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Background: The most recognizable phenotype of severe asthma comprises people who are blood eosinophil and FeNO-high, driven by type-2 (T2) cytokine biology which responds to targeted biological therapies. However, in many people with severe asthma, these T2 biomarkers are suppressed but poorly controlled asthma persists. The mechanisms driving asthma in the absence of T2 biology are poorly understood. Objectives: To explore airway pathology in T2 biomarker-high and -low severe asthma. Methods: T2 biomarker-high severe asthma (T2-high, n=17) was compared to biomarker-intermediate (T2-intermediate, n=21) and biomarker-low (T2-low, n=20) severe asthma, and healthy controls (n=28). Bronchoscopy samples were processed for immunohistochemistry, and sputum for cytokines, PGD2 and LTE4 measurements. Results: Tissue eosinophil, neutrophil and mast cell counts were similar across severe asthma phenotypes and not increased when compared to healthy controls. In contrast, the remodeling features of airway smooth muscle mass and MUC5AC expression were increased in all asthma groups compared to health, but similar across asthma subgroups. Submucosal glands were increased in T2-intermediate and T2-low asthma. In spite of similar tissue cellular inflammation, sputum IL-4, IL-5, and CCL26 were increased in T2-high versus T2-low asthma, and several further T2-associated cytokines, PGD2 and LTE4, were increased in T2-high and T2-intermediate asthma compared to healthy controls. Conclusions: Eosinophilic tissue inflammation within proximal airways is suppressed in T2 biomarker-high and T2-low severe asthma, but inflammatory and structural cell activation is present, with sputum T2-associated cytokines highest in T2 biomarker-high patients. Airway remodeling persists, and may be important for residual disease expression beyond eosinophilic exacerbations.

Chronic rhinosinusitis (CRS) refers to inflammation of the nasal sinuses and mucosa, with persistence of sinus inflammation and clinical manifestations beyond 12 weeks.1 CRS affects between 6 and 12% of adults and is a cause of reduced quality of life (QoL) and high healthcare costs.2 Management consisted of topical and systemic glucocorticoids, antibiotics and often repeated sinus surgery. Over the past 20 years, biologic therapies under investigation for asthma, with overlapping nasal polyposis, have shown significant improvement in sinonasal CRS symptoms and reduced nasal polyp swelling.3 This Cochrane Corner aims to summarize the effectiveness, safety and role of biological therapy in CRS to aid clinicians in the decision-making process.

Background: Non-suppression of fractional exhaled nitric oxide (FeNO) during remotely monitored inhaled corticosteroid (ICS) therapy is associated with persistent symptoms and blood eosinophilia. To provide mechanistic insight, we assessed sputum type 2 cytokines and chemokines before and after a FeNO suppression test. Methods: FeNO suppression was performed in 44 patients with severe asthma and FeNO > 40 ppb. FeNO was monitored for 7 days of 1000μg of fluticasone propionate delivered via an INCATM device, with clinical and sputum sampling on days 0 and 7. FeNO suppression was defined as a 42% reduction in FeNO. Sputum supernatant was analyzed in 15 paired samples by ELISA (Prostaglandin D2, Leukotriene E4) and MSD assays (IL-4,-5,-13,-25,-33, CCL26, TSLP). Results: Suppressors (n=21) vs non-suppressors had a greater drop in ACQ-5 (mean∆: -1.2 vs -0.3, p Conclusion: Failure to suppress FeNO during ICS treatment was associated with steroid-unresponsive sputum PGD2 and LTE4 levels.

