamic cellular responses and their coordination.

There is pressing urgency to better understand the pathogenesis of the severe acute respiratory syndrome (SARS) coronavirus (CoV) clade SARS-CoV-2, which causes the disease known as COVID-19. SARS-CoV-2, like SARS-CoV, utilizes ACE2 to bind host cells. While initial SARS- CoV-2 cell entry and infection depend on ACE2 in concert with the protease TMPRSS2 for spike (S) protein activation, the specific cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human and non- human primate (NHP) single-cell RNA-sequencing (scRNA-seq) datasets to uncover the tissue- resident cell subsets that may serve as the cellular targets of SARS-CoV-2. We identify ACE2 and TMPRSS2 co-expressing cells within type II pneumocytes in NHP lung, absorptive enterocytes in human and NHP terminal ileum, and human nasal goblet secretory cells. Strikingly, we discover, and extensively corroborate using publicly available data sets, that ACE2 is an interferon-stimulated gene (ISG) in human epithelial cells. We further validate this finding in primary upper airway human respiratory epithelial cells. Thus, SARS-CoV-2 may exploit IFN- driven upregulation of ACE2, a key tissue-protective mediator during lung injury, to enhance infection.


In light of the global effort to better understand the new SARS-CoV-2 virus, we and other researchers from the HCA Lung Biological Network and beyond have begun an initiative to investigate datasets from relevant tissues profiled as part of other ongoing studies. These studies represent, for example, large efforts to characterize HIV, Mtb, and influenza infection and allergy in primary human and non-human primate samples. This page serves as a guide to viewing our data interactively on and downloading datasets from our single-cell portal, the Alexandria Project. Alternatively, bulk downloading of our data is available here. For more on research initiatives in COVID-19 being undertaken by the HCA, and the HCA Lung Biological Network in particular, please visit the HCA website here.

We investigated two genes whose protein products are central to the cellular entry of SARS-CoV-2: ACE2 and TMPRSS2. Consistent with previous studies, we found that the gene encoding ACE2, the SARS-CoV-2 entry receptor, is expressed on a subset of lung epithelial cells, type 2 pneumocytes, and a subset of ileal epithelial cells, absorptive enterocytes, across several datasets. The protease TMPRSS2 primes the spike protein of SARS-CoV-2 and is also important for viral entry. Because of this, we identified cells which co-express ACE2 and TMPRSS2 in our datasets, and investigated additional genes enriched within ACE2 and TMPRSS2 co-expressing cells. As we believe this data may prove useful to other researchers investigating similar questions, we have made our datasets public through the interactive Alexandria Project. Here, you can view our annotations of these datasets and investigate which other genes are highly expressed in these cell subsets of interest.

NB None of the datasets presented here were designed to answer specific questions about COVID-19. Additional studies will be required across larger, appropriately structured cohorts. Further, we provide a note of caution when interpreting scRNA-seq data for low abundance transcripts like ACE2 and TMPRSS2 as detection inefficiencies and/or sequencing depth may result in an underestimation of the actual frequencies of ACE2+ or ACE2+/TMPRSS2+ cells in tissues. Moreover, the protein levels of each may differ from their mRNA abundances. We present each data set separately, as each study differed by method of tissue processing and collection protocols, each of which can influence the frequency of recovered cell subsets.


Our pre-print “SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is enriched in specific cell subsets across tissues” can be found here, and the abstract is reproduced below.

There is pressing urgency to better understand the pathogenesis of the severe acute respiratory syndrome (SARS) coronavirus (CoV) clade SARS-CoV-2. SARS-CoV-2, like SARS-CoV, utilizes ACE2 to bind host cells. While initial SARS-CoV-2 cell entry and infection depend on ACE2 in concert with the protease TMPRSS2 for spike (S) protein activation, the specific cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human and non-human primate (NHP) single-cell RNA-sequencing (scRNA-seq) datasets to uncover the cell subsets that may serve as cellular targets of SARS-CoV-2. We identify ACE2/TMPRSS2 co-expressing cells within type II pneumocytes, absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discover that ACE2 is an interferon-stimulated gene (ISG) in human barrier tissue epithelial cells. Thus, SARS-CoV-2 may exploit IFN-driven upregulation of ACE2, a key tissue-protective mediator during lung injury, to enhance infection.


Atlas of ACE2 expression in healthy non-human primate lung and ileum

In this study, we collected cells from various tissues in healthy and SHIV-infected non-human primates using Seq-Well v1. Here we highlight the lung and ileum and show that ACE2 and TMPRSS2 are co-expressed most frequently in type II pneumocytes in the lung and absorptive enterocytes in the ileum.

