Publications by authors named "Bernett Lee"

125 Publications

Large-scale cis- and trans-eQTL analyses identify thousands of genetic loci and polygenic scores that regulate blood gene expression.

Nat Genet 2021 09 2;53(9):1300-1310. Epub 2021 Sep 2.

Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland.

Trait-associated genetic variants affect complex phenotypes primarily via regulatory mechanisms on the transcriptome. To investigate the genetics of gene expression, we performed cis- and trans-expression quantitative trait locus (eQTL) analyses using blood-derived expression from 31,684 individuals through the eQTLGen Consortium. We detected cis-eQTL for 88% of genes, and these were replicable in numerous tissues. Distal trans-eQTL (detected for 37% of 10,317 trait-associated variants tested) showed lower replication rates, partially due to low replication power and confounding by cell type composition. However, replication analyses in single-cell RNA-seq data prioritized intracellular trans-eQTL. Trans-eQTL exerted their effects via several mechanisms, primarily through regulation by transcription factors. Expression of 13% of the genes correlated with polygenic scores for 1,263 phenotypes, pinpointing potential drivers for those traits. In summary, this work represents a large eQTL resource, and its results serve as a starting point for in-depth interpretation of complex phenotypes.
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http://dx.doi.org/10.1038/s41588-021-00913-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432599PMC
September 2021

A subset of Kupffer cells regulates metabolism through the expression of CD36.

Immunity 2021 Sep 31;54(9):2101-2116.e6. Epub 2021 Aug 31.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A(∗)STAR), Singapore 138648, Singapore.

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206ESAM population (KC1) and a minor CD206ESAM population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions.
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http://dx.doi.org/10.1016/j.immuni.2021.08.006DOI Listing
September 2021

Single cell analysis of M. tuberculosis phenotype and macrophage lineages in the infected lung.

J Exp Med 2021 Sep 22;218(9). Epub 2021 Jul 22.

Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY.

In this study, we detail a novel approach that combines bacterial fitness fluorescent reporter strains with scRNA-seq to simultaneously acquire the host transcriptome, surface marker expression, and bacterial phenotype for each infected cell. This approach facilitates the dissection of the functional heterogeneity of M. tuberculosis-infected alveolar (AMs) and interstitial macrophages (IMs) in vivo. We identify clusters of pro-inflammatory AMs associated with stressed bacteria, in addition to three different populations of IMs with heterogeneous bacterial phenotypes. Finally, we show that the main macrophage populations in the lung are epigenetically constrained in their response to infection, while inter-species comparison reveals that most AMs subsets are conserved between mice and humans. This conceptual approach is readily transferable to other infectious disease agents with the potential for an increased understanding of the roles that different host cell populations play during the course of an infection.
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http://dx.doi.org/10.1084/jem.20210615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8302446PMC
September 2021

Non-terminally exhausted tumor-resident memory HBV-specific T cell responses correlate with relapse-free survival in hepatocellular carcinoma.

Immunity 2021 08 15;54(8):1825-1840.e7. Epub 2021 Jul 15.

Singapore Immunology Network, A(∗)STAR, 8A Biomedical Grove, Singapore 138648, Singapore; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109, USA. Electronic address:

Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating T cells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8 T cell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific T cells were enriched for two Trm cell subsets that were PD-1TOX, despite being clonally expanded. High frequencies of intratumoral terminally exhausted T cells were uncommon. Patients with tumor-infiltrating HBV-specific CD8 Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8 Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.
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http://dx.doi.org/10.1016/j.immuni.2021.06.013DOI Listing
August 2021

FUT6 deficiency compromises basophil function by selectively abrogating their sialyl-Lewis x expression.

Commun Biol 2021 07 2;4(1):832. Epub 2021 Jul 2.

Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore.

Sialyl-Lewis x (sLe, CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. Here we show using flow cytometry that sLe expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (FUT6). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the FUT6 gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional FUT6 gene harbor a mixed population of sLe and sLe basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated FUT6-deficient basophils rolling on E-selectin is severely impaired. FUT6 null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites. FUT6-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts.
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http://dx.doi.org/10.1038/s42003-021-02295-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253766PMC
July 2021

Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma.

Int J Mol Sci 2021 Jun 14;22(12). Epub 2021 Jun 14.

Singapore Immunology Network, A*STAR, Singapore, 8A Biomedical Grove Level 3 & 4 Immunos Building, Singapore 138648, Singapore.

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1β inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.
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http://dx.doi.org/10.3390/ijms22126350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231859PMC
June 2021

A multi-omics investigation of the composition and function of extracellular vesicles along the temporal trajectory of COVID-19.

