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Accurate and comprehensive extraction of information from high-dimensional single cell datasets necessitates faithful visualizations to assess biological populations. A state-of-the-art algorithm for non-linear dimension reduction, t-SNE, requires multiple heuristics and fails to produce clear representations of datasets when millions of cells are projected. We develop opt-SNE, an automated toolkit for t-SNE parameter selection that utilizes Kullback-Leibler divergence evaluation in real time to tailor the early exaggeration and overall number of gradient descent iterations in a dataset-specific manner. The precise calibration of early exaggeration together with opt-SNE adjustment of gradient descent learning rate dramatically improves computation time and enables high-quality visualization of large cytometry and transcriptomics datasets, overcoming limitations of analysis tools with hard-coded parameters that often produce poorly resolved or misleading maps of fluorescent and mass cytometry data. In summary, opt-SNE enables superior data resolution in t-SNE space and thereby more accurate data interpretation. Visualisation tools that use dimensionality reduction, such as t-SNE, provide poor visualisation on large data sets of millions of observations. Here the authors present opt-SNE, that automatically finds data set-tailored parameters for t-SNE to optimise visualisation and improve analysis.

Key Points Mammalian BET proteins, a class of transcriptional co-regulators that contain dual, mutually related bromodomain motifs and an extraterminal domain, are important in the control of networks of genes; these proteins bind to acetylated lysines in the histones of nucleosomal chromatin, recruit chromatin-modification enzymes to target promoters and function as co-activators or co-repressors of gene expression, depending on the context. New small-molecule inhibitors have recently been developed that disrupt the binding interface between the bromodomain and the acetylated lysine groups; the inhibitors have remarkable potency, selectivity and are well tolerated. They have recently been used as anticancer and anti-inflammatory agents. These developments are important because chromatin was not considered to be a druggable target; as a result of these new drugs, a whole field of new epigenetically targeted therapeutics has become available for investigation. As this field of therapeutics rapidly expands, several features of BET protein function will need to be considered, including possible redundancy among the closely related family members, the selectivity of next-generation agents for specific BET proteins, and possible undesirable consequences of systemic administration without cellular targeting. These side effects might include uncontrolled transcriptional derepression of genes, altered haematopoiesis, immunosuppression or reactivation of latent viruses. The bromodomain is a highly conserved motif found in proteins that interact with chromatin. Small molecules that inhibit bromodomain and extraterminal (BET) proteins have been described, and this Review examines these developments and discusses the implications for small molecule epigenetic targeting of chromatin networks in cancer. The bromodomain is a highly conserved motif of 110 amino acids that is bundled into four anti-parallel α-helices and found in proteins that interact with chromatin, such as transcription factors, histone acetylases and nucleosome remodelling complexes. Bromodomain proteins are chromatin 'readers'; they recruit chromatin-regulating enzymes, including 'writers' and 'erasers' of histone modification, to target promoters and to regulate gene expression. Conventional wisdom held that complexes involved in chromatin dynamics are not 'druggable' targets. However, small molecules that inhibit bromodomain and extraterminal (BET) proteins have been described. We examine these developments and discuss the implications for small molecule epigenetic targeting of chromatin networks in cancer.

Barrier tissues are populated by functionally plastic CD4 + resident memory T (T RM ) cells. Whether the barrier epithelium regulates CD4 + T RM cell locations, plasticity and activities remains unclear. Here we report that lung epithelial cells, including distinct surfactant protein C (SPC) low MHC high epithelial cells, function as anatomically-segregated and temporally-dynamic antigen presenting cells. In vivo ablation of lung epithelial MHC-II results in altered localization of CD4 + T RM cells. Recurrent encounters with cognate antigen in the absence of epithelial MHC-II leads CD4 + T RM cells to co-express several classically antagonistic lineage-defining transcription factors, changes their cytokine profiles, and results in dysregulated barrier immunity. In addition, lung epithelial MHC-II is needed for surface expression of PD-L1, which engages its ligand PD-1 to constrain lung CD4 + T RM cell phenotypes. Thus, we establish epithelial antigen presentation as a critical regulator of CD4 + T RM cell function and identify epithelial-CD4 + T RM cell immune interactions as core elements of barrier immunity. The maintenance of T resident memory (T RM ) cells within pulmonary tissues is incompletely understood. Here the authors show that antigen presentation by lung epithelial cells maintains function and phenotype of pulmonary T RM cells within specific locational niches.