To visualize these cells in the Alexandria Project, visit this study: Atlas of healthy non-human primate lung and ileum ACE2+ cells

This project contains two UMAP visualizations (one for lung cells and one for ileum cells): toggle between them by clicking on the ‘Explore’ tab, select ‘View Options’ in the top right hand corner, and switch between lung and ileum under the ‘Load Cluster’ dropdown.

For each of the lung and ileum cell UMAPs, we provide subsets of these visualizations which contain only the cell types enriched for double positive cells. By selecting the ‘Load cluster’ options called ‘Lung Epithelial Cells’ or ‘Ileum Absorptive Enterocytes’, you will be able to view gene expression differences between double positive cells and other cells within those cell types. In the lung epithelial cells visualization, the following additional annotations are available under the ‘Select Annotation’ dropdown:

  • ACE2+ – cells expressing ACE2
  • TMPRSS2+ – cells expressing TMPRSS2
  • ACE2_TMPRSS2_double_positive – cells expressing both genes
  • celltype_double_positive – the cell type column and double positive column combined so genes that are differentially expressed between only one cell type may be viewed
  • celltype_ACE2+ – same as above for ACE2 expression

After selecting one of these annotations, you can then search for a gene of interest in the ‘Search Genes’ box in the left corner to view the expression of that gene as a violin plot split by the annotation you select. For example, to reproduce Figure 1D in the manuscript, you would select annotation “ACE2_TMPRSS2_double_positive” and search for gene ‘IFNGR2’ in the ‘Search Genes’ box.

The same options are available for the ileal absorptive enterocytes visualization except that the cell type is combined with the double positive/ACE2 column since this subset only includes one cell type.

Full expression matrices (and therefore gene expression values visible when using the ‘Search Genes’ box) are available only for epithelial cell types as this data is derived from a pre-publication study.

Atlas of ACE2 and TMPRSS2 in human ileum

In this study, samples from human ileum were collected, processed, and run on 10x 3′ v2 show ACE2 and TMPRSS2 co-expression in absorptive enterocytes.

To visualize these cells in the Alexandria Portal, visit this study: ACE2 and TMPRSS2 most enriched within GSTA1+MGST3+ absorptive enterocytes in context of non-inflamed terminal ileum

Two tSNE visualizations are available for this study, one of all cells in the ileum samples “non-inflammed-tsne” and one of just the epithelial cells in the samples “non-inflammed-epth-tsne”. To toggle between these, select the “Explore” tab under the study title, then select ‘View options’ from the upper right corner of the plot and choose the visualization of interest in the ‘Load cluster’ dropdown.

Full expression matrices (and therefore gene expression values visible when using the ‘search genes’ box) are available only for the epithelial cell types as this data is derived from a pre-publication study.

Atlas of ACE2 and TMPRSS2 expression in human HIV- and TB-infected lung

In this study, samples from lung surgeries were run with Seq-Well S^3 and contain a variety of immune and epithelial cell types. We found the majority of ACE2 and TPRSS2 double positive cells in type II pneumocyte cells.

To view these cells in the Alexandria Project, visit this study: Human lung HIV-TB co-infection ACE2+ cells

To visualize the double positive cells in these samples, click the ‘Explore’ tab, then select ‘View Options’ in the top right corner of the plot and choose ‘ACE2_TMPRSS2_double_positive’ under the ‘Select Annotation’ dropdown menu.

Full expression matrices (and therefore gene expression values visible when using the ‘search genes’ box) are available only for epithelial cell types as this data is derived from a pre-publication study.

A subset of ACE2+ secretory cells in human nasal mucosa

In this study, we find a subset of secretory cells which co-express ACE2 and TMPRSS2.

To visualize these cells on the Alexandria Project, visit this study: Allergic inflammatory memory in human respiratory epithelial progenitor cells

To view the tSNE of epithelial cells, select the ‘Explore’ tab, select ‘View Options’ in the right hand corner of the plot and choose ‘Epithelial cells’ in the ‘Load Cluster’ dropdown. To view the cell type annotations in the first panel of the above plot choose ‘subset’ in the ‘Select Annotation’ dropdown menu. To view the ACE2/TMPRSS2 double positive cells, select ‘ACE2_TMPRSS2’ from the ‘Select annotation dropdown menu, and to view the cluster subsets from the third panel of this plot, select ‘res_0_8’ from the ‘Select Annotation Dropdown Menu’.

ACE2 and TPRSS2 co-expressing cells found in non-human primate granulomas and adjacent uninvolved lung tissue

In this study, we collected lung tissue from non-human primates infected with mTB. These tissues come from both mTB granulomas and adjacent uninvolved lung in the same monkey.

These cells, profiled using Seq-Well S^3, can be investigated interactively in the Alexandria Project: Epithelial cells in NHP TB granuloma and uninvolved lung

To view the data colored by granuloma and uninvolved lung choose the “Granuloma” annotation accessible by clicking “View Options” in the top right-hand corner and selecting from the “Select Annotation” dropdown menu.

