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Up to 20% of idiopathic interstitial lung disease is familial, referred to as Familial Pulmonary Fibrosis (FPF). We performed an integrated analysis of FPF genetic risk by comprehensively evaluating for genetic rare variants (RVs) in a large cohort of FPF kindreds.We performed whole-exome sequencing and/or candidate gene sequencing from affected individuals in 569 FPF kindreds, followed by co-segregation analysis in large kindreds, gene burden analysis, gene-based risk scoring, cell type enrichment analysis, and co-expression network construction.We found that 14.9% - 23.4% of genetic risk in kindreds could be explained by RVs in genes previously linked to FPF, predominantly telomere related genes. We identified new candidate genes in a small number of families, including SYDE1, SERPINB8, GPR87, and NETO1, and developed tools for evaluation and prioritization of RV-containing genes across kindreds. Several pathways were enriched for RV-containing genes in FPF, including focal adhesion and mitochondrial complex I assembly. By combining single cell transcriptomics with prioritized candidate genes, we discovered that expression of RV-containing genes was enriched in smooth muscle cells, type II alveolar epithelial cells, and endothelial cells.In the most comprehensive FPF genetic study to date, we defined the prevalence of RVs in known FPF-related genes and identified new candidate genes and pathways relevant to FPF. However, we did not identify new RV-containing genes shared across multiple kindreds, thereby suggesting that heterogeneous genetic variants involving a variety of genes and pathways mediate genetic risk in most FPF kindreds.

Immune cells have been implicated in idiopathic pulmonary fibrosis (IPF), but the phenotypes and effector mechanisms of these cells remain incompletely characterized. We performed mass cytometry to quantify immune cell subsets in lungs of 12 patients with IPF and 15 organ donors without chronic lung disease and used existing single-cell RNA-sequencing data to investigate transcriptional profiles of immune cells overrepresented in IPF. Among myeloid cells, we found increased numbers of alveolar macrophages (AMØs) and dendritic cells (DCs) in IPF, as well as a subset of monocyte-derived DCs. In contrast, monocyte-like cells and interstitial macrophages were reduced in IPF. Transcriptomic profiling identified an enrichment for IFN-γ response pathways in AMØs and DCs from IPF, as well as antigen processing in DCs and phagocytosis in AMØs. Among T cells, we identified three subsets of memory T cells that were increased in IPF, including CD4(+) and CD8(+) resident memory T cells (T(RM)) and CD8(+) effector memory cells. The response to the IFN-γ pathway was enriched in CD4 T(RM) and CD8 T(RM) cells in IPF, together with T cell activation and immune response–regulating signaling pathways. Increased AMØs, DCs, and memory T cells were present in IPF lungs compared with control subjects. In IPF, these cells possess an activation profile indicating increased IFN-γ signaling and upregulation of adaptive immunity in the lungs. Together, these studies highlight critical features of the immunopathogenesis of IPF.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis (PF). Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA-sequencing of lung tissue from 67 PF and 49 unaffected donors. Employing a pseudo-bulk approach, we mapped expression quantitative trait loci (eQTL) across 38 cell types, observing both shared and cell type-specific regulatory effects. Further, we identified disease-interaction eQTL and demonstrated that this class of associations is more likely to be cell-type specific and linked to cellular dysregulation in PF. Finally, we connected PF risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression, and implicates context-specific eQTL as key regulators of lung homeostasis and disease.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. OBJECTIVE: To identify and validate molecular determinants of IPF survival. METHODS: A staged genome-wide association study (GWAS) was performed using paired genomic and survival data. Stage I cases were drawn from centers across the US and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplant-free survival (TFS). Stage I variants with nominal significance (p

