Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

Coexpression of CD71 and CD117 Identifies an Early Unipotent Neutrophil Progenitor Population in Human Bone Marrow.

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (Lin-CD66b+CD117+CD71+). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117+CD71+ eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.

Single-cell Transcriptional Landscape of Temporal Neutrophil Response to Burn Wound in Larval Zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. In this study, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-d time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found that neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds a comparative single-cell transcriptomic framework to identify neutrophil markers of tissue damage using model organisms.

A tessellated lymphoid network provides whole-body T cell surveillance in zebrafish.

Homeostatic trafficking to lymph nodes allows T cells to efficiently survey the host for cognate antigen. Nonmammalian jawed vertebrates lack lymph nodes but maintain diverse T cell pools. Here, we exploit in vivo imaging of transparent zebrafish to investigate how T cells organize and survey for antigen in an animal devoid of lymph nodes. We find that naïve-like T cells in zebrafish organize into a previously undescribed whole-body lymphoid network that supports streaming migration and coordinated trafficking through the host. This network has the cellular hallmarks of a mammalian lymph node, including naïve T cells and CCR7-ligand expressing nonhematopoietic cells, and facilitates rapid collective migration. During infection, T cells transition to a random walk that supports antigen-presenting cell interactions and subsequent activation. Our results reveal that T cells can toggle between collective migration and individual random walks to prioritize either large-scale trafficking or antigen search in situ. This lymphoid network thus facilitates whole-body T cell trafficking and antigen surveillance in the absence of a lymph node system.

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

Isoform-level quantification for single-cell RNA sequencing.

MOTIVATION: RNA expression at isoform level is biologically more informative than at gene level and can potentially reveal cellular subsets and corresponding biomarkers that are not visible at gene level. However, due to the strong 3' bias sequencing protocol, mRNA quantification for high-throughput single-cell RNA sequencing such as Chromium Single Cell 3' 10× Genomics is currently performed at the gene level. RESULTS: We have developed an isoform-level quantification method for high-throughput single-cell RNA sequencing by exploiting the concepts of transcription clusters and isoform paralogs. The method, called Scasa, compares well in simulations against competing approaches including Alevin, Cellranger, Kallisto, Salmon, Terminus and STARsolo at both isoform- and gene-level expression. The reanalysis of a CITE-Seq dataset with isoform-based Scasa reveals a subgroup of CD14 monocytes missed by gene-based methods. AVAILABILITY AND IMPLEMENTATION: Implementation of Scasa including source code, documentation, tutorials and test data supporting this study is available at Github: https://github.com/eudoraleer/scasa and Zenodo: https://doi.org/10.5281/zenodo.5712503. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pattern of F-18 FDG Uptake in Colon Cancer after Bacterial Cancer Therapy Using Engineered Salmonella Typhimurium: A Preliminary In Vivo Study.

Purpose: Bacterial cancer therapy (BCT) research using engineered Salmonella typhimurium has increased in recent years. 2-Deoxy-2[18F] fluoro-D-glucose positron emission tomography (FDG PET) is widely used in cancer patients to detect cancer, monitor treatment responses, and predict prognoses. The aim of this pilot study was to investigate FDG uptake patterns in a mouse tumor model after BCT. Procedures. BCT was performed via the intravenous injection of attenuated S. typhimurium (SLΔppGpp/lux) into female mice bearing a tumor (derived from CT26 murine colon cancer cells) in the right thigh. 18F-FDG PET images acquired before BCT and at different time points after BCT. In vivo bioluminescence imaging confirmed bacterial presence in the tumor. The tumor volume, standardized uptake value (SUV) of FDG (SUVmax and SUVmean), early SUV reduction%, and normalized tumor volume change were analyzed. Results: Early after BCT (1 or 2 days post-injection (dpi)), FDG tumor uptake decreased in 10 out of 11 mice and then increased at later stages. FDG uptake before BCT was correlated with normalized tumor volume change after BCT. Early FDG reduction% after BCT was correlated with normalized volume change after BCT. Conclusions: Early after BCT, FDG tumor uptake decreased and then increased at later stages. The higher the FDG tumor uptake before BCT, the better the BCT response. FDG uptake patterns were related to tumor volume change after BCT. Therefore, FDG uptake was a good candidate for evaluating BCT.