Background Whether the clinical or pathophysiologic significance of the “treatable trait” high blood eosinophil count in COPD is the same as for asthma remains controversial. We sought to determine the relationship between the blood eosinophil count, clinical characteristics and gene expression from bronchial brushings in COPD and asthma. Methods Subjects were recruited into a COPD (emphysema versus airway disease [EvA]) or asthma cohort (Unbiased BIOmarkers in PREDiction of respiratory disease outcomes, U‐BIOPRED). We determined gene expression using RNAseq in EvA (n = 283) and Affymetrix microarrays in U‐BIOPRED (n = 85). We ran linear regression analysis of the bronchial brushings transcriptional signal versus blood eosinophil counts as well as differential expression using a blood eosinophil > 200 cells/μL as a cut‐off. The false discovery rate was controlled at 1% (with continuous values) and 5% (with dichotomized values). Results There were no differences in age, gender, lung function, exercise capacity and quantitative computed tomography between eosinophilic versus noneosinophilic COPD cases. Total serum IgE was increased in eosinophilic asthma and COPD. In EvA, there were 12 genes with a statistically significant positive association with the linear blood eosinophil count, whereas in U‐BIOPRED, 1197 genes showed significant associations (266 positive and 931 negative). The transcriptome showed little overlap between genes and pathways associated with blood eosinophil counts in asthma versus COPD. Only CST1 was common to eosinophilic asthma and COPD and was replicated in independent cohorts. Conclusion Despite shared “treatable traits” between asthma and COPD, the molecular mechanisms underlying these clinical entities are predominately different., In a chronic obstructive pulmonary disease (COPD) cohort (EvA, n = 283), 12 genes, whereas in asthma cohort (UBIOPRED, n = 85), 1197 genes in bronchial epithelial brushes were correlated with a blood eosinophil count. The gene CST1 was common to eosinophilic asthma and COPD and was replicated in independent cohorts. Despite shared “treatable traits” between asthma and COPD, the molecular mechanisms underlying these clinical entities are predominately different.

Vaccines get a help-MAIT Mucosal-associated invariant T (MAIT) cells are a T cell subset important for mucosal homeostasis. These cells recognize derivatives of microbiota-derived vitamin B2 precursors but can also be activated by certain cytokines in the context of viral infections. Provine et al. report that a leading adenoviral vector vaccine, ChAdOx1, activated MAIT cells in immunized mice (see the Perspective by Juno and O'Connor). This activation required interferon-α produced by plasmacytoid dendritic cells as well as monocyte-derived interleukin-18 and tumor necrosis factor. MAIT cell activation positively correlated with vaccine-mediated T cell responses in human subjects, and mice deficient in MAIT cells showed impaired CD8 + T cell immunity to target antigens after vaccination. This work suggests an additional pathway that could be exploited to enhance the efficacy of vaccines. Science , this issue p. 521 ; see also p. 460

Summary Azithromycin (AZM) is a synthetic macrolide antibiotic effective against a broad range of bacterial and mycobacterial infections. Due to an additional range of anti‐viral and anti‐inflammatory properties, it has been given to patients with the coronaviruses SARS‐CoV or MERS‐CoV. It is now being investigated as a potential candidate treatment for SARS‐CoV‐2 having been identified as a candidate therapeutic for this virus by both in vitro and in silico drug screens. To date there are no randomised trial data on its use in any novel coronavirus infection, although a large number of trials are currently in progress. In this review, we summarise data from in vitro, murine and human clinical studies on the anti‐viral and anti‐inflammatory properties of macrolides, particularly AZM. AZM reduces in vitro replication of several classes of viruses including rhinovirus, influenza A, Zika virus, Ebola, enteroviruses and coronaviruses, via several mechanisms. AZM enhances expression of anti‐viral pattern recognition receptors and induction of anti‐viral type I and III interferon responses. Of relevance to severe coronavirus‐19 disease (COVID‐19), which is characterised by an over‐exuberant innate inflammatory response, AZM also has anti‐inflammatory properties including suppression of IL‐1beta, IL‐2, TNF and GM‐CSF. AZM inhibits T cells by inhibiting calcineurin signalling, mammalian target of rapamycin activity and NFκB activation. AZM particularly targets granulocytes where it concentrates markedly in lysosomes, particularly affecting accumulation, adhesion, degranulation and apoptosis of neutrophils. Given its proven safety, affordability and global availability, tempered by significant concerns about antimicrobial stewardship, there is an urgent mandate to perform well‐designed and conducted randomised clinical trials.

