Transient abnormal myelopoiesis (TAM), a preleukemic disorder unique to neonates with Down syndrome (DS), may transform to childhood acute myeloid leukemia (ML-DS). Acquired GATA1 mutations are ...present in both TAM and ML-DS. Current definitions of TAM specify neither the percentage of blasts nor the role of GATA1 mutation analysis. To define TAM, we prospectively analyzed clinical findings, blood counts and smears, and GATA1 mutation status in 200 DS neonates. All DS neonates had multiple blood count and smear abnormalities. Surprisingly, 195 of 200 (97.5%) had circulating blasts. GATA1 mutations were detected by Sanger sequencing/denaturing high performance liquid chromatography (Ss/DHPLC) in 17 of 200 (8.5%), all with blasts >10%. Furthermore low-abundance GATA1 mutant clones were detected by targeted next-generation resequencing (NGS) in 18 of 88 (20.4%; sensitivity ∼0.3%) DS neonates without Ss/DHPLC-detectable GATA1 mutations. No clinical or hematologic features distinguished these 18 neonates. We suggest the term “silent TAM” for neonates with DS with GATA1 mutations detectable only by NGS. To identify all babies at risk of ML-DS, we suggest GATA1 mutation and blood count and smear analyses should be performed in DS neonates. Ss/DPHLC can be used for initial screening, but where GATA1 mutations are undetectable by Ss/DHPLC, NGS-based methods can identify neonates with small GATA1 mutant clones.
Key Points
Summary
Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they ...represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte–macrophage colony‐stimulating factor and interleukin‐4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon‐γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system.
Background
The effects of inhaled corticosteroids (ICS) on healthy airways are poorly defined.
Objectives
To delineate the effects of ICS on gene expression in healthy airways, without confounding ...caused by changes in disease‐related genes and disease‐related alterations in ICS responsiveness.
Methods
Randomized open‐label bronchoscopy study of high‐dose ICS therapy in 30 healthy adult volunteers randomized 2:1 to (i) fluticasone propionate 500 mcg bd daily or (ii) no treatment, for 4 weeks. Laboratory staff were blinded to allocation. Biopsies and brushings were analysed by immunohistochemistry, bulk RNA sequencing, DNA methylation array and metagenomics.
Results
ICS induced small between‐group differences in blood and lamina propria eosinophil numbers, but not in other immunopathological features, blood neutrophils, FeNO, FEV1, microbiome or DNA methylation. ICS treatment upregulated 72 genes in brushings and 53 genes in biopsies, and downregulated 82 genes in brushings and 416 genes in biopsies. The most downregulated genes in both tissues were canonical markers of type‐2 inflammation (FCER1A, CPA3, IL33, CLEC10A, SERPINB10 and CCR5), T cell‐mediated adaptive immunity (TARP, TRBC1, TRBC2, PTPN22, TRAC, CD2, CD8A, HLA‐DQB2, CD96, PTPN7), B‐cell immunity (CD20, immunoglobulin heavy and light chains) and innate immunity, including CD48, Hobit, RANTES, Langerin and GFI1. An IL‐17‐dependent gene signature was not upregulated by ICS.
Conclusions
In healthy airways, 4‐week ICS exposure reduces gene expression related to both innate and adaptive immunity, and reduces markers of type‐2 inflammation. This implies that homeostasis in health involves tonic type‐2 signalling in the airway mucosa, which is exquisitely sensitive to ICS.
4‐week ICS suppressed a broad spectrum of genes associated with adaptive T and B cell, and innate immunity, likely explaining the association between ICS use and pneumonia. Tonic T2 signalling in healthy airways was exquisitively sensitive to ICS, while an IL‐17‐dependent gene signature and CEACAM family members were not upregulated by 4‐week ICS exposure, suggesting their increased expression in severe asthma is disease‐related rather than treatment‐related. 4‐week ICS did not alter the airway microbiome, DNA methylation or airway cellularity. Abbreviations: CCR5, C‐C motif chemokine receptor 5; CLEC10A, C‐type lectin domain containing 10A; CPA3, carboxypeptidase A3; DPI, dry powder inhaler; HLA‐DQB2, major histocompatibility complex, class II, DQ beta 2; ICS, inhaled corticosteroid; IGH, immunoglobulin heavy locus; IGL/K, immunoglobulin lambda/kappa locus; PTPN22, protein tyrosine phosphatase non‐receptor type 22; RNAseq, RNA sequencing; rRNA, ribosomal RNA; SERPINB10, serpin family B member 10; TRAC, T cell receptor alpha constant; TRAP, triiodothyronine receptor auxiliary protein.
Mucosal‐associated invariant T (MAIT) cells are a well‐characterized innate‐like T cell population abundant in the human liver, peripheral tissues and blood. MAIT cells serve in the first line of ...defense against infections, through engagement of their T cell receptor, which recognizes microbial metabolites presented on MR1, and through cytokine‐mediated triggering. Typically, they show a quiescent memory phenotype but can undergo rapid upregulation of effector functions including cytolysis upon stimulation. T cells profoundly change their cellular metabolism during their maturation and activation. We sought to determine how MAIT cell metabolism may facilitate both the long‐term memory phase in tissue and the transition to rapid effector function. Here, we show, by flow cytometric metabolism assays and extracellular flux analysis that, despite an effector‐memory profile, human MAIT cells are metabolically quiescent in a resting state comparable to naïve and central memory T cells. Upon stimulation, they rapidly increase uptake of glucose and show a concomitant upregulation of the effector molecules notably granzyme B, which is impaired by inhibition of glycolysis with 2‐deoxyglucose. These findings suggest that MAIT cells share some metabolic characteristics of both resting and effector T cell subsets, with a rapid transition upon triggering. Metabolic programming of this cell type may be of interest in understanding and modulating their function in infectious diseases and cancer.