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2+ foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4, which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.

Atherosclerosis Impairs Naive CD4 T-Cell Responses via Disruption of Glycolysis.

Objective: CD4 T cells are important regulators of atherosclerotic progression. The metabolic profile of CD4 T cells controls their signaling and function, but how atherosclerosis affects T-cell metabolism is unknown. Here, we sought to determine the impact of atherosclerosis on CD4 T-cell metabolism and the contribution of such metabolic alterations to atheroprogression. Approach and Results: Using PCR arrays, we profiled the expression of metabolism genes in CD4 T cells from atherosclerotic apolipoprotein-E knockout mice fed a Western diet. These cells exhibited dysregulated expression of genes critically involved in glycolysis and fatty acid degradation, compared with those from animals fed a standard laboratory diet. We examined how T-cell metabolism was changed in either Western diet­fed apolipoprotein-E knockout mice or samples from patients with cardiovascular disease by measuring glucose uptake, activation, and proliferation in CD4 T cells. We found that naive CD4 T cells from Western diet­fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet­fed animals. Similarly, we observed that naive CD4 T-cell frequencies were reduced in the circulation of human subjects with high cardiovascular disease compared with low cardiovascular disease. Naive T cells from high cardiovascular disease subjects also showed reduced proliferative capacity. Conclusions: These results highlight the dysfunction that occurs in CD4 T-cell metabolism and immune responses during atherosclerosis. Targeting metabolic pathways within naive CD4 T cells could thus yield novel therapeutic approaches for improving CD4 T-cell responses against atheroprogression.

Learning Biological Dynamics From Spatio-Temporal Data by Gaussian Processes.

Model discovery methods offer a promising way to understand biology from data. We propose a method to learn biological dynamics from spatio-temporal data by Gaussian processes. This approach is essentially "equation free" and hence avoids model derivation, which is often difficult due to high complexity of biological processes. By exploiting the local nature of biological processes, dynamics can be learned with data sparse in time. When the length scales (hyperparameters) of the squared exponential covariance function are tuned, they reveal key insights of the underlying process. The squared exponential covariance function also simplifies propagation of uncertainty in multi-step forecasting. After evaluating the performance of the method on synthetic data, we demonstrate a case study on real image data of E. coli colony.

Gender-related differences in clinical outcomes after either single or double left main bifurcation stenting.

We sought to examine the impact of gender differences in clinical outcomes at 3 years also comparing the role of double versus single stenting approach for the treatment of coronary unprotected LM bifurcation lesions. We retrospectively analyzed both the procedural and medical data of patients referred to our hub center for complex LM bifurcation disease, treated using Crossover provisional stenting, T or T-and-Protrusion (TAP), Culotte, and Nano-inverted-T (NIT) techniques between January 1st, 2008 and May 1st 2018. The main outcome of the study was to evaluate the association between gender and target lesion failure (TLF) based on the different stenting technique used. Five hundred and sixty-seven patients (251 females, mean age 70.0 ± 10 years, mean Syntax score 31.6 ± 6.3) were evaluated. Crossover, T or TAP, culotte and NIT techniques were performed in 171 (30.1%), 61 (10.7%), 98 (17.2%) and 237 (41.8%) patients, respectively with no differences in baseline and peri-procedural items among gender. At a mean follow-up of 37.1 ± 10.8 months (range 22.1-39.3 moths), the overall TLF rate, cardiovascular mortality and stent thrombosis were 12.1%, 3.1% and 1.0%, respectively. Female gender was associated with an increased rate of major bleeding when treated with double stent strategy (p = 0.02). No gender difference in TLF was noted among gender, independently from the stenting approach used. Among patients with ULM bifurcation disease undergoing PCI, TLF rates were not different between genders at 3-year follow-up either using a single or double stent technique.