Full expression matrices (and therefore gene expression values visible when using the ‘Search Genes’ box) are available only for relevant cell types as this data is derived from a pre-publication study.

Comparison of ACE2 and TMPRSS2 expression in human duodenal and ileal tissue and organoid-derived epithelial cells

In this study, samples from adult human duodenum and ileum were collected and split for primary tissue single-cell RNA-seq and organoid culture under several conditions and profiled with Seq-Well S^3. Organoids were cultured and passaged every 6-8 days in Matrigel domes with established media conditions meant to recapitulate the broad diversity of in vivo epithelial cell types (Fujii, M., et al., Cell Stem Cell. 2018). Organoid culture media contained recombinant Noggin, Rspondin-3, FGF2, IGF1, afamin-Wnt3A, in addition to Gastrin and TGF-b inhibitor A83-01 with and without recombinant EGF (E/NR3+F2I1Gi+Af-W3+A83).  Cells co-expressing ACE2 and TMPRSS2 were identified principally within enterocyte clusters of both tissues and organoids.

Visualize these samples interactively and read more about this study on the Alexandria Project: Comparison of ACE2 and TMPRSS2 expression in human duodenal and ileal tissue and organoid-derived epithelial cells

This project contains two UMAP visualizations (one for organoid cells and one for primary tissue cells): toggle between them by clicking on the ‘Explore’ tab, select ‘View Options’ in the top right hand corner, and switch between tissue and organoid under the ‘Load Cluster’ dropdown.

To visualize cells which co-express ACE2 and TMPRSS2, select the ‘ACE2_TMPRSS2’ option under the ‘Select Annotation’ dropdown. You can then search for a gene of interest in the ‘Search Genes’ box in the left corner to view the expression of that gene as a violin plot split by the annotation you select.

Full expression matrices (and therefore gene expression values visible when using the ‘Search Genes’ box) are available only for enterocyte cell types as this data is derived from a pre-publication study. Expression of ACE2 and TMPRSS2 are available for all cells.

Interferon regulation of ACE2 in human and murine basal cells

Analysis of these datasets and others lead to the hypothesis that expression of the ACE2 receptor may be upregulated by interferon. To further interrogate this hypothesis, we cultured basal cells from two primary human donors, one human basal cell line, and one mouse trachea and stimulated them with  IL4, IL17a, IFNgamma, IFNαlpha, IFNbeta for 12 hours overnight. We performed bulk RNA sequencing and differential expression to show a dose dependent upregulation of canonical ISGs (interferon signaling genes). Specifically, we see that ACE2 is most significantly unregulated following IFN alpha stimulation in primary human basal cells, diminished in the BEAS-2B cell line and not seen in mouse cells. 

To visualize these samples, the gene expression data may be viewed interactively in the Alexandria Project: Interferon regulation of ACE2 in human and murine basal cells

Under the ‘Explore’ tab, use the ‘Search genes’ field in the top left corner to visualize log-normalized gene expression. To visualize expression in each sample, select ‘View Options’ in the top left corner of the plot and choose the sample of interest under ‘Load cluster’. The ‘Stim_Dose’ annotation refers to the dose of each stimulation condition applied to that sample. 

Data for all samples in this study can be dowloaded under the ‘Download’ tab.

Murine nasal mucosa after intranasal interferon exposure

We test the impact of IFNalpha stimulation in vivo by treating two mice intranasally with 200 ng of IFNalpha and two with saline. After 12 hours, the nasal mucosa of the respiratory and olfactory epithelia and underlying lamina propria were isolated and prepared for sequencing with Seq-Well S^3.

These cells may be viewed interactively in the Alexandria Project: Murine nasal mucosa after intranasal interferon exposure

Data for all samples in this study can be dowloaded under the ‘Download’ tab.

Alexandria Project Details

Alexandria Documentation

Single Cell Portal Documentation

We thank the Broad Institute Single Cell Portal team for creating the platform that allows Alexandria to exist and for their working tirelessly to help us share our datasets for others to access.