Supplemental Digital Content is available in the text., Background: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. Methods: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary–venous ECs (COL15A1neg) localized to the lung parenchyma and systemic–venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic–venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). Conclusions: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Summary Lack of diversity in human genomics limits our understanding of the genetic underpinnings of complex traits, hinders precision medicine, and contributes to health disparities. To map genetic effects on gene regulation in the underrepresented Indonesian population, we have integrated genotype, gene expression, and CpG methylation data from 115 participants across three island populations that capture the major sources of genomic diversity in the region. In a comparison with European datasets, we identify eQTLs shared between Indonesia and Europe as well as population-specific eQTLs that exhibit differences in allele frequencies and/or overall expression levels between populations. By combining local ancestry and archaic introgression inference with eQTLs and methylQTLs, we identify regulatory loci driven by modern Papuan ancestry as well as introgressed Denisovan and Neanderthal variation. GWAS colocalization connects QTLs detected here to hematological traits, and further comparison with European datasets reflects the poor overall transferability of GWAS statistics across diverse populations. Our findings illustrate how population-specific genetic architecture, local ancestry, and archaic introgression drive variation in gene regulation across genetically distinct and in admixed populations and highlight the need for performing association studies on non-European populations., Graphical abstract

Stem cells undergo dynamic changes in response to injury to regenerate lost cells. However, the identity of transitional states and the mechanisms that drive their trajectories remain understudied. Using lung organoids, multiple in vivo repair models, single-cell transcriptomics and lineage tracing, we find that alveolar type-2 epithelial cells undergoing differentiation into type-1 cells acquire pre-alveolar type-1 transitional cell state (PATS) en route to terminal maturation. Transitional cells undergo extensive stretching during differentiation, making them vulnerable to DNA damage. Cells in the PATS show an enrichment of TP53, TGFβ, DNA-damage-response signalling and cellular senescence. Gain and loss of function as well as genomic binding assays revealed a direct transcriptional control of PATS by TP53 signalling. Notably, accumulation of PATS-like cells in human fibrotic lungs was observed, suggesting persistence of the transitional state in fibrosis. Our study thus implicates a transient state associated with senescence in normal epithelial tissue repair and its abnormal persistence in disease conditions.

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages – wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

BackgroundLoss of secretory immunoglobulin A (SIgA) is common in COPD small airways and likely contributes to disease progression. We hypothesized loss of SIgA results from reduced expression of pIgR, a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium.MethodspIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in-situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice utilized immunostaining, primary murine tracheal epithelial cell (MTEC) culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from non-typeable Haemophilus influenzae (NTHi) was evaluated in vitro.ResultsWe found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and NTHi-secreted proteases are both capable of degrading SIgA in vitro and may also contribute to a deficient SIgA immunobarrier in COPD.InterpretationLoss of the SIgA immunobarrier in small airways of patients severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.Key MessagesWhat is the key question? Localized SIgA deficiency in small airways is an established driver of COPD pathogenesis, but the mechanism of loss remains unclear. We hypothesized loss of SIgA is due to reduced numbers of pIgR-expressing cells in SIgA-deficient small airways.What is the bottom line? pIgR is primarily expressed by secretory cells in human and murine airways. Although numbers of secretory cells are similar between SIgA-deficient and SIgA-replete airways in COPD, there is reduced expression of pIgR protein, but not mRNA, in SIgA-deficient airways. Additionally, host and bacterial proteases degrade SIgA in vitro, suggesting loss of SIgA may relate to both impaired transcytosis and increased degradation.Why read on? This study highlights the complexity of SIgA immunobarrier maintenance and suggests that strategies aimed at restoring the SIgA immunobarrier will need to account for both impaired transcytosis and degradation by host and/or bacterial proteases.

Precision-cut lung slices (PCLS) are increasingly utilized for ex vivo disease modeling, but a high-resolution characterization of cellular phenotype stability in PCLS has not been reported. Comparing the single-cell transcriptomic profile of human PCLS after five days of culture to freshly isolated human lung tissue, we found striking changes in endothelial cell and alveolar epithelial cell programs, reflecting both injury and pathways activated in static culture, while immune cell frequencies and programs remained largely intact and similar to the native lung. These cellular dynamics should be considered when utilizing PCLS as a model of the human lung.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection., Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 and poor outcomes. Here the authors compare the transcriptomes of single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in these patients.