[Ortho-geriatric rehabilitation: challenges and impact]. / Réadaptation ortho-gériatrique : enjeux et impact.

The risk of developing functional dependence rises with age. Following an acute event, a rehabilitation stay is often needed to restore functional capacities and consider home discharge. The geriatric rehabilitation process usually involves standardized multidisciplinary management, setting and frequent reviewing of the goals, and a discharge plan. In a retrospective observational study conducted in orthogeriatric rehabilitation conducted in Geneva, comorbidities, functional status at admission, and length of stay appear to have a significant impact on recovery potential and destination at discharge, whereas the intensity of the rehabilitation program (number of therapies per week) does not influence patient outcome.

Le risque de développer une dépendance fonctionnelle augmente avec l'âge. À la suite d'un événement aigu, un séjour en réadaptation est souvent nécessaire pour restaurer les capacités fonctionnelles et permettre un retour à domicile. Le processus de réadaptation gériatrique comporte une prise en charge standardisée pluridisciplinaire, la fixation et la révision régulière d'objectifs et la planification de la sortie. Dans une étude observationnelle rétrospective en réadaptation ortho-gériatrique menée à Genève, les comorbidités, l'état fonctionnel à l'admission et la durée de séjour prédisent les chances de récupération fonctionnelle et le retour à domicile, tandis que l'intensité du programme (nombre de thérapies par semaine) n'influence pas le devenir du patient.

Effect of a Surfactant Mixture on Coalescence Occurring in Concentrated Emulsions: The Hole Nucleation Theory Revisited.

By conducting both a bottle test and isolate drop-drop experiments, we determine the coalescence rates of water droplets within water-in-oil emulsions stabilized by a large amount of Span 80 in the presence of Tween 20, a surfactant that acts as a demulsifier. Using a microscopic model based on a theory of hole nucleation, we establish an analytical formula that quantitatively predicts the coalescence frequency per unit area of droplets whose interfaces are fully covered by surfactant molecules. Despite its simplicity and the strong assumptions made for its derivation, this formula captures our experimental findings on Span 80-stabilized emulsions as well as other results, found in the literature, remarkably well on a wide range of water-in-crude oil systems.

Naive CD8+ T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

OBJECTIVE: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4+ T cells have been extensively studied in CVD, the importance of CD8+ T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8+ T (TN) cell population expressing CD95 (termed CD95+CD8+ stem cell memory T [CD8 TSCM] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95lo cells within the TN compartment. We found that CD8 TSCM cells positively correlated with CVD risk in humans, while CD8+ TN cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice also displayed respective 7- and 2-fold increases in CD8+ TSCM frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8+ TSCM cells were 1.7-fold increased in aortas from western diet fed ApoE-/- mice compared with normal laboratory diet-fed ApoE-/- mice. Importantly, transfer of TSCM cells into immune-deficient Rag.Ldlr recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis. CONCLUSIONS: CD8+ TSCM cells are increased in humans with high CVD. As these TSCM cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.

A physical phantom for amine chemical exchange saturation transfer (CEST) MRI.