Whether cultured in vitro or part of a complex tissue in vivo, a cell’s phenotype and function are significantly influenced by dynamic interactions with its microenvironment. To explicitly examine how a cell’s spatiotemporal activity impacts its behavior, we developed and validated a strategy termed SPACECAT – Spatially PhotoActivatable Color Encoded Cell Address Tags – to annotate, track, and isolate specific cells in a non-destructive, viability-preserving manner. In SPACECAT, a biological sample is immersed in a photocaged fluorescent molecule, and cells within a location of interest are labeled for further study by uncaging that molecule with user-patterned near-UV light. SPACECAT offers high spatial precision and temporal stability across diverse cell and tissue types, and is compatible with common downstream assays, including flow cytometry and single-cell RNA-Seq. Illustratively, we leveraged this approach in patient-derived intestinal organoids, a spatially complex system less amenable to genetic manipulations, to select for crypt-like regions enriched in stem-like and actively mitotic cells. Moreover, we demonstrate its applicability and utility on ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model, uncovering spatially-biased gene expression patterns among immune cell subsets and identifying rare myeloid phenotypes enriched around tumor/healthy border regions. In sum, our method provides a minimally invasive and broadly applicable approach to link cellular spatiotemporal features and/or behavioral phenotypes with diverse downstream assays, enabling fundamental insights into the connections between tissue microenvironments and biological (dys)function.

The full expression matrix and associated metadata from the publication “Integrated Single-Cell Analysis of Multicellular Immune Dynamics during Hyperacute HIV-1 Infection” can be downloaded from the Alexandria Project hosted through the Single Cell Portal at the Broad Institute. Please visit the interactive dataset here to access and explore the data online. See below for an extended Supplementary Discussion contextualizing our findings in the single-cell transcriptomics and HIV literature. Supplementary Software is available for download on this page.

Memories of previous immune events enable barrier tissues to rapidly recall distinct environmental exposures. To effectively inform future responses, these past experiences can be stored in cell types that are long-term residents or essential constituents of tissues. There is an emerging understanding that, in addition to antigen-specific immune cells, diverse haematopoietic, stromal, parenchymal and neuronal cell types can store inflammatory memory. Here, we explore the impact of previous immune activity on various cell lineages with the goal of presenting a unified view of inflammatory memory to environmental exposures (such as allergens, antigens, noxious agents and microorganisms) at barrier tissues. We propose that inflammatory memory is distributed across diverse cell types and stored through shifts in cell states, and we provide a framework to guide future experiments. This distribution and storage may promote adaptation or maladaptation in homeostatic, maintenance and disease settings — especially if the distribution of memory favours cellular cooperation during storage or recall.

The induction of broadly neutralizing antibodies (bnAbs) is highly desired for an effective vaccine against HIV-1. Typically, bnAbs develop in patients with high viremia, but they can also evolve in some untreated HIV-1 controllers with low viral loads. Here, we identify a subgroup of neutralizer-controllers characterized by myeloid DCs (mDCs) with a distinct inflammatory signature and a superior ability to prime T follicular helper (Tfh)-like cells in an STAT4-dependent fashion. This distinct immune profile is associated with a higher frequency of Tfh-like cells in peripheral blood (pTfh) and an enrichment for Tfh-defining genes in circulating CD4+ T cells. Correspondingly, monocytes from this neutralizer controller subgroup upregulate genes encoding for chemotaxis and inflammation, and they secrete high levels of IL-12 in response to TLR stimulation. Our results suggest the existence of multi-compartment immune networks between mDCs, Tfh, and monocytes that may facilitate the development of bnAbs in a subgroup of HIV-1 controllers.

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.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide. The only available vaccine, BCG (Bacillus Calmette–Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography–computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

By affinity capture and amplification of TCR transcripts from whole-transcriptome libraries, TCR CDR3 sequences can be recovered from 3′-barcoded scRNA-seq libraries (e.g. Seq-Well, Drop-seq, etc.). This method can be applied post-hoc, allowing for the capture of additional information from archived samples. The protocol can also be found here.

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. The protocol can be found here.

Advances in single-cell RNA sequencing (scRNA-seq) allow the characterization of cellular states across human tissues with unprecedented granularity, creating opportunities to reassess the diversity of cell states found in health and the molecular networks that drive inflammatory disease. Type 2 inflammation is classically characterized by the recruitment and/or hyperplasia of TH2 cells, type 2 innate lymphoid cells (ILC2s), mast cells, and eosinophils, which promote the generation of IgE and the type 2 cytokines IL-4, IL-5, and IL-13. Although decades of research have elucidated pathobiologic roles for these hematopoietic cells in airway disease and linked them to the development of goblet cell metaplasia, we can now define their maturation and activation in human tissue, as well as the cellular neighborhoods in which they function. Although few in number, recent scRNA-seq studies in mice and human subjects have highlighted the remarkable cellular diversity of the airway, especially among constituent epithelial cells (EpCs), implicating them in novel proinflammatory pathways that support immune function and promote inflammatory disease.