Multiple myeloma (MM) is a malignancy of plasma cells in the bone marrow and the second most common hematological malignancy. MM is characterized by a high degree of heterogeneity and has a hereditary genetic component with relatives of MM patients having a higher risk of disease development. Additionally, MM displays a disparity in occurrence and mortality among sexes with males having a higher risk than females. Although new therapies have significantly improved survival rates, MM remains incurable as most patients experience relapses. Therefore, understanding the genetic control underlying complex outcomes is important for the development of better treatments for MM patients. While the majority of research has been focusing on protein-coding regions of the genome, recent studies have started to illuminate the role of non-coding variations on MM. For example, many of the 176 GWAS risk loci associated with MM susceptibility are located within or adjacent to regulatory regions, indicating a role in transcriptional regulation. However, how the non-coding genetic variations affect gene expression, tumor etiology and outcome is poorly understood. To better understand the genetic and biological underpinnings of MM, we utilize genomic approaches to examine non-coding germline and somatic effects on gene expression and cancer outcome. First, we analyzed whole-genome sequencing (WGS) from peripheral blood and WGS and RNA-seq from baseline tumor specimens of 607 participants from the CoMMpass longitudinal study of the Multiple Myeloma Research Foundation. Using both joint and sex-stratified analyses, we detected 6,504 unique germline variants associated with changes in gene expression, i.e. expression quantitative trait loci (eQTLs), corresponding to 4,598 unique eGenes. Among the identified eQTLs, 33.76% exhibit sex-specific effects and 654 variants are associated with genes that influence survival in the CoMMpass cohort. In addition to the germline eQTLs, we identified 266 somatic eQTLs, corresponding to 208 eGenes. Among the identified eQTLs, 566 germline and 88 somatic eQTLs overlap with an ATACseq peak in MM cell lines. Second, we selected a set of eQTLs to validate their functions in transcriptional activity in MM cell lines (2 females, 2 males) using CRISPR interference screens (CRISPRi) with a scRNA-seq readout. By analyzing global transcriptional changes, we identify potential downstream targets that may play essential roles in MM genesis and progression. In conclusion, using computational and functional genomics approaches, we have identified germline and somatic eQTLs that are associated with gene expression and survival in MM, thus providing insights into how these non-coding variations contribute to tumor etiology and outcome. Citation Format: Linh T. Bui, Heini M. Natri, Lance M. Peter, Bianca Argente, Austin J. Gutierrez, Arnold Federico, Mei-I Chung, Jonathan Keats, Nicholas E. Banovich. Functions of genetic variation on gene expression and survival in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5780.

One life-threatening outcome of cardiovascular disease is myocardial infarction, where cardiomyocytes are deprived of oxygen. To study inter-individual differences in response to hypoxia, we established an in vitro model of induced pluripotent stem cell-derived cardiomyocytes from 15 individuals. We measured gene expression levels, chromatin accessibility, and methylation levels in four culturing conditions that correspond to normoxia, hypoxia and short or long-term re-oxygenation. We characterized thousands of gene regulatory changes as the cells transition between conditions. Using available genotypes, we identified 1,573 genes with a cis expression quantitative locus (eQTL) in at least one condition, as well as 367 dynamic eQTLs, which are classified as eQTLs in at least one, but not in all conditions. A subset of genes with dynamic eQTLs is associated with complex traits and disease. Our data demonstrate how dynamic genetic effects on gene expression, which are likely relevant for disease, can be uncovered under stress.

SummaryLung organogenesis requires precisely timed shifts in the spatial organization and function of parenchymal cells, especially during the later stages of lung development. To investigate the mechanisms governing lung parenchymal dynamics during development, we performed a single cell RNA sequencing (scRNA-seq) time-series yielding 92,238 epithelial, endothelial, and mesenchymal cells across 8 time points from embryonic day 12 (E12) to postnatal day 14 (P14) in mice. We combined new computational analyses with RNAin situhybridization to explore transcriptional velocity, fate likelihood prediction, and spatiotemporal localization of cell populations during the transition between the saccular and alveolar stages. We interrogated this atlas to illustrate the complexity of type 1 pneumocyte function during the saccular and alveolar stages, and we demonstrate an integrated view of the cellular dynamics during lung development.