Nat Metab 2021 07 22;3(7):909-922. Epub 2021 Jun 22.

State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

Exosomes represent a subtype of extracellular vesicle that is released through retrograde transport and fusion of multivesicular bodies with the plasma membrane. Although no perfect methodologies currently exist for the high-throughput, unbiased isolation of pure plasma exosomes, investigation of exosome-enriched plasma fractions of extracellular vesicles can confer a glimpse into the endocytic pathway on a systems level. Here we conduct high-coverage lipidomics with an emphasis on sterols and oxysterols, and proteomic analyses of exosome-enriched extracellular vesicles (EVs hereafter) from patients at different temporal stages of COVID-19, including the presymptomatic, hyperinflammatory, resolution and convalescent phases. Our study highlights dysregulated raft lipid metabolism that underlies changes in EV lipid membrane anisotropy that alter the exosomal localization of presenilin-1 (PS-1) in the hyperinflammatory phase. We also show in vitro that EVs from different temporal phases trigger distinct metabolic and transcriptional responses in recipient cells, including in alveolar epithelial cells, which denote the primary site of infection, and liver hepatocytes, which represent a distal secondary site. In comparison to the hyperinflammatory phase, EVs from the resolution phase induce opposing effects on eukaryotic translation and Notch signalling. Our results provide insights into cellular lipid metabolism and inter-tissue crosstalk at different stages of COVID-19 and are a resource to increase our understanding of metabolic dysregulation in COVID-19.
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http://dx.doi.org/10.1038/s42255-021-00425-4DOI Listing
July 2021

Immune cell phenotypes associated with disease severity and long-term neutralizing antibody titers after natural dengue virus infection.

Cell Rep Med 2021 May 18;2(5):100278. Epub 2021 May 18.

Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore.

Prior immunological exposure to dengue virus can be both protective and disease-enhancing during subsequent infections with different dengue virus serotypes. We provide here a systematic, longitudinal analysis of B cell, T cell, and antibody responses in the same patients. Antibody responses as well as T and B cell activation differentiate primary from secondary responses. Hospitalization is associated with lower frequencies of activated, terminally differentiated T cells and higher percentages of effector memory CD4 T cells. Patients with more severe disease tend to have higher percentages of plasmablasts. This does not translate into long-term antibody titers, since neutralizing titers after 6 months correlate with percentages of specific memory B cells, but not with acute plasmablast activation. Overall, our unbiased analysis reveals associations between cellular profiles and disease severity, opening opportunities to study immunopathology in dengue disease and the potential predictive value of these parameters.
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http://dx.doi.org/10.1016/j.xcrm.2021.100278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8149372PMC
May 2021

Persistent Symptoms and Association With Inflammatory Cytokine Signatures in Recovered Coronavirus Disease 2019 Patients.

Open Forum Infect Dis 2021 Jun 2;8(6):ofab156. Epub 2021 Apr 2.

National Centre for Infectious Diseases, Singapore.

Background: The complications and sequelae of coronavirus disease 2019 (COVID-19) and their effect on long-term health are unclear, and the trajectory of associated immune dysregulation is poorly understood.

Methods: We conducted a prospective longitudinal multicenter cohort study at 4 public hospitals in Singapore. Patients with COVID-19 were monitored for a median of 6 months after recovery from acute infection. Clinical symptoms and radiologic data were collected, along with plasma samples for quantification of immune mediators. The relationship between clinical symptoms and immune cytokine profiles was investigated.

Results: Two hundred eighty-eight participants were recruited, and follow-up data were available for 183, 175, and 120 participants at days 30, 90, and 180 postsymptom onset, respectively. Symptoms related to COVID-19 were present in 31 (16.9%), 13 (7.4%), and 14 (11.7%) at days 30, 90, and 180. In a multivariable model, age >65 years, non-Chinese ethnicity, and the severity of acute infection were associated with increased likelihood of persistent symptoms. Recovered COVID-19 patients had elevated levels of proinflammatory interleukin (IL)-17A, stem cell factor, IL-12p70, and IL-1β and pro-angiogenic macrophage inflammatory protein 1β, brain-derived neurotrophic factor, and vascular endothelial growth factor at day 180 compared with healthy controls. Higher levels of monocyte chemoattractant protein-1 and platelet-derived growth factor-BB were detected in patients with persistent symptoms, versus symptom-free patients.

Conclusions: Approximately 10% of recovered patients had persistent symptoms 6 months after initial infection. Immune cytokine signatures of the recovered patients reflected ongoing chronic inflammation and angiogenesis. Patients with COVID-19 should be monitored closely for emerging long-term health consequences.
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http://dx.doi.org/10.1093/ofid/ofab156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083585PMC
June 2021

Asymptomatic COVID-19: disease tolerance with efficient anti-viral immunity against SARS-CoV-2.