Immune responses within barrier tissues are regulated, in part, by nociceptors, specialized peripheral sensory neurons that detect noxious stimuli. Previous work has shown that nociceptor ablation not only alters local responses to immune challenge at peripheral sites, but also within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Indeed, although sympathetic innervation of LNs is well documented, it has been unclear whether the LN parenchyma itself is innervated by sensory neurons. Here, using a combination of high-resolution imaging, retrograde viral tracing, single-cell transcriptomics (scRNA-seq), and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is preferentially located in the outermost cortex of skin-draining LNs. Transcriptomic profiling revealed that there are at least four discrete subsets of sensory neurons that innervate LNs with a predominance of peptidergic nociceptors, and an innervation pattern that is distinct from that in the surrounding skin. To uncover potential LN-resident communication partners for LN-innervating sensory neurons, we employed scRNA-seq to generate a draft atlas of all murine LN cells and, based on receptor-ligand expression patterns, nominated candidate target populations among stromal and immune cells. Using selective optogenetic stimulation of LN-innervating sensory axons, we directly experimentally tested our inferred connections. Acute neuronal activation triggered rapid transcriptional changes preferentially within our top-ranked putative interacting partners, principally endothelium and other nodal stroma cells, as well as several innate leukocyte populations. Thus, LNs are monitored by a unique population of sensory neurons that possesses immunomodulatory potential.

Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. However, it has been challenging to identify the cell types associated with AMD given the genetic complexity of the disease. Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the first single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Heterogeneity is observed within macroglia, suggesting that human retinal glia are more diverse than previously thought. Finally, GWAS-based enrichment analysis identifies glia, vascular cells, and cone photoreceptors to be associated with the risk of AMD. These data provide a detailed analysis of the human retina, and show how scRNA-seq can provide insight into cell types involved in complex, inflammatory genetic diseases.

Transitional B cells must actively undergo selection for self-tolerance before maturing into their resting follicular B cell successors. We found that metabolic quiescence was acquired at the follicular B cell stage in both humans and mice. In follicular B cells, the expression of genes involved in ribosome biogenesis, aerobic respiration, and mammalian target of rapamycin complex 1 (mTORC1) signaling was reduced when compared to that in transitional B cells. Functional metabolism studies, profiling of whole-cell metabolites, and analysis of cell surface proteins in human B cells suggested that this transition was also associated with increased extracellular adenosine salvage. Follicular B cells increased the abundance of the cell surface ectonucleotidase CD73, which coincided with adenosine 5′-monophosphate–activated protein kinase (AMPK) activation. Differentiation to the follicular B cell stage in vitro correlated with surface acquisition of CD73 on human transitional B cells and was augmented with the AMPK agonist, AICAR. Last, individuals with gain-of-function PIK3CD (PI3Kdelta) mutations and increased pS6 activation exhibited a near absence of circulating follicular B cells. Together, our data suggest that mTORC1 attenuation may be necessary for human follicular B cell development. These data identify a distinct metabolic switch during human B cell development at the transitional to follicular stages, which is characterized by an induction of extracellular adenosine salvage, AMPK activation, and the acquisition of metabolic quiescence.

Immunity celebrates its 25th anniversary at an exciting time in immunology, marked by the advent of new, door-opening approaches and a deeper understanding of the centrality of the immune system to both health and disease. We asked 25 investigators to look forward and share a vision of the next quarter century of immunology research.

To mark the 15th anniversary of Nature Methods, we asked scientists from across diverse fields of basic biology research for their views on the most exciting and essential methodological challenges that their communities are poised to tackle in the near future.

Antibody paratopes are formed by hypervariable complementarity-determining regions (CDRH3s) and variable gene-encoded CDRs. The latter show biased usage in human broadly neutralizing antibodies (bnAbs) against both HIV and influenza virus, suggesting the existence of gene-endowed targeting solutions that may be amenable to pathway amplification. To test this, we generated transgenic mice with human CDRH3 diversity but simultaneously constrained to individual user-defined human immunoglobulin variable heavy-chain (VH) genes, including IGHV1-69, which shows biased usage in human bnAbs targeting the hemagglutinin stalk of group 1 influenza A viruses. Sequential immunization with a stalk-only hemagglutinin nanoparticle elicited group 1 bnAbs, but only in IGHV1-69 mice. This VH-endowed response required minimal affinity maturation, was elicited alongside pre-existing influenza immunity, and when IGHV1-69 B cells were diluted to match the frequency measured in humans. These results indicate that the human repertoire could, in principle, support germline-encoded bnAb elicitation using a single recombinant hemagglutinin immunogen.

Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the de novo methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the de novo knockouts. Furthermore, we found unexpected transcriptional repression upon either loss of the de novo methyltransferase DNMT3A or the double knockout of DNMT3A/3B that is further propagated upon differentiation to mesoderm and ectoderm. Taken together, our single-cell RNA-sequencing data provide a high-resolution view into the consequences of depleting the three catalytically active DNMTs in human pluripotent stem cells.

Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.