Intro: Prior metagenomic studies in asthma are limited by inconsistent clinical phenotyping and inadequate sequencing depth for species-level bacterial identification. We hypothesise chronic bacterial infection is i) a ‘treatable trait’ whose prevalence, clinical phenotype and reliable biomarkers need definition and ii) is best characterised using Nanopore sequencing. Aims/Methods: To compare performance of Illumina MiSeq, Nanopore sequencing, and RT-qPCR on total DNA extracts against culture/MALDI-TOF for analysis of induced sputum samples from well-phenotyped severe asthma. Results: In 23 patients with with severe asthma Haemophilus influenzae was commonly cultured (n=8/23), and identified as the dominant bacterial species by metagenomic sequencing using MiSeq and Nanopore. Nanopore provided superior operational characterisitics and longer read lengths allowing whole bacterial genome reconstruction and greater resolution between species. Clinically significant infection was confirmed with validated H.inf plasmid-based RT-qPCR assay. H.inf culture positive patients had sputum neutrophilia and lower FeNO. Conclusions:H.inf is a clinically-relevant pathogen in severe ashma and is identified reliably using molecular microbiological methods. Application of these optimised protocols in ongoing analysis of 3 large patient cohorts is allowing full characterisation of this clinical phenotype.

To-date, there has not been a clear signal suggesting that asthma or treatment with inhaled steroids are a risk factor for severe COVID-19 disease. We have therefore explored ACE2 receptor mRNA expression, and co-factors for Sars-CoV-2 infectivity (TMPRSS2 and furin) in bronchial brushes and biopsies from people with asthma and healthy controls, and looked for relationships between asthma severity, Th2- and IL-17 dependent gene signatures, and clinical demographics (age, sex). We have looked at a cohort of 356 research participants from previously described studies. The only significant association was a positive correlation between ACE2 and IL-17-dependent gene expression, and an inverse correlation between ACE2 and Th2-cytokine-dependent gene expression. These data suggest that differences in ACE2, TMPRSS2 and furin epithelial and airway gene expression are unlikely to confer enhanced COVID-19 pneumonia risk in patients with asthma across all treatment intensities and severity., Expression of mRNA for ACE2, the Sars-CoV-2 receptor, is similar in the lower airways of healthy controls and people with mild-severe asthma. Altered ACE2 expression is unlikely to confer enhanced COVID-19 pneumonia risk in asthma.

Monoclonal antibodies targeting IgE or the type-2 cytokines interleukin (IL)-4, IL-5 and IL-13 are proving highly effective in reducing exacerbations and symptoms in people with severe allergic and eosinophilic asthma, respectively. However, these therapies are not appropriate for 30–50% of patients in severe asthma clinics who present with non-allergic, non-eosinophilic, “type-2 low” asthma. These patients constitute an important and common clinical asthma phenotype, driven by distinct, yet poorly understood pathobiological mechanisms. In this review we describe the heterogeneity and clinical characteristics of type-2 low asthma and summarise current knowledge on the underlying pathobiological mechanisms, which includes neutrophilic airway inflammation often associated with smoking, obesity and occupational exposures and may be driven by persistent bacterial infections and by activation of a recently described IL-6 pathway. We review the evidence base underlying existing treatment options for specific treatable traits that can be identified and addressed. We focus particularly on severe asthma as opposed to difficult-to-treat asthma, on emerging data on the identification of airway bacterial infection, on the increasing evidence base for the use of long-term low-dose macrolides, a critical appraisal of bronchial thermoplasty, and evidence for the use of biologics in type-2 low disease. Finally, we review ongoing research into other pathways including tumour necrosis factor, IL-17, resolvins, apolipoproteins, type I interferons, IL-6 and mast cells. We suggest that type-2 low disease frequently presents opportunities for identification and treatment of tractable clinical problems; it is currently a rapidly evolving field with potential for the development of novel targeted therapeutics.