EMBO Mol Med 2021 06 27;13(6):e14045. Epub 2021 May 27.

A*STAR Infectious Diseases Labs (A*STAR ID Labs), Agency for Science, Technology and Research, Singapore City, Singapore.

The immune responses and mechanisms limiting symptom progression in asymptomatic cases of SARS-CoV-2 infection remain unclear. We comprehensively characterized transcriptomic profiles, cytokine responses, neutralization capacity of antibodies, and cellular immune phenotypes of asymptomatic patients with acute SARS-CoV-2 infection to identify potential protective mechanisms. Compared to symptomatic patients, asymptomatic patients had higher counts of mature neutrophils and lower proportion of CD169 expressing monocytes in the peripheral blood. Systemic levels of pro-inflammatory cytokines were also lower in asymptomatic patients, accompanied by milder pro-inflammatory gene signatures. Mechanistically, a more robust systemic Th2 cell signature with a higher level of virus-specific Th17 cells and a weaker yet sufficient neutralizing antibody profile against SARS-CoV-2 was observed in asymptomatic patients. In addition, asymptomatic COVID-19 patients had higher systemic levels of growth factors that are associated with cellular repair. Together, the data suggest that asymptomatic patients mount less pro-inflammatory and more protective immune responses against SARS-CoV-2 indicative of disease tolerance. Insights from this study highlight key immune pathways that could serve as therapeutic targets to prevent disease progression in COVID-19.
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http://dx.doi.org/10.15252/emmm.202114045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185544PMC
June 2021

Association of SARS-CoV-2 clades with clinical, inflammatory and virologic outcomes: An observational study.

EBioMedicine 2021 Apr 8;66:103319. Epub 2021 Apr 8.

Singapore Ministry of Health, Singapore; Saw Swee Hock School of Public Health, National University of Singapore, Singapore. Electronic address:

Background: Host determinants of severe coronavirus disease 2019 include advanced age, comorbidities and male sex. Virologic factors may also be important in determining clinical outcome and transmission rates, but limited patient-level data is available.

Methods: We conducted an observational cohort study at seven public hospitals in Singapore. Clinical and laboratory data were collected and compared between individuals infected with different SARS-CoV-2 clades. Firth's logistic regression was used to examine the association between SARS-CoV-2 clade and development of hypoxia, and quasi-Poisson regression to compare transmission rates. Plasma samples were tested for immune mediator levels and the kinetics of viral replication in cell culture were compared.

Findings: 319 patients with PCR-confirmed SARS-CoV-2 infection had clinical and virologic data available for analysis. 29 (9%) were infected with clade S, 90 (28%) with clade L/V, 96 (30%) with clade G (containing D614G variant), and 104 (33%) with other clades 'O' were assigned to lineage B.6. After adjusting for age and other covariates, infections with clade S (adjusted odds ratio (aOR) 0·030 (95% confidence intervals (CI): 0·0002-0·29)) or clade O (B·6) (aOR 0·26 (95% CI 0·064-0·93)) were associated with lower odds of developing hypoxia requiring supplemental oxygen compared with clade L/V. Patients infected with clade L/V had more pronounced systemic inflammation with higher concentrations of pro-inflammatory cytokines, chemokines and growth factors. No significant difference in the severity of clade G infections was observed (aOR 0·95 (95% CI: 0·35-2·52). Though viral loads were significantly higher, there was no evidence of increased transmissibility of clade G, and replicative fitness in cell culture was similar for all clades.

Interpretation: Infection with clades L/V was associated with increased severity and more systemic release of pro-inflammatory cytokines. Infection with clade G was not associated with changes in severity, and despite higher viral loads there was no evidence of increased transmissibility.
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http://dx.doi.org/10.1016/j.ebiom.2021.103319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027908PMC
April 2021

Bayesian analysis of cytokines and chemokine identifies immune pathways of HBsAg loss during chronic hepatitis B treatment.

Sci Rep 2021 04 2;11(1):7455. Epub 2021 Apr 2.

Translational Immunology Programme, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology, and Research, Singapore (A*STAR) Research Entities (RE), 61 Biopolis Drive, Proteos, Singapore, 138673, Singapore.