While transient fibroblast activation is a normal and adaptive aspect of injury-repair in many contexts, persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF) and other chronic fibrotic lung diseases. The mechanisms regulating persistent fibroblast activation in IPF have not been fully elucidated. In the lungs of IPF patients and in mice with experimental lung fibrosis, we observed that expression of the thromboxane-prostanoid receptor (TBXA2R) was increased in fibroblasts. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, suggesting an alternative ligand activates profibrotic TBXA2R signaling. We found that F2-isoprostanes (F2-IsoPs), nonezymatic products of arachidonic acid metabolism generated in the setting of reactive oxygen species (ROS), are persistently elevated in experimental lung fibrosis and act as an alternative TBXA2R ligand. Further studies demonstrated that F2-isoprostanes signal through TBXA2R to activate Smad signaling, revealing TBXA2R as a previously unrecognized regulator of the transforming growth factor beta (TGF-β) pathway. Further, treatment with the small-molecule TBXA2R antagonist ifetroban protected mice from lung fibrosis in three pre-clinical models: bleomycin treatment, Hermansky-Pudlak Syndrome, and radiation-induced fibrosis. Importantly, treatment with ifetroban during the fibrotic phase of bleomycin injury markedly enhanced fibrotic resolution. Together, these studies implicate TBXA2R signaling as a crucial mediator linking oxidative stress to fibroblast activation and indicate that TBXA2R antagonists could be efficacious for treatment of IPF and other chronic fibrotic disorders.

Previously published comparative functional genomic data sets from primates using frozen tissue samples, including many data sets from our own group, were often collected and analyzed using nonoptimal study designs and analysis approaches. In addition, when samples from multiple tissues were studied in a comparative framework, individuals and tissues were confounded. We designed a multitissue comparative study of gene expression and DNA methylation in primates that minimizes confounding effects by using a balanced design with respect to species, tissues, and individuals. We also developed a comparative analysis pipeline that minimizes biases attributable to sequence divergence. Thus, we present the most comprehensive catalog of similarities and differences in gene expression and DNA methylation levels between livers, kidneys, hearts, and lungs, in humans, chimpanzees, and rhesus macaques. We estimate that overall, interspecies and inter-tissue differences in gene expression levels can only modestly be accounted for by corresponding differences in promoter DNA methylation. However, the expression pattern of genes with conserved inter-tissue expression differences can be explained by corresponding interspecies methylation changes more often. Finally, we show that genes whose tissue-specific regulatory patterns are consistent with the action of natural selection are highly connected in both gene regulatory and protein-protein interaction networks., L.E.B. was supported by the National Science Foundation Graduate Research Fellowship (DGE-1144082). Additionally, L.E.B. and I.E. were funded by the National Institutes of Health (NIH) Genetics and Gene Regulation Training Grant (T32 GM07197). J.R. was funded by a Swiss National Science Foundation postdoc mobility fellowship (PBLAP3- 134342) and the FP7 Marie Curie International Outgoing Fellowship PRIMATE_REG_ EVOL. This project was funded in part by the Office of Research Infrastructure/Office of the Director (ORIP/OD) P51OD011132 grant. T.M.-B. is supported by BFU2017-86471-P (Ministry of Economy and Competitiveness/ European Regional Development Fund, European Union), NIH U01 MH106874 grant, Howard Hughes Medical Institute: International Early Career, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca and Centres de Recerca de Catalunya Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya

Recent advances in immunotherapy, particularly chimeric antigen receptor (CAR)-engineered T cell therapy, have shown promise for the treatment of many tumor types including progressive recurrent glioblastoma (GBM). While early phase clinical trials have illuminated the potential for CAR T cell therapy to effectively treat GBM, they have also highlighted the unique challenges regarding the efficacy and safety of immunotherapy for brain tumors, and many patients continue to progress during therapy. We seek to overcome these challenges and ultimately extend the time of survival for patients diagnosed with GBM by investigating the immune- and tumor-mediated mechanisms driving variation in response to CAR T cell therapy. We generated the first multi-omics time-series dataset of CAR T cells, endogenous immune cells, and tumor cells from 59 GBM patients treated with CAR T cell therapy. Using single cell RNA-sequencing and simultaneous quantification of nearly 200 cell surface proteins, we comprehensively profiled the cellular phenotypes and signaling pathways within tumor and circulating immune cells that are associated with treatment response. The combination of mRNA and protein expression allowed us to resolve cell states beyond what either modality was capable of alone. Additionally, we found differentially expressed genes and proteins between tumor biopsies collected before and after CAR T cell therapy as well as differential expression between pre-infusion CAR T cells and those identified within the tumor following infusion. By evaluating the CAR T cell phenotypes prior to and during treatment we sought to address the outstanding question of how intrinsic variability impacts the activity and persistence of CAR T cells and to determine the phenotypes that confer the greatest therapeutic benefit for patients with GBM. Our results have direct implications for precision medicine and future clinical trials investigating the use of CAR T cell therapy for GBM as well as other solid tumors.