Mucosal associated invariant T (MAIT) cells are striking in their abundance and their strict conservation across 150 million years of mammalian evolution, implying they must fulfil critical immunological function(s). MAIT cells are defined by their expression of a semi-invariant αβ TCR which recognises biosynthetic derivatives of riboflavin synthesis presented on MR1. Initial studies focused on their role in detecting predominantly intracellular bacterial and mycobacterial infections. However, it is now recognised that there are several modes of MAIT cell activation and these are related to activation of distinct transcriptional programmes, each associated with distinct functional roles. In this minireview, we summarise current knowledge from human and animal studies of MAIT cell activation induced 1) in an MR1-TCR dependent manner in the context of inflammatory danger signals and associated with antibacterial host defence; 2) in an MR1-TCR independent manner by the cytokines interleukin(IL)-12 / -15 / -18 and type I interferon, which is associated with antiviral responses; and 3) a recently-described TCR-dependent ‘tissue repair’ programme which is associated with accelerated wound healing in the context of commensal microbiota. Because of this capability for diverse functional responses in diverse immunological contexts, these intriguing cells now appear to be multifunctional effectors central to the interface of innate and adaptive immunity.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a new rapidly spreading infectious disease. Current guidance from the World Health Organization (WHO) highlights asthmatics as a high-risk group for severe illness from COVID-19. Viruses are common triggers of asthma exacerbations and the current SARS-CoV-2 pandemic raises several questions regarding the optimum management strategies. Here, we discuss the contentious issue of whether the mainstay therapy systemic corticosteroids should be used in the routine management of COVID-19-associated asthma exacerbations. Recent guidance from the WHO has advised against the use of corticosteroids if COVID-19 is suspected due to concerns that these agents may impair protective innate antiviral immune responses. This may not be appropriate in the unique case of asthma exacerbation, a syndrome associated with augmented type 2 inflammation, a disease feature that is known to directly inhibit antiviral immunity. Corticosteroids, through their suppressive effects on type 2 inflammation, are thus likely to restore impaired antiviral immunity in asthma and, in contrast to non-asthmatic subjects, have beneficial clinical effects in the context of SARS-CoV-2 infection.

While there now exist effective treatments for type 2 high, eosinophilic asthma, there are no specific therapies for 40–50% of people with asthma with other phenotypes, which result from poorly understood underlying pathological mechanisms. One such pathology is neutrophilic inflammation, which has been associated with interleukin (IL)-17 family cytokines. Human genetic studies identified IL-17 polymorphisms associated with asthma; in murine models of allergic airways disease, IL-17A contributes to airway hyperresponsiveness, and in humans, elevated airway IL-17A levels are repeatedly observed in severe asthma. However, the directionality of this association is unknown, and the assumption that IL-17 cytokines drive disease pathology remains speculative. Here, we explore the evidence underlying the relationship between IL-17 and asthma, we review lessons learned from investigating IL-17 in other inflammatory diseases, and discuss the possibility that IL-17 may even be protective in asthma rather than pathogenic. We also critically examine the newly proposed paradigm of a reciprocal relationship between type 2 and type 17 airways inflammation. In summary, we suggest an association between IL-17 and asthma, but research is needed examining the diverse functions of these cytokines, their longitudinal stability, their response to clinical interventions, and for mechanistic studies determining whether they are protective or pathogenic., IL-17 cytokines have been implicated in neutrophilic asthma by genetic, murine and human data. Here, previous studies are critiqued and the assumption their dominant role is pathogenic rather than protective of airway epithelial barrier integrity is challenged. http://bit.ly/3axB4Zs

Sensing of influenza A virus (IAV) infection by pattern recognition receptors can occur by either direct infection of lung epithelial cells or uptake of virus-infected cells by innate cells such as dendritic cells/monocytes. This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells. These cells include innate lymphocytes such as MAIT, NKT, NK, and γδ T cells. Here we describe a method used to allow activation of human innate lymphocytes in co-culture with an IAV-infected human lung epithelial cell line (A549) to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment. We describe (1) infection of the human lung epithelial cell line, (2) co-culture with PBMC, and (3) measurement of activation using intracellular cytokine staining.

The complexity of asthma is underscored by the number of cell types and mediators implicated in the pathogenesis of this heterogeneous syndrome. Type 2 CD4+ T-cells (Th2) and more recently, type 2 innate lymphoid cells dominate current descriptions of asthma pathogenesis. However, another important source of these type 2 cytokines, especially interleukin (IL)-5 and IL-13, are CD8+ T-cells, which are increasingly proposed to play an important role in asthma pathogenesis, because they are abundant and are comparatively insensitive to corticosteroids. Many common triggers of asthma exacerbations are mediated via corticosteroid-resistant pathways involving neutrophils and CD8+ T-cells. Extensive murine data reveal the plasticity of CD8+ T-cells and their capacity to enhance airway inflammation and airway dysfunction. In humans, Tc2 cells are predominant in fatal asthma, while in stable state, severe eosinophilic asthma is associated with greater numbers of Tc2 than Th2 cells in blood, bronchoalveolar lavage fluid and bronchial biopsies. Tc2 cells strongly express CRTH2, the receptor for prostaglandin D2, the cysteinyl leukotriene receptor 1 and the leukotriene B4 receptor. When activated, these elicit Tc2 cell chemotaxis and production of chemokines and type 2 and other cytokines, resulting directly or indirectly in eosinophil recruitment and survival. These factors position CD8+ Tc2 cells as important and underappreciated effector cells contributing to asthma pathogenesis. Here, we review recent advances and new insights in understanding the pro-asthmatic functions of CD8+ T-cells in eosinophilic asthma, especially corticosteroid-resistant asthma, and the molecular mechanisms underlying their pathologic effector function.

