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Tobacco smoking doubles the risk of active tuberculosis (TB) and accounts for up to 20% of all active TB cases globally. How smoking promotes lung microenvironments permissive to Mycobacterium tuberculosis ( Mtb ) growth remains incompletely understood. We investigated primary bronchoalveolar lavage cells from current and never smokers by performing single-cell RNA sequencing (scRNA-seq), flow cytometry, and functional assays. We observed the enrichment of immature inflammatory monocytes in the lungs of smokers compared with nonsmokers. These monocytes exhibited phenotypes consistent with recent recruitment from blood, ongoing differentiation, increased activation, and states similar to those with chronic obstructive pulmonary disease. Using integrative scRNA-seq and flow cytometry, we identified CD93 as a marker for a subset of these newly recruited smoking-associated lung monocytes and further provided evidence that the recruitment of monocytes into the lung was mediated by CCR2-binding chemokines, including CCL11. We also show that these cells exhibit elevated inflammatory responses upon exposure to Mtb and accelerated intracellular growth of Mtb compared with mature macrophages. This elevated Mtb growth could be inhibited by anti-inflammatory small molecules, providing a connection between smoking-induced pro-inflammatory states and permissiveness to Mtb growth. Our findings suggest a model in which smoking leads to the recruitment of immature inflammatory monocytes from the periphery to the lung, which results in the accumulation of these Mtb -permissive cells in the airway. This work defines how smoking may lead to increased susceptibility to Mtb and identifies host-directed therapies to reduce the burden of TB among those who smoke.

Background: Endothelial cells (ECs) are capable of quickly responding in a coordinated manner to a wide array of stresses to maintain vascular homeostasis. Loss of EC cellular adaptation may be a potential marker for cardiovascular disease and a predictor of poor response to endovascular pharmacological interventions such as drug-eluting stents. Here, we report single-cell transcriptional profiling of ECs exposed to multiple stimulus classes to evaluate EC adaptation., Methods: Human aortic ECs were costimulated with both pathophysiological flows mimicking shear stress levels found in the human aorta (laminar and turbulent, ranging from 2.5 to 30 dynes/cm 2 ) and clinically relevant antiproliferative drugs, namely paclitaxel and rapamycin. EC state in response to these stimuli was defined using single-cell RNA sequencing., Results: We identified differentially expressed genes and inferred the TF (transcription factor) landscape modulated by flow shear stress using single-cell RNA sequencing. These flow-sensitive markers differentiated previously identified spatially distinct subpopulations of ECs in the murine aorta. Moreover, distinct transcriptional modules defined flow- and drug-responsive EC adaptation singly and in combination. Flow shear stress was the dominant driver of EC state, altering their response to pharmacological therapies., Conclusions: We showed that flow shear stress modulates the cellular capacity of ECs to respond to paclitaxel and rapamycin administration, suggesting that while responding to different flow patterns, ECs experience an impairment in their transcriptional adaptation to other stimuli., Competing Interests: Disclosures A.S. Kalluri reports compensation for consulting from nference, inc. A.K. Shalek reports compensation for consulting and scientific advisory board membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Ochre Bio, and Dahlia Biosciences. The other authors report no conflicts.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9 + TREM2 + macrophages that express SPP1 , GPNMB , FABP5 , and CD63 . In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-β1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-β1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-β1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-β1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

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 key contributing factor to childhood malnutrition, growth stunting, and diminished oral vaccine responses. Although EE has been shown to be the by-product of a recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE by performing high-throughput, single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE in Lusaka, Zambia (eight HIV-negative and three HIV-positive), six adults without EE in Boston, United States, and two adults in Durban, South Africa, which we complemented with published data from three additional individuals from the same clinical site. We analyzed previously defined bulk-transcriptomic signatures of reduced villus height and decreased microbial translocation in EE and showed 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 determined cell subsets whose fractional abundances associate with EE severity, small intestinal region, and HIV infection. Furthermore, by comparing duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and increased proinflammatory cytokine gene expression in a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells in the EE cohort. Together, our work elucidates epithelial and immune correlates of EE and nominates cellular and molecular targets for intervention.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-β1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation., Competing Interests: The handling editor [AH] declared a shared affiliation with the author(s) [AW, NY] at the time of review. BR, FB, MK and HD were employed by CommaSoft. CW and PB were employed by Boehringer Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Baßler, Fujii, Kapellos, Dudkin, Reusch, Horne, Reiz, Luecken, Osei-Sarpong, Warnat-Herresthal, Bonaguro, Schulte-Schrepping, Wagner, Günther, Pizarro, Schreiber, Knoll, Holsten, Kröger, De Domenico, Becker, Händler, Wohnhaas, Baumgartner, Köhler, Theis, Kraut, Wadsworth, Hughes, Ferreira, Hinkley, Kaltheuner, Geyer, Thiele, Shalek, Feißt, Thomas, Dickten, Beyer, Baum, Yosef, Aschenbrenner, Ulas, Hasenauer, Theis, Skowasch and Schultze.)