BackgroundDespite its importance in health and disease, the cellular diversity of the lung endothelium has not been systematically characterized in humans. Here we provide a reference atlas of human lung endothelial cells (ECs), to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium, both in health and disease.MethodsWe reprocessed control single cell RNA sequencing (scRNAseq) data from five datasets of whole lungs that were used for the analysis of pan-endothelial markers, we later included a sixth dataset of sorted control EC for the vascular subpopulation analysis. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by immunohistochemistry andin situhybridization. Signaling network between different lung cell types was studied using connectomic analysis. For cross species analysis we applied the same methods to scRNAseq data obtained from mouse lungs.ResultsThe six lung scRNAseq datasets were reanalyzed and annotated to identify over 15,000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including pan-endothelial, pan-vascular and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial and venous ECs we found previously indistinguishable subpopulations; among venous EC we identified two previously indistinguishable populations, pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary EC we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1 and TBX2 and general capillary EC. We confirmed that all six endothelial cell types, including the systemic-venous EC and aerocytes are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-Receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. Our manuscript is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com).ConclusionOur integrated analysis provides the comprehensive and well-crafted reference atlas of lung endothelial cells in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Lack of diversity in human genomics limits our understanding of the genetic underpinnings of complex traits, hinders precision medicine, and contributes to health disparities. To map genetic effects on gene regulation in the underrepresented Indonesian population, we have integrated genotype, gene expression, and CpG methylation data from 115 participants across three island populations that capture the major sources of genomic diversity on the region. In a comparison with a European dataset, we identify 166 uniquely Indonesia-specific eQTLs, highlighting the benefits of performing association studies on non-European populations. By combining local ancestry and archaic introgression inference eQTLs and methylQTLs, we identify regulatory loci driven by modern Papuan ancestry as well as introgressed Denisovan and Neanderthal variation. GWAS colocalization connects QTLs detected here to hematological traits. Our findings illustrate how local ancestry and archaic introgression drive variation in gene regulation across genetically distinct and in admixed populations.

Single-cell RNA sequencing provides new insights into pathologic epithelial and mesenchymal remodeling in the human lung., Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5−/KRT17+ pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.

The SARS-CoV-2 novel coronavirus global pandemic (COVID-19) has led to millions of cases and hundreds of thousands of deaths around the globe. While the elderly appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of the developing mouse lung with temporally-resolved RNA-in-situ hybridization (ISH) in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primerTMPRSS2was highest in ciliated cells and type I alveolar epithelial cells (AT1), andTMPRSS2expression was increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases revealed SARS-CoV-2 RNA was detected most frequently in ciliated and secretory cells in the airway epithelium and AT1 cells in the peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressingTMPRSS2.Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in the lung epithelium, and suggest that developmental regulation ofTMPRSS2may underlie the relative protection of infants and children from severe respiratory illness.

BackgroundSingle-cell RNA-sequencing (scRNA-seq) is a rapidly evolving technology that enables measurement of gene expression levels at an unprecedented resolution. Despite the explosive growth in the number of cells that can be assayed by a single experiment, scRNA-seq still has several limitations, including high rates of dropouts, which result in a large number of genes having zero read count in the scRNA-seq data, and complicate downstream analyses.MethodsTo overcome this problem, we treat zeros as missing values and develop nonparametric deep learning methods for imputation. Specifically, our LATE (Learning with AuToEncoder) method trains an autoencoder with random initial values of the parameters, whereas our TRANSLATE (TRANSfer learning with LATE) method further allows for the use of a reference gene expression data set to provide LATE with an initial set of parameter estimates.ResultsOn both simulated and real data, LATE and TRANSLATE outperform existing scRNA-seq imputation methods, achieving lower mean squared error in most cases, recovering nonlinear gene-gene relationships, and better separating cell types. They are also highly scalable and can efficiently process over 1 million cells in just a few hours on a GPU.ConclusionsWe demonstrate that our nonparametric approach to imputation based on autoencoders is powerful and highly efficient.