Our objective was to examine differences in cytokine/chemokine response in chronic hepatitis B(CHB) patients to understand the immune mechanism of HBsAg loss (functional cure) during antiviral therapy. We used an unbiased machine learning strategy to unravel the immune pathways in CHB nucleo(t)side analogue-treated patients who achieved HBsAg loss with peg-interferon-α(peg-IFN-α) add-on or switch treatment in a randomised clinical trial. Cytokines/chemokines from plasma were compared between those with/without HBsAg loss, at baseline, before and after HBsAg loss. Peg-IFN-α treatment resulted in higher levels of IL-27, IL-12p70, IL-18, IL-13, IL-4, IL-22 and GM-CSF prior to HBsAg loss. Probabilistic network analysis of cytokines, chemokines and soluble factors suggested a dynamic dendritic cell driven NK and T cell immune response associated with HBsAg loss. Bayesian network analysis showed a dominant myeloid-driven type 1 inflammatory response with a MIG and I-TAC central module contributing to HBsAg loss in the add-on arm. In the switch arm, HBsAg loss was associated with a T cell activation module exemplified by high levels of CD40L suggesting T cell activation. Our findings show that more than one immune pathway to HBsAg loss was found with peg-IFN-α therapy; by myeloid-driven Type 1 response in one instance, and T cell activation in the other.
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http://dx.doi.org/10.1038/s41598-021-86836-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018960PMC
April 2021

Use of nanopore sequencing to characterize african horse sickness virus (AHSV) from the African horse sickness outbreak in thailand in 2020.

Transbound Emerg Dis 2021 Mar 7. Epub 2021 Mar 7.

Center for Animal & Veterinary Sciences, Professional and Scientific Services, Animal and Veterinary Service, National Parks Board (NParks), Singapore.

African horse sickness (AHS) is a highly infectious and deadly disease despite availability of vaccines. Molecular characterization of African horse sickness virus (AHSV) detected from the March 2020 Thailand outbreak was carried out by whole-genome sequencing using Nanopore with a Sequence-Independent Single Primer Amplification (SISPA) approach. Nucleotide sequence of the whole genome was compared with closest matching AHSV strains using phylogenetic analyses and the AHSV-1 virus shared high sequence identity with isolates from the same outbreak. Substitution analysis revealed non-synonymous and synonymous substitutions in the VP2 gene as compared to circulating South African strains. The use of sequencing technologies, such as Nanopore with SISPA, has enabled rapid detection, identification and detailed genetic characterization of the AHS virus for informed decision-making and implementation of disease control measures. Active genetic information sharing has also allowed emergence of AHSV to be better monitored on a global basis.
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http://dx.doi.org/10.1111/tbed.14056DOI Listing
March 2021

Human neutralising antibodies elicited by SARS-CoV-2 non-D614G variants offer cross-protection against the SARS-CoV-2 D614G variant.

Clin Transl Immunology 2021 22;10(2):e1241. Epub 2021 Feb 22.

ASTAR Infectious Diseases Labs Agency for Science, Technology and Research (A*STAR) Singapore.

Objectives: The emergence of a SARS-CoV-2 variant with a point mutation in the spike (S) protein, D614G, has taken precedence over the original Wuhan isolate by May 2020. With an increased infection and transmission rate, it is imperative to determine whether antibodies induced against the D614 isolate may cross-neutralise against the G614 variant.

Methods: Antibody profiling against the SARS-CoV-2 S protein of the D614 variant by flow cytometry and assessment of neutralising antibody titres using pseudotyped lentiviruses expressing the SARS-CoV-2 S protein of either the D614 or G614 variant tagged with a luciferase reporter were performed on plasma samples from COVID-19 patients with known D614G status ( = 44 infected with D614,  = 6 infected with G614,  = 7 containing all other clades: O, S, L, V, G, GH or GR).

Results: Profiling of the anti-SARS-CoV-2 humoral immunity reveals similar neutralisation profiles against both S protein variants, albeit waning neutralising antibody capacity at the later phase of infection. Of clinical importance, patients infected with either the D614 or G614 clade elicited a similar degree of neutralisation against both pseudoviruses, suggesting that the D614G mutation does not impact the neutralisation capacity of the elicited antibodies.

Conclusions: Cross-reactivity occurs at the functional level of the humoral response on both the S protein variants, which suggests that existing serological assays will be able to detect both D614 and G614 clades of SARS-CoV-2. More importantly, there should be negligible impact towards the efficacy of antibody-based therapies and vaccines that are currently being developed.
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http://dx.doi.org/10.1002/cti2.1241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899292PMC
February 2021

A constant pool of Lgr5 intestinal stem cells is required for intestinal homeostasis.