Rheumatoid arthritis (RA) is an autoimmune disease affecting synovial joints where different CD4 + T cell subsets may contribute to pathology. Here, we perform single cell sequencing on synovial CD4 + T cells from anti-citrullinated protein antibodies (ACPA)+ and ACPA- RA patients and identify two peripheral helper T cell (T PH ) states and a cytotoxic CD4 + T cell subset. We show that the adhesion G-protein coupled receptor 56 (GPR56) delineates synovial CXCL13 high T PH CD4 + T cells expressing LAG-3 and the tissue-resident memory receptors CXCR6 and CD69. In ACPA- SF, T PH cells display lower levels of GPR56 and LAG-3. Further, most expanded T cell clones in the joint are within CXCL13 high T PH CD4 + T cells. Finally, RNA-velocity analyses suggest a common differentiation pathway between the two T PH clusters and effector CD4 + T cells. Our study provides comprehensive immunoprofiling of the synovial CD4 + T cell subsets in ACPA+ and ACPA- RA., (© 2022. The Author(s).)

Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years and reactivate to cause recurrent blood-stage infection. Although they are an important target for malaria eradication, little is known about the molecular features of replicative and non-replicative intracellular liver-stage parasites and their host cell dependence. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites for transcriptional profiling. Coupling enrichment strategies with bulk and single-cell analyses, we capture both parasite and host transcripts in individual hepatocytes throughout the course of infection. We define host- and state-dependent transcriptional signatures and identify unappreciated populations of replicative and non-replicative parasites that share features with sexual transmissive forms. We find that infection suppresses the transcription of key hepatocyte function genes and elicits an anti-parasite innate immune response. Our work provides a foundation for understanding host-parasite interactions and reveals insights into the biology of P. vivax dormancy and transmission., Competing Interests: Declaration of interests A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Ochre Bio, Third Rock Ventures, Relation Therapeutics, Empress Therapeutics, FL82, and Dahlia Biosciences. S.N.B. reports compensation for cofounding, consulting, and/or board membership in Glympse Bio, Satellite Bio, CEND Therapeutics, Xilio Therapeutics, Catalio Capital, Intergalactic Therapeutics, Port Therapeutics, Vertex Pharmaceuticals, Danaher, and Moderna and receives sponsored research funding from Johnson & Johnson, Revitope, and Owlstone., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB., Competing Interests: Declaration of interests A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Third Rock Ventures, Hovione, Relation Therapeutics, FL82, Empress Therapeutics, Ochre Bio, and Dahlia Biosciences.C.L. is a shareholder and consultant for Honeycomb Biotechnologies. T.K.H. is a shareholder and consultant for nference, inc., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 perform single-cell RNA-sequencing on prostate biopsies, prostatectomy specimens, and patient-derived organoids from localized prostate cancer patients. We uncover heterogeneous cellular states in prostate epithelial cells marked by high androgen signaling states that are enriched in prostate cancer and identify a population of tumor-associated club cells that may be associated with prostate carcinogenesis. ERG-negative tumor cells, compared to ERG-positive 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 harbor 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., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Leptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire prognosis and no effective systemic treatment options. Over the past decade, the incidence of LMD has steadily increased due to therapeutics that have extended the survival of cancer patients, highlighting the need for new interventions. To examine the efficacy of immune checkpoint inhibitors (ICI) in patients with LMD, we completed two phase II clinical trials. Here, we investigate the cellular and molecular features underpinning observed patient trajectories in these trials by applying single-cell RNA and cell-free DNA profiling to longitudinal cerebrospinal fluid (CSF) draws from enrolled patients. We recover immune and malignant cell types in the CSF, characterize cell behavior changes following ICI, and identify genomic features associated with relevant clinical phenomena. Overall, our study describes the liquid LMD tumor microenvironment prior to and following ICI treatment and demonstrates clinical utility of cell-free and single-cell genomic measurements for LMD research., (© 2021. The Author(s).)