Stem cell senescence is often seen as an age associated pathological state in which cells acquire an abnormal and irreversible state. Here, we show that alveolar stem cell differentiation during lung regeneration involves a unique previously uncharacterized transitional state that exhibits cardinal features normally associated with cell senescence. Specifically, using organoid cultures, multiple in vivo injury models coupled with single cell transcriptomics and lineage tracing analysis, we find that alveolar stem cell differentiation involves a novel, pre-alveolar type-1 transitional state (PATS) en route to their terminal maturation. PATS can be distinguished based on their unique transcriptional signatures, including enrichment for TP53, TGFβ, and DNA damage repair signaling, and cellular senescence in both in vivo and ex vivo regenerating tissues. Significantly, PATS undergo extensive cell stretching, which makes them vulnerable to DNA damage, a feature commonly associated with most degenerative lung diseases. Importantly, we find enrichment of PATS-like state in human fibrotic lung tissues, suggesting that persistence of such transitional states underlies the pathogenesis of pulmonary fibrosis. Our study thus redefines senescence as a state that can occur as part of a normal tissue maintenance program, and can be derailed in human disease, notably fibrosis.

The phylogenetic and adaptive factors that cause variation in primate facial form—including differences among the major primate clades and variation related to feeding and/or social behavior—are relatively well understood. However, comparatively little is known about the genetic mechanisms that underlie diversity in facial form in primates. Because it is essential for osteoblastic differentiation and skeletal development, the runt-related transcription factor 2 (Runx2) is one gene that may play a role in these genetic mechanisms. Specifically, polymorphisms in the QA ratio (determined by the ratio of the number of polyglutamines to polyalanines in one functional domain of Runx2) have been shown to be correlated with variation in facial length and orientation in other mammal groups. However, to date, the relationship between variation in this gene and variation in facial form in primates has not been explicitly tested. To test the hypothesis that the QA ratio is correlated with facial form in primates, the current study quantified the QA ratio, facial length, and facial angle in a sample of 33 primate species and tested for correlation using phylogenetic generalized least squares. The results indicate that the QA ratio of the Runx2 gene is positively correlated with variation in relative facial length in anthropoid primates. However, no correlation was found in strepsirrhines, and there was no correlation between facial angle and the QA ratio in any groups. These results suggest that, in primates, the QA ratio of the Runx2 gene may play a role in modulating facial size, but not facial orientation. This study therefore provides important clues about the genetic and developmental mechanisms that may underlie variation in facial form in primates.

CAR T cell therapy is emerging as a promising strategy to treat cancer and may offer new therapeutic options for individuals diagnosed with glioblastoma (GBM) and other solid tumors. While early clinical studies evaluating CAR T cell therapy in GBM have established evidence of safety and bioactivity, objective clinical responses have been limited. It remains unclear whether productive CAR T cell therapy for solid tumors requires solely CAR T cell engagement with tumor antigen, or if it also necessitates the stimulation of a patient's endogenous immune response. Focusing on our preclinical and clinical program evaluating IL13Rα2-targeted CAR-T cells for the treatment of IL13Rα2-positive glioblastoma (GBM), we set out to mechanistically interrogate the interplay between CAR T cell therapy and the host tumor microenvironment. We designed a murine CAR T cell syngeneic platform in C57BL/6 immunocompetent mice and demonstrate that single intratumoral infusion of IL13Rα2-CAR T cells mediate potent antitumor activity against established KR158 tumors, a highly invasive and poorly immunogenic murine glioma model. We demonstrate that CAR T cell treatment of mouse syngeneic GBM alters the tumor immune landscape, activates intratumoral myeloid cells and induces endogenous T cell memory responses coupled with feed forward propagation of CAR T responses. IFNγ production by CAR T cells and IFNγ-responsiveness of host immune cells is critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these findings was explored in patient samples from our on-going IL13Rα2-CAR T cell phase I clinical trial [NCT02208362]. Consistent with our preclinical findings, we show that locoregional CAR T cell infusions result in spikes in inflammatory cytokines and an influx of endogenous immune cells into the cerebrospinal fluid (CSF) and resected tumor cavity. Single cell RNA-sequencing revealed unique genes upregulated in immune cells from blood and CSF samples after treatment. One patient of particular interest, who presented with recurrent multifocal GBM, remarkably achieved a complete response (CR) following locoregional delivery of IL13Rα2-CAR T cells, despite heterogeneous IL13Rα2 tumor expression (PMID: 28029927). In this responding patient, we now show the induction of endogenous tumor-specific T cell reactivity and T cell clones whose dynamics contracted with tumor volume following IL13Rα2-targeted CAR T therapy. These studies establish that CAR T cell therapy has the potential to re-shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity and emphasize the importance of the host innate and adaptive immunity in productive CAR T cell therapy of solid tumors. Citation Format: Christine E. Brown, Darya Alizadeh, Vanessa Jonsson, Jonathan Hibbard, Stephanie Yahn, Robyn A. Wong, Xin Yang, Rachel Ng, Natalie Dullerud, Madeleine Maker, Sharahreh Gholamin, Renate Starr, Nicholas Banovich, Stephen J. Forman, Behnam Badie. CAR T cell therapy reshapes the tumor microenvironment to promote host antitumor immune repsonses in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 59.