Asthma arises from the complex interplay of inflammatory pathways in diverse cell types and tissues. We sought to undertake a comprehensive transcriptomic assessment of the epithelium and airway T cells that remain understudied in asthma and investigate interactions between multiple cells and tissues. Epithelial brushings and flow-sorted CD3+ T cells from sputum and BAL were obtained from healthy subjects (n = 19) and patients with asthma (mild, moderate, and severe asthma; n = 46). Gene expression was assessed using Affymetrix HT HG-U133+ PM GeneChips, and results were validated by real-time quantitative PCR. In the epithelium, IL-13 response genes (POSTN, SERPINB2, and CLCA1), mast cell mediators (CPA3 and TPSAB1), inducible nitric oxide synthase, and cystatins (CST1, CST2, and CST4) were upregulated in mild asthma, but, except for cystatins, were suppressed by corticosteroids in moderate asthma. In severe asthma—with predominantly neutrophilic phenotype—several distinct processes were upregulated, including neutrophilia (TCN1 and MMP9), mucins, and oxidative stress responses. The majority of the disease signature was evident in sputum T cells in severe asthma, where 267 genes were differentially regulated compared with health, highlighting compartmentalization of inflammation. This signature included IL-17–inducible chemokines (CXCL1, CXCL2, CXCL3, IL8, and CSF3) and chemoattractants for neutrophils (IL8, CCL3, and LGALS3), T cells, and monocytes. A protein interaction network in severe asthma highlighted signatures of responses to bacterial infections across tissues (CEACAM5, CD14, and TLR2), including Toll-like receptor signaling. In conclusion, the activation of innate immune pathways in the airways suggests that activated T cells may be driving neutrophilic inflammation and steroid-insensitive IL-17 response in severe asthma.

Rationale: Severe asthma is a heterogeneous disease with different inflammatory phenotypes based on blood or sputum measures. Increasingly blood eosinophils (Eos) are used to direct therapy choice but there is limited information how stable inflammatory measures in sputum and blood are over time and how they relate. Method: Sputum was successfully induced in 48 severe asthma patients at 2 timepoints within 2 years. At each timepoint a full blood count was completed. Patients were at least 4-weeks clear of exacerbation and considered stable. The percentage of granulocytes were reported and the stability at these two timepoints was assessed using Spearman’s rho. Results: In this cohort the sputum Eos and blood Eos significantly correlated between the two timepoints (sputum r=0.739,p= Discussion: These results demonstrate that airway neutrophilic inflammation is more dynamic than eosinophilic inflammation and poorly reflected in peripheral blood. A greater understanding of the underlying factors responsible for airway neutrophilic changes is needed if progress is to be made in addressing this severe asthma phenotype.

Background: Severe asthma with persistent airway eosinophilia despite high-intensity inhaled corticosteroids is a phenotype associated with high exacerbation risk. A major barrier to progress is poor understanding of the pathogenic mechanisms and cell types contributing to the disease. Aims and objectives: To determine the function of type-2 cytokine producing CD8+ T cells in severe eosinophilic asthma, and investigate their contribution to pathogenesis of the disease. Methods: Profiles of type-2 CD8+ T cells (Tc2) and type-2 T helper (Th2), in different asthma phenotypes were analysed with flow cytometry. Tc2 cells were isolated and cultured in vitro. Effects of prostaglandin D2 (PGD2) and leukotriene E4 (LTE4) on Tc2 cells were defined using chemotaxis assays, Luminex, quantitative RT-PCR, PrimeFlow RNA assay and microarray. Effects of Tc2 supernatants were determined on eosinophil shape change and apoptosis, and on eosinophil chemokine production from airway epithelial cells. Results: In two independent patient cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ Tc2 cells are enriched in blood and airways, and concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways in severe eosinophilic asthma. In vitro PGD2 and LTE4 function synergistically to enhance Tc2 cell recruitment and activation. They regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. Conclusions: These finding are consistent with an important role for human Tc2 cells in severe eosinophilic asthma.