Cell Rep 2021 01;34(4):108633

A(∗)STAR Institute of Medical Biology, Singapore, Singapore; A(∗)STAR Institute of Molecular and Cell Biology, Singapore, Singapore; Division of Epithelial Stem Cell Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. Electronic address:

Lgr5 crypt base columnar cells, the operational intestinal stem cells (ISCs), are thought to be dispensable for small intestinal (SI) homeostasis. Using a Lgr5-2A-DTR (diphtheria toxin receptor) model, which ablates Lgr5 cells with near-complete efficiency and retains endogenous levels of Lgr5 expression, we show that persistent depletion of Lgr5 ISCs in fact compromises SI epithelial integrity and reduces epithelial turnover in vivo. In vitro, Lgr5-2A-DTR SI organoids are unable to establish or survive when Lgr5 ISCs are continuously eliminated by adding DT to the media. However, transient exposure to DT at the start of culture allows organoids to form, and the rate of outgrowth reduces with the increasing length of DT presence. Our results indicate that intestinal homeostasis requires a constant pool of Lgr5 ISCs, which is supplied by rapidly reprogrammed non-Lgr5 crypt populations when preexisting Lgr5 ISCs are ablated.
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http://dx.doi.org/10.1016/j.celrep.2020.108633DOI Listing
January 2021

Sensitive detection of total anti-Spike antibodies and isotype switching in asymptomatic and symptomatic individuals with COVID-19.

Cell Rep Med 2021 Feb 16;2(2):100193. Epub 2021 Jan 16.

Infectious Diseases Laboratories (ID Labs), Agency for Science, Technology and Research (A∗STAR), Immunos, Biopolis, Singapore 138648, Singapore.

Early detection of infection is crucial to limit the spread of coronavirus disease 2019 (COVID-19). Here we develop a flow cytometry-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein antibodies in individuals with COVID-19. The assay detects specific immunoglobulin M (IgM), IgA, and IgG in individuals with COVID-19 and also acquisition of all IgG subclasses, with IgG1 being the most dominant. The antibody response is significantly higher at a later stage of infection. Furthermore, asymptomatic individuals with COVID-19 also develop specific IgM, IgA, and IgG, with IgG1 being the most dominant subclass. Although the antibody levels are lower in asymptomatic infection, the assay is highly sensitive and detects 97% of asymptomatic infections. These findings demonstrate that the assay can be used for serological analysis of symptomatic and asymptomatic infections, which may otherwise remain undetected.
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http://dx.doi.org/10.1016/j.xcrm.2021.100193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816583PMC
February 2021

T-Cell Lymphoma Clonality by Copy Number Variation Analysis of T-Cell Receptor Genes.

Cancers (Basel) 2021 Jan 19;13(2). Epub 2021 Jan 19.

Department of Pathology, National University Hospital, National University Health System, Singapore 119074, Singapore.

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, = 44) and extranodal NK/T-cell lymphomas (ENKTLs, = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.
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http://dx.doi.org/10.3390/cancers13020340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832336PMC
January 2021

Human Tumor-Infiltrating MAIT Cells Display Hallmarks of Bacterial Antigen Recognition in Colorectal Cancer.

Cell Rep Med 2020 Jun 23;1(3):100039. Epub 2020 Jun 23.

Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Growing evidence indicates a role for the gut microbiota in modulating anti-tumor treatment efficacy in human cancer. Here we study mucosa-associated invariant T (MAIT) cells to look for evidence of bacterial antigen recognition in human colon, lung, and kidney carcinomas. Using mass cytometry and single-cell mRNA sequencing, we identify a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3 and observe high expression of CD39 on MAIT cells from colorectal cancer (CRC) only, which we show to be expressed specifically after TCR stimulation. We further reveal that these cells are phenotypically and functionally exhausted. Sequencing data show high bacterial infiltration in CRC tumors and highlight an enriched species, with capability to activate MAIT cells in a TCR-dependent way. Our results provide evidence of a MAIT cell response to microbial antigens in CRC and could pave the way for manipulating MAIT cells or the microbiome for cancer therapy.
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http://dx.doi.org/10.1016/j.xcrm.2020.100039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659584PMC
June 2020

Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early marker for severe COVID-19.

Nat Commun 2020 10 16;11(1):5243. Epub 2020 Oct 16.

Infectious Disease Horizontal Technology Center, Agency for Science, Technology and Research, Immunos, Biopolis, 138648, Singapore.

SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.
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http://dx.doi.org/10.1038/s41467-020-19080-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568554PMC
October 2020

Metformin enhances anti-mycobacterial responses by educating CD8+ T-cell immunometabolic circuits.

Nat Commun 2020 10 16;11(1):5225. Epub 2020 Oct 16.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, 138648, Singapore.