Low levels of type I interferon (IFN-I) are thought to be a driving force for immune activation and T-cell exhaustion in HIV-1 infected individuals on combination antiretroviral therapy (cART), though the causative mechanisms for persistent IFN-I signaling have remained unclear. Here, we show Rev–CRM1-dependent nuclear export and peripheral membrane association of intron-containing HIV-1 RNA, independent of primary viral sequence or viral protein expression, is subject to sensing and signaling via MAVS, resulting in IFN-I-dependent pro-inflammatory responses in macrophages. Additionally, HIV-1 intron-containing-RNA-induced innate immune activation of macrophages leads to upregulation of inhibitory receptor expression and functional immune exhaustion of co-cultured T cells. Our findings suggest that persistent expression of HIV-1 intron-containing RNA in macrophages contributes to chronic immune activation and T-cell dysfunction and that use of HIV RNA expression inhibitors as adjunct therapy might abrogate aberrant inflammation and restore immune function in HIV-infected individuals on cART. Type I Interferon is thought to be a driving force for immune activation and T cell exhaustion during HIV infection. Here the authors show that intron-containing HIV RNA induces innate immune activation resulting in associated T cell dysfunction.

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

Displacement of Bromodomain and Extra-Terminal (BET) proteins from chromatin has promise for cancer and inflammatory disease treatments, but roles of BET proteins in metabolic disease remain unexplored. Small molecule BET inhibitors, such as JQ1, block BET protein binding to acetylated lysines, but lack selectivity within the BET family (Brd2, Brd3, Brd4, Brdt), making it difficult to disentangle contributions of each family member to transcriptional and cellular outcomes. Here, we demonstrate multiple improvements in pancreatic β-cells upon BET inhibition with JQ1 or BET-specific siRNAs. JQ1 (50-400 nM) increases insulin secretion from INS-1 cells in a concentration dependent manner. JQ1 increases insulin content in INS-1 cells, accounting for increased secretion, in both rat and human islets. Higher concentrations of JQ1 decrease intracellular triglyceride stores in INS-1 cells, a result of increased fatty acid oxidation. Specific inhibition of both Brd2 and Brd4 enhances insulin transcription, leading to increased insulin content. Inhibition of Brd2 alone increases fatty acid oxidation. Overlapping yet discrete roles for individual BET proteins in metabolic regulation suggest new isoform-selective BET inhibitors may be useful to treat insulin resistant/diabetic patients. Results imply that cancer and diseases of chronic inflammation or disordered metabolism are related through shared chromatin regulatory mechanisms.

Pulmonary TB that develops in immunocompetent adult humans is responsible for approximately 85% of the disease burden and is central for Mtb transmission. Most humans contain Mtb infection within primary granulomatous lesions, but in certain immunocompetent humans, containment fails, leading to hematogenous spread and active pulmonary disease with the formation of cavities that enable Mtb transmission via aerosols. To reveal lung-specific microenvironments conducive for Mtb survival and replication despite systemic immunity, we use fluorescence multiplex immunohistochemistry and spatial transcriptomic analyses of heterogenous TB lesions that uniquely form in the lungs of immunocompetent but TB-susceptible B6.Sst1S mice after hematogenous spread from the primary lesion. Initially, these secondary lung lesions manifested local adoptive immunity featuring tertiary lymphoid follicles similar to resistant B6 mice and contained primarily non- replicating bacilli. Following these early events, however, the B6.Sst1S mice uniquely demonstrate expansion of myeloid cell populations with the appearance of alternatively activated macrophages, dissolution of lymphoid follicles, and the accumulation of de- differentiated lung epithelial cells. These processes led to bronchogenic expansion, broncho- occlusion, and necrosuppurative pneumonia closely resembling advanced pulmonary tuberculosis in humans. To determine whether lung parenchymal cells or lung oxygenation were necessary for the pulmonary TB progression, we implanted lung and spleen fragments subcutaneously prior to the infection. The lung implants uniquely displayed the formation of the characteristic organized granulomas with necrosis and Mtb replication that paralleled TB progression in native lungs, demonstrating that the cellular composition of inflamed lung tissue, not oxygenation, is a critical determinant of pulmonary TB progression. Our data demonstrate that deleterious bi-directional interactions of aberrantly activated macrophages with the inflammation-injured lung resident cells determine lung vulnerability to virulent Mtb in immunocompetent hosts. Because these mechanisms enable Mtb transmission among humans via aerosols, they are likely evolutionary conserved and, therefore, represent appealing targets for host-directed TB therapies.