Mucosal‐associated invariant T (MAIT) cells are an innate‐like T cell population abundant in humans. T cell immunometabolism has been studied before in memory cell populations showing distinct usage of metabolic pathways according to activation status. Here, we show for the first time that upon stimulation, MAIT cells engage their glycolytic pathway along with an upregulation of the effector molecule granzyme B.
See also: Special Feature on MAIT cells
Human type-2 CD8
T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia-a ...phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8
CRTH2
(Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D
(PGD
) and cysteinyl leukotriene E
(LTE
) are also increased in the airways of the same group of patients. In vitro PGD
and LTE
function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.
Summary
Dendritic cells (
DC
) are the main immune mediators inducing primary immune responses.
DC
generated from monocytes (
M
o
DC
) are a model system to study the biology of
DC
in vitro
, as they ...represent inflammatory
DC
in vivo
. Previous studies on the generation of
M
o
DC
in horses indicated that there was no distinct difference between immature and mature
DC
and that the expression profile was distinctly different from humans, where
CD
206 is expressed on immature
M
o
DC
whereas
CD
83 is expressed on mature
M
o
DC
. Here we describe the kinetics of equine
M
o
DC
differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte–macrophage colony‐stimulating factor and interleukin‐4 generating immature
DC
(i
M
o
DC
). These cells were further activated with a cocktail of cytokines including interferon‐γ) but not
CD
40 ligand to obtain mature
DC
(m
M
o
DC
). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative
PCR
analysis were performed to carry out gene expression analysis. This study demonstrates that equine i
M
o
DC
and m
M
o
DC
can be distinguished both phenotypically and functionally but the expression pattern of some markers including
CD
206 and
CD
83 is dissimilar to the human system.
Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune ...and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.
Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been ...described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies. To characterise cDC populations in porcine blood, DC were enriched from PBMC by CD14 depletion and CD172a enrichment then stained with lineage mAbs (Lin; CD3, CD8α, CD14 and CD21) and mAbs specific for CD172a, CD1 and CD4. Two distinct porcine cDC subpopulations were FACSorted CD1
cDC (Lin
CD172
CD1
CD4
) and CD1
cDC (Lin
CD172a
CD1
CD4
), and characterised by phenotypic and functional analyses. CD1
cDC were distinct from CD1
cDC, expressing higher levels of CD172a, MHC class II and CD11b. Following TLR stimulation, CD1
cDC produced IL-8 and IL-10 while CD1
cDC secreted IFN-α, IL-12 and TNF-α. CD1
cDC were superior in stimulating allogeneic T cell responses and in cross-presenting viral antigens to CD8 T cells. Comparison of transcriptional profiles further suggested that the CD1
and CD1
populations were enriched for the orthologues of cDC1 and cDC2 subsets respectively.
Background:
First-line defence against viral infection is contingent upon rapid detection of conserved viral structural and genomic motifs by pattern recognition receptors, followed by activation of ...the type I IFN response and establishment of an antiviral state. Novel antiviral functions of bone morphogenetic protein and related activin cytokines, acting in conjunction with, and independently of, type I IFN, have recently been described. How these antiviral effects are mediated and triggered by viral infection has not been defined.
Methods:
Microarray and RNAseq data from hepatoma-derived cell lines stimulated with Activin A
in vitro
were interrogated both by pathway analysis and for evidence of IFN-stimulated gene induction. Liver tissue obtained from patients with chronic HCV were examined by real-time quantitative polymerase chain reaction (RT-qPCR) for evidence of Activin A induction. Activin expression by peripheral blood mononuclear cells exposed to nucleic acid analogues was quantified by RT-qCR, whereas induction dynamics in acute infection was investigated in
in vitro
Sendai virus infection and a murine influenza A.
Results:
Transcriptomic analyses delineated strikingly congruent patterns of gene regulation in hepatocytes stimulated with recombinant Activin A and IFNα
in vitro
. Activin A mRNA, encoded by
INHBA
, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors
in vitro
, across multiple cell lines and in human peripheral blood mononuclear cells.
In vivo
, imurine influenza A also upregulated
Inhba
mRNA in the lung; this local upregulation of
Inhba
is retained in MAVS knockout mice, indicating roles for non-RIG-I-like receptors in its induction. Activin induction and signalling were also detectable in patients with chronic viral hepatitis.
Conclusions:
These data suggest Activin A is triggered in parallel with type I IFN responses and can trigger related antiviral effector functions, with implications for the development of targeted antiviral therapies and revealing novel facets of Activin biology.
Abstract
The advancements of high-throughput genomics have unveiled much about the human genome highlighting the importance of variations between individuals and their contribution to disease. Even ...though numerous software have been developed to make sense of large genomics datasets, a major short falling of these has been the inability to cope with repetitive regions, specifically to validate structural variants and accordingly assess their role in disease. Here we describe our program STEAK, a massively parallel software designed to detect chimeric reads in high-throughput sequencing data for a broad number of applications such as identifying presence/absence, as well as discovery of transposable elements (TEs), and retroviral integrations. We highlight the capabilities of STEAK by comparing its efficacy in locating HERV-K HML-2 in clinical whole genome projects, target enrichment sequences, and in the 1000 Genomes CEU Trio to the performance of other TE and virus detecting tools. We show that STEAK outperforms other software in terms of computational efficiency, sensitivity, and specificity. We demonstrate that STEAK is a robust tool, which allows analysts to flexibly detect and evaluate TE and retroviral integrations in a diverse range of sequencing projects for both research and clinical purposes.
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