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-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 can each contribute to the antimicrobial response.

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., Competing Interests: Declaration of Interests A.K.S. has received compensation for consulting and SAB membership from Honeycomb Biotechnologies, Cellarity, Ochre Bio, Repertoire, and Dahlia Biosciences., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 S 3 ("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 S 3 increased 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 S 3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation., Competing Interests: Declaration of Interests A.K.S. and J.C.L. have received compensation for consulting and SAB membership from Honeycomb Biotechnologies. A.K.S. has received compensation for consulting and SAB membership from Cellarity, Repertoire Immune Medicines, Orche Bio, and Dahlia Biosciences. T.M.G., T.K.H., M.H.W., A.K.S., and J.C.L. are co-inventors on a provisional patent application filed by MIT relating to the improved methodology described in this manuscript., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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 angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (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 among 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 discovered 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., Competing Interests: Declaration of Interests A.R. is an SAB member of ThermoFisher Scientific, Neogene Therapeutics, Asimov, and Syros Pharmaceuticals; a co-founder of and equity holder in Celsius Therapeutics; and an equity holder in Immunitas Therapeutics. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Cogen Therapeutics, Orche Bio, and Dahlia Biosciences. L.S.K. is on the SAB for HiFiBio; she reports research funding from Kymab Limited, Bristol Meyers Squibb, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals and consulting fees from Equillium, FortySeven, Inc, Novartis, Inc, EMD Serono, Gilead Sciences, and Takeda Pharmaceuticals. A.S. is an employee of Johnson and Johnson. N.K. is an inventor on a patent using thyroid hormone mimetics in acute lung injury that is now being considered for intervention in COVID-19 patients. J.L. is a scientific consultant for 10X Genomics, Inc. O.R.R, is a co-inventor on patent applications filed by the Broad Institute to inventions relating to single-cell genomics applications, such as in PCT/US2018/060860 and US Provisional Application No. 62/745,259. S.T. in the last three years was a consultant at Genentech, Biogen, and Roche and is a member of the SAB of Foresite Labs. M.H.W. is now an employee of Pfizer. F.J.T. reports receiving consulting fees from Roche Diagnostics GmbH and ownership interest in Cellarity, Inc. P.H. is a co-inventor on a patent using artificial intelligence and high-resolution microscopy for COVID-19 infection testing based on serology., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide 1 . 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 1,2 . 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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Seq-Well is a low-cost picowell platform that can be used to simultaneously profile the transcriptomes of thousands of cells from diverse, low input clinical samples. In Seq-Well, uniquely barcoded mRNA capture beads and cells are co-confined in picowells that are sealed using a semipermeable membrane, enabling efficient cell lysis and mRNA capture. The beads are subsequently removed and processed in parallel for sequencing, with each transcript's cell of origin determined via the unique barcodes. Due to its simplicity and portability, Seq-Well can be performed almost anywhere.

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases 1 . Specialized subsets of epithelial cells-including secretory and ciliated cells-differentiate from basal stem cells to collectively protect the upper airway 2-4 . Allergic inflammation can develop from persistent activation 5 of type 2 immunity 6 in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps 7 . Basal cell hyperplasia is a hallmark of severe disease 7-9 , but it is not known how these progenitor cells 2,10,11 contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing 12 , report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic 13 , epigenetic 14,15 and extrinsic factors 11,16,17 that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories.

Background: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity., Results: Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology., Conclusions: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.

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.

To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies., (Copyright © 2016, American Association for the Advancement of Science.)

An innovative, microwell-based platform for single-cell RNA sequencing (RNA-seq) combines cost efficiency, scalability and parallelizability, and will enable many new avenues of biological inquiry.See related Research article: http://dx.doi.org/10.1186/s13059-015-0684-3.