The human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression reveals a generalizable signature defining functional fetal liver hematopoietic stem cells and allows for the stratification of enrichment strategies with high translational potential. More precisely, our integrated analysis identifies CD201 (endothelial protein C receptor (EPCR), encoded by PROCR ) as a marker that can specifically enrich for engraftment potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers. As such, it serves as a useful resource for the field and forms the basis for further biological exploration of strategies to retain the engraftment potential of hematopoietic stem cells ex vivo or induce this potential during in vitro hematopoietic stem cell generation. During human embryonic development, haematopoietic stem cells of the foetal liver undergo expansion, while retaining engraftment capacity. Here the authors apply CITE-seq to profile single cells from a human foetal liver, identifying a molecular signature of engraftment potential

Adipose tissue fibrosis is regulated by the chronic and progressive metabolic imbalance caused by differences in caloric intake and energy expenditure. By exploring the cellular heterogeneity within fibrotic adipose tissue, we demonstrate that early adipocyte progenitor cells expressing both platelet-derived growth factor receptor (PDGFR) α and β are the major contributors to extracellular matrix deposition. We show that the fibrotic program is promoted by senescent macrophages. These macrophages were enriched in the fibrotic stroma and exhibit a distinct expression profile. Furthermore, we demonstrate that these cells display a blunted phagocytotic capacity and acquire a senescence-associated secretory phenotype. Finally, we determined that osteopontin, which was expressed by senescent macrophages in the fibrotic environment promoted progenitor cell proliferation, fibrotic gene expression, and inhibited adipogenesis. Our work reveals that obesity promotes macrophage senescence and provides a conceptual framework for the discovery of rational therapeutic targets for metabolic and inflammatory disease associated with obesity.

Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to COVID-19-ARDS. This secondary tissue injury arises from dysregulated neutrophils and neutrophil extracellular traps (NETs) intended to kill pathogens, but instead cause cell-injury. Insufficiency of pleiotropic therapeutic approaches delineate the need for inhibitors of dysregulated neutrophil-subset(s) that induce subset-specific apoptosis critical for neutrophil function-shutdown. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/VEGF-signal peptide receptor, DEspR, are apoptosis-resistant like DEspR+ cancer-cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID-19-ARDS. Here, we report the significant association of increased peripheral DEspR+CD11b+ neutrophil-counts with severity and mortality in ARDS and COVID-19-ARDS, and intravascular NET-formation, in contrast to DEspR[-] neutrophils. We detect DEspR+ neutrophils and monocytes in lung tissue patients in ARDS and COVID-19-ARDS, and increased neutrophil RNA-levels of DEspR ligands and modulators in COVID-19-ARDS scRNA-seq data-files. Unlike DEspR[-] neutrophils, DEspR+CD11b+ neutrophils exhibit delayed apoptosis, which is blocked by humanized anti-DEspR-IgG4 S228P antibody, hu6g8, in ex vivo assays. Ex vivo live-cell imaging of Rhesus-derived DEspR+CD11b+ neutrophils showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data identify DEspR+CD11b+ neutrophils as a targetable ‘rogue’ neutrophil-subset associated with severity and mortality in ARDS and COVID-19-ARDS.