Patients with type 2 diabetes (T2D) have a lower risk of Mycobacterium tuberculosis infection, progression from infection to tuberculosis (TB) disease, TB morality and TB recurrence, when being treated with metformin. However, a detailed mechanistic understanding of these protective effects is lacking. Here, we use mass cytometry to show that metformin treatment expands a population of memory-like antigen-inexperienced CD8CXCR3 T cells in naive mice, and in healthy individuals and patients with T2D. Metformin-educated CD8 T cells have increased (i) mitochondrial mass, oxidative phosphorylation, and fatty acid oxidation; (ii) survival capacity; and (iii) anti-mycobacterial properties. CD8 T cells from Cxcr3 mice do not exhibit this metformin-mediated metabolic programming. In BCG-vaccinated mice and guinea pigs, metformin enhances immunogenicity and protective efficacy against M. tuberculosis challenge. Collectively, these results demonstrate an important function of CD8 T cells in metformin-derived host metabolic-fitness towards M. tuberculosis infection.
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http://dx.doi.org/10.1038/s41467-020-19095-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567856PMC
October 2020

Monocytic Myeloid-Derived Suppressor Cells Underpin Resistance to Adoptive T Cell Therapy in Nasopharyngeal Carcinoma.

Mol Ther 2021 02 30;29(2):734-743. Epub 2020 Sep 30.

Institute of Molecular and Cell Biology, A∗STAR, Singapore 138673, Singapore; Tessa Therapeutics, Singapore 038982, Singapore; Institute of Biomedical Studies, Baylor University, Waco, TX 76712, USA. Electronic address:

Advanced, late-stage Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) is incurable, and its treatment remains a clinical and therapeutic challenge. Results from a phase II clinical trial in advanced NPC patients employing a combined chemotherapy and EBV-specific T cell (EBVST) immunotherapy regimen showed a response rate of 71.4%. Longitudinal analysis of patient samples showed that an increase in EBV DNA plasma concentrations and the peripheral monocyte-to-lymphocyte ratio negatively correlated with overall survival. These parameters were combined into a multivariate analysis to stratify patients according to risk of death. Immunophenotyping at serial time points showed that low-risk individuals displayed significantly decreased amounts of monocytic myeloid-derived suppressor cells postchemotherapy, which subsequently influenced successful cytotoxic T-lymphocyte (CTL) immunotherapy. Examination of the low-risk group, 2 weeks post-EBVST infusion, showed that individuals with a greater overall survival possessed an increased frequency of CD8 central and effector memory T cells, together with higher levels of plasma interferon (IFN)-γ, and cytotoxic lymphocyte-associated transcripts. These results highlight the importance of the rational selection of chemotherapeutic agents and consideration of their impact on both systemic immune responses and downstream cellular immunotherapy outcomes.
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http://dx.doi.org/10.1016/j.ymthe.2020.09.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854281PMC
February 2021

Systematic analysis of disease-specific immunological signatures in patients with febrile illness from Saudi Arabia.

Clin Transl Immunology 2020 22;9(8):e1163. Epub 2020 Aug 22.

Singapore Immunology Network Agency for Science, Technology and Research (ASTAR) Singapore.

Objectives: Little is known about the prevalence of febrile illness in the Arabian region as clinical, laboratory and immunological profiling remains largely uncharacterised.

Methods: A total of 2018 febrile patients from Jazan, Saudi Arabia, were recruited between 2014 and 2017. Patients were screened for dengue and chikungunya virus, , , , group A streptococcus and . Clinical history and biochemical parameters from blood tests were collected. Patient sera of selected disease-confirmed infections were quantified for immune mediators by multiplex microbead-based immunoassays.

Results: Approximately 20% of febrile patients were tested positive for one of the pathogens, and they presented overlapping clinical and laboratory parameters. Nonetheless, eight disease-specific immune mediators were identified as potential biomarkers for dengue (MIP-1α, MCP-1), malaria (TNF-α), streptococcal and meningococcal (eotaxin, GRO-α, RANTES, SDF-1α and PIGF-1) infections, with high specificity and sensitivity profiles. Notably, based on the conditional inference model, six of these mediators (MIP-1α, TNF-α, GRO-α, RANTES, SDF-1α and PIGF-1) were revealed to be 68.4% accurate in diagnosing different febrile infections, including those of unknown diseases.

Conclusions: This study is the first extensive characterisation of the clinical analysis and immune biomarkers of several clinically important febrile infections in Saudi Arabia. Importantly, an immune signature with robust accuracy, specificity and sensitivity in differentiating several febrile infections was identified, providing useful insights into patient disease management in the Arabian Peninsula.
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http://dx.doi.org/10.1002/cti2.1163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443187PMC
August 2020

Viral dynamics and immune correlates of COVID-19 disease severity.