Indonesia is the world’s fourth most populous country, host to striking levels of human diversity, regional patterns of admixture, and varying degrees of introgression from both Neanderthals and Denisovans. However, it has been largely excluded from the human genomics sequencing boom of the last decade. To serve as a benchmark dataset of molecular phenotypes across the region, we generated genome-wide CpG methylation and gene expression measurements in over 100 individuals from three locations that capture the major genomic and geographical axes of diversity across the Indonesian archipelago. Investigating between- and within-island differences, we find up to 10.55% of tested genes are differentially expressed between the islands of Sumba and New Guinea. Variation in gene expression is closely associated with DNA methylation, with expression levels of 9.80% of genes correlating with nearby promoter CpG methylation, and many of these genes being differentially expressed between islands. Genes identified in our differential expression and methylation analyses are enriched in pathways involved in immunity, highlighting Indonesia's tropical role as a source of infectious disease diversity and the strong selective pressures these diseases have exerted on humans. Finally, we identify robust within-island variation in DNA methylation and gene expression, likely driven by fine-scale environmental differences across sampling sites. Together, these results strongly suggest complex relationships between DNA methylation, transcription, archaic hominin introgression and immunity, all jointly shaped by the environment. This has implications for the application of genomic medicine, both in critically understudied Indonesia and globally, and will allow a better understanding of the interacting roles of genomic and environmental factors shaping molecular and complex phenotypes., Author summary Understanding how gene expression varies across individuals and environments is fundamental for the success of molecular medicine. However, the vast majority of studies of gene expression, particularly in healthy individuals, have primarily involved populations of European descent. It is unclear whether these findings are generalizable to the rest of humanity. We examined gene expression and DNA methylation in 116 individuals across Indonesia, the world's fourth most populous country. Our three populations–Mentawai off the western coast of Sumatra, Sumba in central Indonesia, and a group of hunter-gatherers, the Korowai, living in the province of Indonesian New Guinea–span the main axes of genetic and geographic diversity in the region. Up to 10% of genes show differences in expression and methylation patterns between islands, with many involved in immune function. Variation between villages within each location can likely be attributed to small-scale environmental differences. These findings emphasise the need to consider a broad range of genetic and environmental backgrounds when examining how molecular patterns and processes are shared across human populations.

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is required for understanding skewed immune responses and finding outcome predictors. Here, we performed a longitudinal multi-omics study using a two-centre cohort of 14 patients. We analysed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of IFN-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signalling. Megakaryocyte- and erythroid cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond classical immune cells and may serve as an entry point to develop biomarkers and targeted treatments of patients with COVID-19., Graphical Abstract, Highlights • SARS-CoV2 infection elicits dynamic changes of circulating cells in the blood • Severe COVID-19 is characterized by increased metabolically active plasmablasts • Elevation of IFN-activated megakaryocytes and erythroid cells in severe COVID-19 • Cell-type specific expression signatures are associated with a fatal COVID-19 outcome, Bernardes et al. explore COVID-19 disease trajectories by performing longitudinal multi-omics analyses in peripheral blood samples from hospitalized patients. The analyses identify increased numbers of plasmablasts, interferon-activated megakaryocytes and erythroid cells as hallmarks of severe disease, and define molecular signatures linked to a fatal COVID-19 disease outcome.