Genomic medicine has paved the way for identifying biomarkers and therapeutically actionable targets for complex diseases, but is complicated by the involvement of thousands of variably expressed genes across multiple cell types. Single-cell RNA-sequencing study (scRNA-seq) allows the characterization of such complex changes in whole organs. The study is based on applying network tools to organize and analyze scRNA-seq data from a mouse model of arthritis and human rheumatoid arthritis, in order to find diagnostic biomarkers and therapeutic targets. Diagnostic validation studies were performed using expression profiling data and potential protein biomarkers from prospective clinical studies of 13 diseases. A candidate drug was examined by a treatment study of a mouse model of arthritis, using phenotypic, immunohistochemical, and cellular analyses as read-outs. We performed the first systematic analysis of pathways, potential biomarkers, and drug targets in scRNA-seq data from a complex disease, starting with inflamed joints and lymph nodes from a mouse model of arthritis. We found the involvement of hundreds of pathways, biomarkers, and drug targets that differed greatly between cell types. Analyses of scRNA-seq and GWAS data from human rheumatoid arthritis (RA) supported a similar dispersion of pathogenic mechanisms in different cell types. Thus, systems-level approaches to prioritize biomarkers and drugs are needed. Here, we present a prioritization strategy that is based on constructing network models of disease-associated cell types and interactions using scRNA-seq data from our mouse model of arthritis, as well as human RA, which we term multicellular disease models (MCDMs). We find that the network centrality of MCDM cell types correlates with the enrichment of genes harboring genetic variants associated with RA and thus could potentially be used to prioritize cell types and genes for diagnostics and therapeutics. We validated this hypothesis in a large-scale study of patients with 13 different autoimmune, allergic, infectious, malignant, endocrine, metabolic, and cardiovascular diseases, as well as a therapeutic study of the mouse arthritis model. Overall, our results support that our strategy has the potential to help prioritize diagnostic and therapeutic targets in human disease.

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 development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies has empowered the characterization of complex biological samples by dramatically increasing the number of constituent cells that can be examined concurrently. Nevertheless, these approaches typically recover substantially less information per-cell as compared to lower-throughput microtiter plate-based strategies. To uncover critical phenotypic differences among cells and effectively link scRNA-Seq observations to legacy datasets, reliable detection of phenotype-defining transcripts – such as transcription factors, affinity receptors, and signaling molecules – by these methods is essential. Here, we describe a substantially improved massively-parallel scRNA-Seq protocol we term Seq-Well S^3 (“Second-Strand Synthesis”) that increases the efficiency of transcript capture and gene detection by up to 10- and 5-fold, respectively, relative to previous iterations, surpassing best-in-class commercial analogs. We first characterized the performance of Seq-Well S^3 in cell lines and PBMCs, and then examined five different inflammatory skin diseases, illustrative of distinct types of inflammation, to explore the breadth of potential immune and parenchymal cell states. Our work presents an essential methodological advance as well as a valuable resource for studying the cellular and molecular features that inform human skin inflammation.

Cellular immunity is critical for controlling intracellular pathogens, but the dynamics and cooperativity of the evolving host response to infection are not well defined. Here, we apply single-cell RNA-sequencing to longitudinally profile pre- and immediately post-HIV infection peripheral immune responses of multiple cell types in four untreated individuals. Onset of viremia induces a strong transcriptional interferon response integrated across most cell types, with subsequent pro-inflammatory T cell differentiation, monocyte MHC-II upregulation, and cytolytic killing. With longitudinal sampling, we nominate key intra- and extracellular drivers that induce these programs, and assign their multi-cellular targets, temporal ordering, and duration in acute infection. Two individuals studied developed spontaneous viral control, associated with initial elevated frequencies of proliferating cytotoxic cells, inclusive of a previously unappreciated proliferating natural killer (NK) cell subset. Our study presents a unified framework for characterizing immune evolution during a persistent human viral infection at single-cell resolution, and highlights programs that may drive response coordination and influence clinical trajectory.

Sustained viremia after acute HIV infection is associated with profound CD4+ T cell loss and exhaustion of HIV-specific CD8+ T cell responses. To determine the impact of combination antiretroviral therapy (cART) on these processes, we examined the evolution of immune responses in acutely infected individuals initiating treatment before peak viremia. Immediate treatment of Fiebig stages I and II infection led to a rapid decline in viral load and diminished magnitude of HIV-specific (tetramer+) CD8+ T cell responses compared to untreated donors. There was a strong positive correlation between cumulative viral antigen exposure before full cART-induced suppression and immune responses measured by MHC class I tetramers, IFN-γ ELISPOT, and CD8+ T cell activation. HIV-specific CD8+ T responses of early treated individuals were characterized by increased CD127 and BCL-2 expression, greater in vitro IFN-γ secretion, and enhanced differentiation into effector memory (Tem) cells. Transcriptional analysis of tetramer+ CD8+ T cells from treated persons revealed reduced expression of genes associated with activation and apoptosis, with concurrent up-regulation of prosurvival genes including BCL-2AXL, and SRC. Early treatment also resulted in robust HIV-specific CD4+ T cell responses compared to untreated HIV-infected individuals. Our data show that limiting acute viremia results in enhanced functionality of HIV-specific CD4+ and CD8+ T cells, preserving key antiviral properties of these cells.