OBJECTIVE: To develop a robust amine chemical exchange saturation transfer (CEST) physical phantom, validate the temporal stability, and create a supporting software for automatic image processing and quality assurance. MATERIALS AND METHODS: The phantom was designed as an assembled laser-cut acrylic rack and 18 vials of phantom solutions, prepared with different pHs, glycine concentrations, and gadolinium concentrations. We evaluated glycine concentrations using ultraviolet absorbance for 70 days and measured the pH, relaxation rates, and CEST contrast for 94 days after preparation. We used Spearman's correlation to determine if glycine degraded over time. Linear regression and Bland-Altman analysis were performed between baseline and follow-up measurements of pH and MRI properties. RESULTS: No degradation of glycine was observed (p > 0.05). The pH and MRI measurements stayed stable for 3 months and showed high consistency across time points (R2 = 1.00 for pH, R1, R2, and CEST contrast), which was further validated by the Bland-Altman plots. Examples of automatically generated reports are provided. DISCUSSION: We designed a physical phantom for amine CEST-MRI, which is easy to assemble and transfer, holds 18 different solutions, and has excellent short-term chemical and MRI stability. We believe this robust phantom will facilitate the development of novel sequences and cross-scanners validations.

Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma.

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

RATIONALE: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. OBJECTIVE: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis. METHODS AND RESULTS: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. CONCLUSIONS: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

Coalescence in concentrated emulsions: theoretical predictions and comparison with experimental bottle test behaviour.

Fusion between emulsion drops, also called coalescence, may be undesirable for storage or sought after depending on the desired application. In this latter case, a complete separation of the two liquids composing the emulsion is required. The same objective may be applicable to foams. We have performed bottle test experiments on a model system of water in oil (w/o) emulsion stabilized by high amounts of hydrophobic surfactant Span 80. We observe two regimes for emulsion separation: the first regime, which is fast and includes sedimentation of the water droplets, and the second regime, which exhibits a very dense and stable emulsion zone. We predict the initial thickness of the dense zone as a simple function of surfactant concentration and mean droplet size. From the assumption that the coalescence rate depends only on the area of the thin film between two contacted droplets, we quantitatively model the separation kinetics of the dense emulsion zone. Our results give rise to a simple method that allows measuring the coalescence frequency per unit area, only by monitoring bottle test experiments.

Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry.

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16+ nonclassical monocyte population and 4 subsets belong to the CD14+ classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan+CXCR6+ nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.

Identification of distinct mutational patterns and new driver genes in oesophageal squamous cell carcinomas and adenocarcinomas.

OBJECTIVES: Oesophageal squamous cell carcinoma (OSCC) and adenocarcinoma (OAC) are distinct cancers in terms of a number of clinical and epidemiological characteristics, complicating the design of clinical trials and biomarker developments. We analysed 1048 oesophageal tumour-germline pairs from both subtypes, to characterise their genomic features, and biological and clinical significance. DESIGN: Previously exome-sequenced samples were re-analysed to identify significantly mutated genes (SMGs) and mutational signatures. The biological functions of novel SMGs were investigated using cell line and xenograft models. We further performed whole-genome bisulfite sequencing and chromatin immunoprecipitation (ChIP)-seq to characterise epigenetic alterations. RESULTS: OSCC and OAC displayed nearly mutually exclusive sets of driver genes, indicating that they follow independent developmental paths. The combined sample size allowed the statistical identification of a number of novel subtype-specific SMGs, mutational signatures and prognostic biomarkers. Particularly, we identified a novel mutational signature similar to Catalogue Of Somatic Mutations In Cancer (COSMIC)signature 16, which has prognostic value in OSCC. Two newly discovered SMGs, CUL3 and ZFP36L2, were validated as important tumour-suppressors specific to the OSCC subtype. We further identified their additional loss-of-function mechanisms. CUL3 was homozygously deleted specifically in OSCC and other squamous cell cancers (SCCs). Notably, ZFP36L2 is associated with super-enhancer in healthy oesophageal mucosa; DNA hypermethylation in its super-enhancer reduced active histone markers in squamous cancer cells, suggesting an epigenetic inactivation of a super-enhancer-associated SCC suppressor. CONCLUSIONS: These data comprehensively contrast differences between OSCC and OAC at both genomic and epigenomic levels, and reveal novel molecular features for further delineating the pathophysiological mechanisms and treatment strategies for these cancers.