Clin Infect Dis 2020 Aug 28. Epub 2020 Aug 28.

National Centre for Infectious Diseases, Singapore.

Background: Key knowledge gaps remain in the understanding of viral dynamics and immune response of SARS-CoV-2 infection.

Methods: We evaluated these characteristics and established their association with clinical severity in a prospective observational cohort study of 100 patients with PCR-confirmed SARS-CoV-2 infection (mean age 46 years, 56% male, 38% with comorbidities). Respiratory samples (n=74) were collected for viral culture, serum samples for measurement of IgM/IgG levels (n=30), and plasma samples for levels of inflammatory cytokines and chemokines (n=81). Disease severity was correlated with results from viral culture, serologic testing, and immune markers.

Results: 57 (57%) patients developed viral pneumonia, of whom 20 (20%) required supplemental oxygen including 12 (12%) invasive mechanical ventilation. Viral culture from respiratory samples was positive for 19 of 74 patients (26%). No virus was isolated when the PCR cycle threshold (Ct) value was >30 or >14 days after symptom onset. Seroconversion occurred at a median of 12.5 days (IQR 9-18) for IgM and 15.0 days (IQR 12-20) for IgG; 54/62 patients (87.1%) sampled at day 14 or later seroconverted. Severe infections were associated with earlier seroconversion and higher peak IgM and IgG levels. Levels of IP-10, HGF, IL-6, MCP-1, MIP-1α, IL-12p70, IL-18, VEGF-A, PDGF-BB and IL-1RA significantly correlated with disease severity.

Conclusion: We found virus viability was associated with lower PCR Ct value in early illness. A stronger antibody response was associated with disease severity. The overactive proinflammatory immune signatures offers targets for host-directed immunotherapy which should be evaluated in randomised controlled trials.
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http://dx.doi.org/10.1093/cid/ciaa1280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499509PMC
August 2020

Immunohistochemical scoring of CD38 in the tumor microenvironment predicts responsiveness to anti-PD-1/PD-L1 immunotherapy in hepatocellular carcinoma.

J Immunother Cancer 2020 08;8(2)

Division of Pathology, Singapore General Hospital, Singapore

Introduction: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality globally. Immune-checkpoint blockade (ICB) is one of the systemic therapy options for HCC. However, response rates remain low, necessitating robust predictive biomarkers. In the present study, we examined the expression of CD38, a molecule involved in the immunosuppressive adenosinergic pathway, on immune cells present in the tumor microenvironment. We then investigated the association between CD38 and ICB treatment outcomes in advanced HCC.

Methods: Clinically annotated samples from 49 patients with advanced HCC treated with ICB were analyzed for CD38 expression using immunohistochemistry (IHC), multiplex immunohistochemistry/immunofluorescence (mIHC/IF) and multiplex cytokine analysis.

Results: IHC and mIHC/IF analyses revealed that higher intratumoral CD38 cell proportion was strongly associated with improved response to ICB. The overall response rates to ICB was significantly higher among patients with high proportion of total CD38cells compared with patients with low proportion (43.5% vs 3.9%, p=0.019). Higher responses seen among patients with a high intratumoral CD38cell proportion translated to a longer median progression-free survival (mPFS, 8.21 months vs 1.64 months, p=0.0065) and median overall survival (mOS, 19.06 months vs 9.59 months, p=0.0295). Patients with high CD38CD68macrophage density had a better mOS of 34.43 months compared with 9.66 months in patients with low CD38CD68 macrophage density. CD38 macrophages produce more interferon γ (IFN-γ) and related cytokines, which may explain its predictive value when treated with ICB.

Conclusions: A high proportion of CD38 cells, determined by IHC, predicts response to ICB and is associated with superior mPFS and OS in advanced HCC.
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http://dx.doi.org/10.1136/jitc-2020-000987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451957PMC
August 2020

Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study.

Lancet 2020 08 18;396(10251):603-611. Epub 2020 Aug 18.

Infectious Diseases Horizontal Technology Centre, Agency for Science, Technology, and Research, Singapore; Singapore Immunology Network, Agency for Science, Technology, and Research, Singapore. Electronic address:

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection.

Methods: We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study-a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed.

Findings: Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14-28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00-0·48]) compared with infection with wild-type virus only.

Interpretation: The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines.

Funding: National Medical Research Council Singapore.
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http://dx.doi.org/10.1016/S0140-6736(20)31757-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434477PMC
August 2020

Linear B-cell epitopes in the spike and nucleocapsid proteins as markers of SARS-CoV-2 exposure and disease severity.