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.

Mice engrafted with components of a human immune system have become widely-used models for studying aspects of human immunity and disease. However, a defined methodology to objectively measure and compare the quality of the human immune response in different models is lacking. Here, by taking advantage of the highly immunogenic live-attenuated yellow fever virus vaccine YFV-17D, we provide an in-depth comparison of immune responses in human vaccinees, conventional humanized mice, and second generation humanized mice. We demonstrate that selective expansion of human myeloid and natural killer cells promotes transcriptomic responses akin to those of human vaccinees. These enhanced transcriptomic profiles correlate with the development of an antigen-specific cellular and humoral response to YFV-17D. Altogether, our approach provides a robust scoring of the quality of the human immune response in humanized mice and highlights a rational path towards developing better pre-clinical models for studying the human immune response and disease.

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.

We introduce a microfluidic platform that enables single-cell mass and growth rate measurements upstream of single-cell RNA-sequencing (scRNA-seq) to generate paired single-cell biophysical and transcriptional data sets. Biophysical measurements are collected with a serial suspended microchannel resonator platform (sSMR) that utilizes automated fluidic state switching to load individual cells at fixed intervals, achieving a throughput of 120 cells per hour. Each single-cell is subsequently captured downstream for linked molecular analysis using an automated collection system. From linked measurements of a murine leukemia (L1210) and pro-B cell line (FL5.12), we identify gene expression signatures that correlate significantly with cell mass and growth rate. In particular, we find that both cell lines display a cell-cycle signature that correlates with cell mass, with early and late cell-cycle signatures significantly enriched amongst genes with negative and positive correlations with mass, respectively. FL5.12 cells also show a significant correlation between single-cell growth efficiency and a G1-S transition signature, providing additional transcriptional evidence for a phenomenon previously observed through biophysical measurements alone. Importantly, the throughput and speed of our platform allows for the characterization of phenotypes in dynamic cellular systems. As a proof-ofprinciple, we apply our system to characterize activated murine CD8+ T cells and uncover two unique features of CD8+ T cells as they become proliferative in response to activation: i) the level of coordination between cell cycle gene expression and cell mass increases, and ii) translation-related gene expression increases and shows a correlation with single-cell growth efficiency. Overall, our approach provides a new means of characterizing the transcriptional mechanisms of normal and dysfunctional cellular mass and growth rate regulation across a range of biological contexts


Human immunity relies on the coordinated responses of many cellular subsets and functional states. Inter-individual variations in cellular composition and communication could thus potentially alter host protection. Here, we explore this hypothesis by applying single-cell RNA-sequencing to examine viral responses among the dendritic cells (DCs) of three elite controllers (ECs) of HIV-1 infection.


To overcome the potentially confounding effects of donor-to-donor variability, we present a generally applicable computational framework for identifying reproducible patterns in gene expression across donors who share a unifying classification. Applying it, we discover a highly functional antiviral DC state in ECs whose fractional abundance after in vitro exposure to HIV-1 correlates with higher CD4+ T cell counts and lower HIV-1 viral loads, and that effectively primes polyfunctional T cell responses in vitro. By integrating information from existing genomic databases into our reproducibility-based analysis, we identify and validate select immunomodulators that increase the fractional abundance of this state in primary peripheral blood mononuclear cells from healthy individuals in vitro.


Overall, our results demonstrate how single-cell approaches can reveal previously unappreciated, yet important, immune behaviors and empower rational frameworks for modulating systems-level immune responses that may prove therapeutically and prophylactically useful.


Complete information about the scRAD R package is available on the Shalek Lab Resources page.

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.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

We develop single-cell genomic approaches to comprehensively profile complex biological ensembles. To date, the majority of our work has focused on establishing, validating, and scaling single-cell transcriptomics, often through the development of microdevices to enable genome-wide identification of the cell types/states that comprise functional or dysfunctional biological samples.

Most recently, we have developed Seq-Well, a portable, low-cost platform for high-throughput single-cell RNA-Seq (scRNA-Seq). By providing open access to resources and protocols, we hope to democratize access to cutting-edge approaches in single-cell genomics.

