Tobacco smoke exposure recruits inflammatory airspace monocytes that establish permissive lung niches for Mycobacterium tuberculosis.

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.

CD8+ lymphocytes are critical for early control of tuberculosis in macaques.

The functional role of CD8+ lymphocytes in tuberculosis remains poorly understood. We depleted innate and/or adaptive CD8+ lymphocytes in macaques and showed that loss of all CD8α+ cells (using anti-CD8α antibody) significantly impaired early control of Mycobacterium tuberculosis (Mtb) infection, leading to increased granulomas, lung inflammation, and bacterial burden. Analysis of barcoded Mtb from infected macaques demonstrated that depletion of all CD8+ lymphocytes allowed increased establishment of Mtb in lungs and dissemination within lungs and to lymph nodes, while depletion of only adaptive CD8+ T cells (with anti-CD8ß antibody) worsened bacterial control in lymph nodes. Flow cytometry and single-cell RNA sequencing revealed polyfunctional cytotoxic CD8+ lymphocytes in control granulomas, while CD8-depleted animals were unexpectedly enriched in CD4 and γδ T cells adopting incomplete cytotoxic signatures. Ligand-receptor analyses identified IL-15 signaling in granulomas as a driver of cytotoxic T cells. These data support that CD8+ lymphocytes are required for early protection against Mtb and suggest polyfunctional cytotoxic responses as a vaccine target.

Single-Cell RNA Sequencing Reveals That Adaptation of Human Aortic Endothelial Cells to Antiproliferative Therapies Is Modulated by Flow-Induced Shear Stress.

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/cm2) 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.

Protective intravenous BCG vaccination induces enhanced immune signaling in the airways.

Intradermal (ID) Bacillus Calmette-Guérin (BCG) is the most widely administered vaccine in the world. However, ID-BCG fails to achieve the level of protection needed in adults to alter the course of the tuberculosis epidemic. Recent studies in non-human primates have demonstrated high levels of protection against Mycobacterium tuberculosis ( Mtb ) following intravenous (IV) administration of BCG. However, the protective immune features that emerge following IV BCG vaccination remain incompletely defined. Here we used single-cell RNA-sequencing (scRNAseq) to transcriptionally profile 157,114 unstimulated and purified protein derivative (PPD)-stimulated bronchoalveolar lavage (BAL) cells from 29 rhesus macaques immunized with BCG across routes of administration and doses to uncover cell composition-, gene expression-, and biological network-level signatures associated with IV BCG-mediated protection. Our analyses revealed that high-dose IV BCG drove an influx of polyfunctional T cells and macrophages into the airways. These macrophages exhibited a basal activation phenotype even in the absence of PPD-stimulation, defined in part by IFN and TNF-α signaling up to 6 months following BCG immunization. Furthermore, intercellular immune signaling pathways between key myeloid and T cell subsets were enhanced following PPD-stimulation in high-dose IV BCG-vaccinated macaques. High-dose IV BCG also engendered quantitatively and qualitatively stronger transcriptional responses to PPD-stimulation, with a robust Th1-Th17 transcriptional phenotype in T cells, and augmented transcriptional signatures of reactive oxygen species production, hypoxia, and IFN-γ response within alveolar macrophages. Collectively, this work supports that IV BCG immunization creates a unique cellular ecosystem in the airways, which primes and enables local myeloid cells to effectively clear Mtb upon challenge.

Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation.

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-ß1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-ß1 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-ß1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-ß1 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.

Alveolar macrophages in early stage COPD show functional deviations with properties of impaired immune activation.

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-ß1 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.

Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation.

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.

Single cell sequencing identifies clonally expanded synovial CD4+ TPH cells expressing GPR56 in rheumatoid arthritis.

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 (TPH) states and a cytotoxic CD4+ T cell subset. We show that the adhesion G-protein coupled receptor 56 (GPR56) delineates synovial CXCL13high TPH CD4+ T cells expressing LAG-3 and the tissue-resident memory receptors CXCR6 and CD69. In ACPA- SF, TPH cells display lower levels of GPR56 and LAG-3. Further, most expanded T cell clones in the joint are within CXCL13high TPH CD4+ T cells. Finally, RNA-velocity analyses suggest a common differentiation pathway between the two TPH clusters and effector CD4+ T cells. Our study provides comprehensive immunoprofiling of the synovial CD4+ T cell subsets in ACPA+ and ACPA- RA.

A single-cell liver atlas of Plasmodium vivax infection.

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.

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

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

PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment.

BACKGROUND: Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood. METHODS: We use an in vivo expression screen of known cancer-associated somatic mutations in mouse syngeneic tumor models treated with checkpoint blockade to identify oncogenes that promote immune evasion. We then validated candidates from this screen in vivo and analyzed the tumor immune microenvironment of tumors expressing mutant protein to identify mechanisms of immune evasion. RESULTS: We found that expression of a catalytically active mutation in phospho-inositol 3 kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers a selective growth advantage to tumors treated with immunotherapy that is reversed by pharmacological PI3K inhibition. PIK3CA H1047R-expression in tumors decreased the number of CD8+ T cells but increased the number of inhibitory myeloid cells following immunotherapy. Inhibition of myeloid infiltration by pharmacological or genetic modulation of Ccl2 in PIK3CA H1047R tumors restored sensitivity to programmed cell death protein 1 (PD-1) checkpoint blockade. CONCLUSIONS: PI3K activation enables tumor immune evasion by promoting an inhibitory myeloid microenvironment. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. Our data suggest that some oncogenes promote tumorigenesis by enabling tumor cells to avoid clearance by the immune system. Identification of those mechanisms can advance rational combination strategies to increase the efficacy of immunotherapy.

Single-cell analysis of human primary prostate cancer reveals the heterogeneity of tumor-associated epithelial cell states.

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.

Genomic and transcriptomic correlates of immunotherapy response within the tumor microenvironment of leptomeningeal metastases.

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.

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

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

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

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

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

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 S3 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 S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

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.

Systematic comparison of single-cell and single-nucleus RNA-sequencing methods.

The scale and capabilities of single-cell RNA-sequencing methods have expanded rapidly in recent years, enabling major discoveries 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 representative 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.

Author Correction: Systematic comparison of single-cell and single-nucleus RNA-sequencing methods.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.