Background: Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. However, whilst they are also activated during human viral infections, it is unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Aims and objectives: To determine whether MAIT cells play a significant role – either protective or detrimental – during influenza A infection in vivo. Methods: We used major histocompatibility complex–related protein 1 (MR1) tetramers and intracellular cytokine staining to track MAIT cell frequencies and activation during in vivo murine experimental challenge with two strains of influenza A virus in immunocompetent (C57BL/6), MAIT-cell deficient (MR1-/-) and immunodeficient (Rag2-/-γC-/-) mice. Results: MAIT cells accumulated and were activated early in infection, with upregulation of CD25, CD69 and Granzyme B peaking at 5 days post infection. Activation was modulated via cytokines interleukin (IL)-12, -15, -18 and type I interferon, independent of MR1. MR1-/- mice, which lack MAIT cells, showed enhanced body weight loss and mortality to severe (H1N1) influenza. This was ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient Rag2-/-γC-/- mice which lack T, B and NK cells. Conclusions: MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.

Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infectionsin vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulated and were activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post infection. Activation was modulated via cytokines independently of MR1. MAIT cell-deficient MR1−/−mice showed enhanced weight loss and mortality to severe (H1N1) influenza. This was ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2−/−γC−/−mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.

The functions and in vivo roles of type-2 CD8+ T cells in humans have not been well defined and this cell type has been largely overlooked in models of 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 D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 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.

RATIONALE: Mucosal-associated invariant T (MAIT) cells are a recently described abundant, proinflammatory T-cell subset with unknown roles in pulmonary immunity. Nontypeable Haemophilus influenzae (NTHi) is the leading bacterial pathogen during chronic obstructive pulmonary disease (COPD) exacerbations and is a plausible target for MAIT cells. OBJECTIVES: To investigate whether MAIT cells respond to NTHi and the effects of inhaled corticosteroids (ICS) on their frequency and function in COPD. METHODS: Eleven subjects with COPD receiving ICS, 8 steroid-naive subjects with COPD, and 21 healthy control subjects underwent phlebotomy, sputum induction, bronchoalveolar lavage, and endobronchial biopsy. Pulmonary and monocyte-derived macrophages were cultured in vitro with NTHi. MEASUREMENTS AND MAIN RESULTS: Frequencies of Vα7.2(+)CD161(+) MAIT cells, surface expression of the major histocompatibility complex-related protein 1 (MR1), and intracellular IFN-γ expression were measured by flow cytometry. MAIT-cell frequencies were reduced in peripheral blood of ICS-treated subjects with COPD (median 0.38%; interquartile range [IQR], 0.25-0.96) compared with healthy control subjects (1.8%; IQR, 1.4-2.5; P = 0.001) or steroid-naive patients with COPD (1.8%; IQR, 1.2-2.3; P = 0.04). MAIT cells were reduced in bronchial biopsies from subjects with COPD treated with steroids (0.73%; IQR, 0.46-1.3) compared with healthy control subjects (4.0%; IQR, 1.6-5.0; P = 0.02). Coculture of live NTHi increased macrophage surface expression of MR1 and induced IFN-γ from CD4 cells and CD8 cells, but most potently from MAIT cells (median IFN-γ-positive frequencies, 2.9, 8.6, and 27.6%, respectively). In vitro fluticasone and budesonide reduced MR1 surface expression twofold and decreased NTHi-induced IFN-γ secretion eightfold. CONCLUSIONS: MAIT cells are deficient in blood and bronchial tissue in steroid-treated, but not steroid-naive, COPD. NTHi constitutes a target for pulmonary MAIT-cell immune responses, which are significantly impaired by corticosteroids.

Individuals with self-healed tuberculosis from the preantibiotic era offer a unique insight into the natural history of and protective immunity to tuberculosis. In 27 such persons whose tuberculosis self-healed >50 years earlier, circulating Mycobacterium tuberculosis antigen-specific interferon γ (IFN-γ)- and interleukin 2 (IL-2)-secreting T cells were detected ex vivo in 16 and 19 individuals, respectively. The M. tuberculosis-specific T cell cytokine profile was dominated by effector memory T cells that secrete both IFN-γ and IL-2 and included T cells that secrete only IFN-γ or IL-2, suggesting persistence of antigen secreted by viable bacilli. Of 10 individuals with no M. tuberculosis antigen-specific IFN-γ-secreting T cells detectable ex vivo, 7 had evidence of central memory T cells, consistent with clearance of infection.