To complement and inform the analysis of scRNA-Seq datasets, we create methods to simultaneously profile additional cellular characteristics of interest (e.g. genome, epigenome, or proteome), independently, or in combination with, scRNA-Seq. For a given technique or system, we ask what additional information would help us better interpret our scRNA-Seq results and develop methods to collect these data. These novel methods often map ancillary information into a DNA-based readout that can be coanalyzed with cellular mRNA or developing/applying microdevices. Recently, we have developed a method for integrated mRNA and protein detection that leverages proximity extension assays (Genshaft et al. 2016). To extract the information content from these novel datasets more effectively, we also formulate new computational methods and analyses.

We use microdevices, coupled with functional signal readouts, to create and study defined cell-cell interactions. By explicitly enumerating cell type, number, and additional functional properties (e.g., cytokine secretion), we model ensemble behaviors, looking for synergies and antagonisms­. These genetic signatures, along with those collected via our other platforms, provide a unique and essential reference for deconvolving behaviors in complex ensembles. We are also using genetic tracing strategies to examine differences between interacting and random cell pairs in vivo, and are developing computational methods (Tirosh et al., 2016) to identify putative interactions from scRNA-Seq data.

As the amount of data we have relating to cells, properties, surroundings, and interactions increases exponentially, we are motivated to develop pan-system measurements and analyses to paint comprehensive pictures of immune response in health and disease. Relying on massive transcriptomic datasets generated from complex tissues, like melanoma tumors, inflamed human gut, M. tuberculosis (MTB)-induced granulomas, and healthy or SHIV-infected monkey tissues, we have begun to construct social networks of integrated responses to physiological perturbations. The technologies outlined above uniquely enable us to generate foundational datasets (e.g., transcriptomes from interacting cell pairs) for deconvolving and interpreting the potential drivers of observed ensemble behaviors, as well as for identifying which properties we cannot explain, and thus need to study. To date, our lab has generated over 2 million single-cell transcriptomes across multiple tissues, individuals, and species; we are utilizing this data, paired with metadata and additional characteristics, to look for common cellular network motifs, such as division of labor, quorum sensing, persistence, or bet-hedging.

Vaccines remain the most effective tool to prevent infectious diseases. Here, we introduce an in vitro booster vaccination approach that relies on antigen-dependent activation of human memory B cells in culture. This stimulation induces antigen-specific B cell proliferation, differentiation of B cells into plasma cells, and robust antibody secretion after a few days of culture. We validated this strategy using cells from healthy donors to retrieve human antibodies against tetanus toxoid and influenza hemagglutinin (HA) from H1N1 and newly emergent subtypes such as H5N1 and H7N9. Anti-HA antibodies were cross-reactive against multiple subtypes, and some showed neutralizing activity. Although these antibodies may have arisen as a result of previous influenza infection, we also obtained gp120-reactive antibodies from non–HIV-infected donors, indicating that we can generate antibodies without prior antigenic exposure. Overall, our novel approach can be used to rapidly produce therapeutic antibodies and has the potential to assess the immunogenicity of candidate antigens, which could be exploited in future vaccine development.

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.

Recent advances in cellular profiling have demonstrated substantial heterogeneity in the behaviour of cells once deemed ‘identical’, challenging fundamental notions of cell ‘type’ and ‘state’. Not surprisingly, these findings have elicited substantial interest in deeply characterizing the diversity, interrelationships and plasticity among cellular phenotypes. To explore these questions, experimental platforms are needed that can extensively and controllably profile many individual cells. Here, microfluidic structures — whether valve-, droplet- or nanowell-based — have an important role because they can facilitate easy capture and processing of single cells and their components, reducing labour and costs relative to conventional plate-based methods while also improving consistency. In this article, we review the current state-of-the-art methodologies with respect to microfluidics for mammalian single-cell ‘omics’ and discuss challenges and future opportunities.

Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.


The immune system plays an important role in regulating homeostatic balance across tissues and individuals in the face of changing and challenging environments. Given the pivotal and outsized impact cell subsets (e.g., rare precocious DCs) can have on ensemble dynamics (e.g., global activation of an antiviral response and deactivation of inflammation), we aim to understand the functional consequences of variation in cellular composition across tissues, as well as how different immune cells adapt to changing environmental conditions.

Motivating questions in the lab include:

  1. How can we perform observational and experimental studies to understand the fundamental units of tissues structure and function?
  2. Can we derive basic principles governing homeostatic and pathogenic immune responses within tissues?
  3. What dictates the evolution of clonal antigen-specific T & B cell responses?

To this end, we are several multiple tissues from multiple organisms across common sources of variation. By examining consistent and unique themes that emerge across these systems, we aim to extract basic principles that govern homeostatic and pathogenic immune responses within tissues. Ultimately, we intend to leverage this information to rationally engineer immune responses (e.g., in vaccines and immunotherapies).