EBioMedicine 2020 Aug 22;58:102911. Epub 2020 Jul 22.

Infectious Diseases Horizontal Technology Centre (ID HTC), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos #04-06, Biopolis, Singapore 138648, Singapore; Laboratory of Microbial Immunity, Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos #04-06, Biopolis, Singapore 138648, Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; National Institute of Health Research, Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool, Liverpool, United Kingdom; Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom. Electronic address:

Background: Given the unceasing worldwide surge in COVID-19 cases, there is an imperative need to develop highly specific and sensitive serology assays to define exposure to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Methods: Pooled plasma samples from PCR positive COVID-19 patients were used to identify linear B-cell epitopes from a SARS-CoV-2 peptide library of spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins by peptide-based ELISA. Hit epitopes were further validated with 79 COVID-19 patients with different disease severity status, 13 seasonal human CoV, 20 recovered SARS patients and 22 healthy donors.

Findings: Four immunodominant epitopes, S14P5, S20P2, S21P2 and N4P5, were identified on the S and N viral proteins. IgG responses to all identified epitopes displayed a strong detection profile, with N4P5 achieving the highest level of specificity (100%) and sensitivity (>96%) against SARS-CoV-2. Furthermore, the magnitude of IgG responses to S14P5, S21P2 and N4P5 were strongly associated with disease severity.

Interpretation: IgG responses to the peptide epitopes can serve as useful indicators for the degree of immunopathology in COVID-19 patients, and function as higly specific and sensitive sero-immunosurveillance tools for recent or past SARS-CoV-2 infections. The flexibility of these epitopes to be used alone or in combination will allow for the development of improved point-of-care-tests (POCTs).

Funding: Biomedical Research Council (BMRC), the A*ccelerate GAP-funded project (ACCL/19-GAP064-R20H-H) from Agency of Science, Technology and Research (A*STAR), and National Medical Research Council (NMRC) COVID-19 Research fund (COVID19RF-001) and CCGSFPOR20002. ATR is supported by the Singapore International Graduate Award (SINGA), A*STAR.
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http://dx.doi.org/10.1016/j.ebiom.2020.102911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375792PMC
August 2020

Macrophage polarisation associated with atherosclerosis differentially affects their capacity to handle lipids.

Atherosclerosis 2020 07 10;305:10-18. Epub 2020 Jun 10.

Department of Infection, Immunity & Cardiovascular Disease, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK. Electronic address:

Background And Aims: Lipid-rich foam cell macrophages drive atherosclerosis via several mechanisms, including inflammation, lipid uptake, lipid deposition and plaque vulnerability. The atheroma environment shapes macrophage function and phenotype; anti-inflammatory macrophages improve plaque stability while pro-inflammatory macrophages promote rupture. Current evidence suggests a variety of macrophage phenotypes occur in atherosclerotic plaques with local lipids, cytokines, oxidised phospholipids and pathogenic stimuli altering their phenotype. In this study, we addressed differential functioning of macrophage phenotypes via a systematic analysis of in vitro polarised, human monocyte-derived macrophage phenotypes, focussing on molecular events that regulate foam-cell formation.

Methods: We examined transcriptomes, protein levels and functionally determined lipid handling and foam cell formation capacity in macrophages polarised with IFNγ+LPS, IL-4, IL-10, oxPAPC and CXCL4.

Results: RNA sequencing of differentially polarised macrophages revealed distinct gene expression changes, with enrichment in atherosclerosis and lipid-associated pathways. Analysis of lipid processing activity showed IL-4 and IL-10 macrophages have higher lipid uptake and foam cell formation activities, while inflammatory and oxPAPC macrophages displayed lower foam cell formation. Inflammatory macrophages showed low lipid uptake, while higher lipid uptake in oxPAPC macrophages was matched by increased lipid efflux capacity.

Conclusions: Atherosclerosis-associated macrophage polarisation dramatically affects lipid handling capacity underpinned by major transcriptomic changes and altered protein levels in lipid-handling gene expression. This leads to phenotype-specific differences in LDL uptake, cellular cholesterol levels and cholesterol efflux, informing how the plaque environment influences atherosclerosis progression by influencing macrophage phenotypes.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.05.003DOI Listing
July 2020

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Immunity 2020 08 23;53(2):303-318.e5. Epub 2020 Jun 23.

Singapore Immunology Network (SIgN), A∗STAR (Agency for Science, Technology and Research), Biopolis, Singapore 138648, Singapore.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.
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http://dx.doi.org/10.1016/j.immuni.2020.06.005DOI Listing
August 2020
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