The majority of individuals infected withMycobacterium tuberculosisachieve lifelong immune containment of the bacillus. What constitutes this effective host immune response is poorly understood. We compared the frequencies of gamma interferon (IFN-γ)-secreting T cells specific for five region of difference 1 (RD1)-encoded antigens and one DosR-encoded antigen in 205 individuals either with active disease (n= 167), whose immune responses had failed to contain the bacillus, or with remotely acquired latent infection (n= 38), who had successfully achieved immune control, and a further 149 individuals with recently acquired asymptomatic infection. When subjects with an IFN-γ enzyme-linked immunospot (ELISpot) assay response to one or more RD1-encoded antigens were analyzed, T cells from subjects with active disease recognized more pools of peptides from these antigens than T cells from subjects with nonrecent latent infection (P= 0.002). The T-cell frequencies for peptide pools were greater for subjects with active infection than for subjects with nonrecent latent infection for summed RD1 peptide pools (P≤ 0.006) and culture filtrate protein 10 (CFP-10) antigen (P= 0.029). Individuals with recently acquired (6 months) latent infection did not differ in numbers of peptide pools recognized, proportions recognizing any individual antigen or peptide pool, or antigen-specific T-cell frequencies (P≥ 0.11). The hierarchy of immunodominance for different antigens was purified protein derivative (PPD) > CFP-10 > early secretory antigenic target 6 > Rv3879c > Rv3878 > Rv3873 > Acr1, and the hierarchies were very similar for active and remotely acquired latent infections. Responses to the DosR antigen α-crystallin were not associated with latency (P= 0.373). In contrast to the RD1-specific responses, the responses to PPD were not associated with clinical status (P> 0.17) but were strongly associated with positive tuberculin skin test results (≥15-mm induration;P≤ 0.01). Our results suggest that RD1-specific IFN-γ-secreting T-cell frequencies correlate with the presence of disease rather than with protective immunity inM. tuberculosis-infected individuals and do not distinguish recently acquired asymptomatic infection from remotely acquired latent infection.

Summary New T cell-based blood tests for tuberculosis infection could improve diagnosis of tuberculosis but their clinical utility remains unknown. We describe the role of the ELISpot test in the diagnostic work-up of 13 patients presenting with suspected tuberculosis in routine practice. Of the seven patients with a final diagnosis of active tuberculosis, all were positive by ELISpot including three with false-negative tuberculin skin test results. Rapid determination of tuberculosis infection by ELISpot accelerated the diagnosis of tuberculosis, enabling early treatment initiation.

Introduction: Sputum inflammometry has been used to describe asthma phenotypes and severe asthma is associated with neutrophilia based on the percentage of cells present. Sputum density is not accounted for in clinical practice. Methods: Induced sputum was obtained from 110 severe asthmatics (BTS step 4/5) of the Wessex Severe Asthma Cohort. Inflammatory biological markers were measured in sputum supernatant with Multiplex ELISA. Biomarker concentrations were compared to sputum neutrophil density using Spearmans correlation. Results: The median [IQR] cell number (x 106/g sputum) was significantly lower in P phenotype (n=41 0.64 \[0.96]) than N (n=29 2.1 [2.6\] (p

Background: studies with monocyte-derived macrophages (MDMs) and animal models have suggested a role for alternatively activated (M2) macrophages in asthmatic inflammation, but in vivo evidence for this phenotype in human asthma is lacking.Objective: to characterize the phenotype of lung macrophages from asthmatic patients in relation to disease severity and treatment.Methods: M2 biomarkers were first identified by using MDMs exposed to T(H)2 cytokines and then used to phenotype sputum and bronchoalveolar lavage (BAL) macrophages from 12 healthy control subjects, 12 patients with mild asthma, and 14 patients with moderate asthma and to assess the effects of corticosteroids and phosphatidylinositol 3-kinase (PI3K) inhibitors.Results: sputum macrophages from asthmatic patients expressed significantly more CCL17 mRNA but less CD163 than macrophages from healthy subjects. However, none of the other M2 biomarkers were differentially expressed in asthmatic patients, and ex vivo BAL cells spontaneously produced similar amounts of M2 cytokines/chemokines (IL-10, CCL17, and CCL22). CCL17 mRNA overexpression correlated weakly but significantly with sputum eosinophilia (P = .0252) and was also observed in macrophages from patients with moderate asthma treated with inhaled steroids, suggesting relative insensitivity to inhibition by corticosteroids. The PI3K inhibitor LY294002 inhibited basal CCL17 release from BAL cells and IL-4-stimulated release from MDMs.Conclusions: this study does not support the existence in human asthma of the full M2 phenotype described to date but points to upregulation of CCL17 in both patients with mild and those with moderate asthma, providing a further source for this ligand of CCR4(+) cells that contributes to airways inflammation. CCL17 expression is corticosteroid resistant but suppressed by PI3K enzyme inhibitors

Central nervous system tuberculosis remains a clinical diagnostic challenge. The ex vivo Mycobacterium tuberculosis-specific enzyme-linked immunospot assay (ELISPOT) is a novel assay for the rapid detection of M. tuberculosis-specific T-lymphocytes in the peripheral blood. However, when performed on peripheral blood, this assay cannot distinguish between active tuberculosis or latent tuberculosis infection. On the assumption that M. tuberculosis-specific T-lymphocytes migrate to sites of infection, we were able to demonstrate high levels of M. tuberculosis-specific cells by ELISPOT in the cerebrospinal fluid of a patient with tuberculous meningitis and intracerebral tuberculoma four weeks before cerebrospinal fluid culture became positive for M. tuberculosis by culture.

Background: The role of new T-cell–based blood tests for tuberculosis in the diagnosis of active tuberculosis is unclear. Objective: To compare the performance of 2 interferon-? assays and tuberculin skin testing in adults with suspected tuberculosis. Design: Prospective study conducted in routine practice. Setting: 2 urban hospitals in the United Kingdom. Patients: 389 adults, predominantly of South Asian and black ethnicity, with moderate to high clinical suspicion of active tuberculosis. Intervention: Tuberculin skin testing, the enzyme-linked immunospot assay (ELISpot) incorporating early secretory antigenic target-6 and culture filtrate protein-10 (standard ELISpot), and ELISpot incorporating a novel antigen, Rv3879c (ELISpotPLUS) were performed during diagnostic assessment by independent persons who were blinded to results of the other test. Measurements: Sensitivity, specificity, predictive values, and likelihood ratios. Results: 194 patients had a final diagnosis of active tuberculosis, of which 79% were culture-confirmed. Sensitivity for culture confirmed and highly probable tuberculosis was 89% (95% CI, 84% to 93%) with ELISpotPLUS, 85% (CI, 79% to 90%) with standard ELISpot, 79% (CI, 72% to 85%) with 15-mm threshold tuberculin skin testing, and 83% (CI, 77% to 89%) with stratified thresholds of 15 and 10 mm in vaccinated and unvaccinated patients, respectively. The ELISpotPLUS assay was more sensitive than tuberculin skin testing with 15-mm cutoff points (P = 0.01) but not with stratified cutoff points (P = 0.10). The ELISpotPLUS assay had 4% higher diagnostic sensitivity than standard ELISpot (P = 0.02). Combined sensitivity of ELISpotPLUS and tuberculin skin testing was 99% (CI, 95% to 100%), conferring a negative likelihood ratio of 0.02 (CI, 0 to 0.06) when both test results were negative. Limitations: Local standards for tuberculin skin testing differed from others used internationally. The study sample included few immunosuppressed patients. Conclusion: The ELISpotPLUS assay is more sensitive than standard ELISpot and, when used in combination with tuberculin skin testing, enables rapid exclusion of active infection in patients with moderate to high pretest probability of tuberculosis.

Distinct IFN-γ and IL-2 profiles of Ag-specific CD4+ T cells have recently been associated with different clinical disease states and Ag loads in viral infections. We assessed the kinetics and functional profile of Mycobacterium tuberculosis Ag-specific T cells secreting IFN-γ and IL-2 in 23 patients with untreated active tuberculosis when bacterial and Ag loads are high and after curative treatment, when Ag load is reduced. The frequencies of M. tuberculosis Ag-specific IFN-γ-secreting T cells declined during 28 mo of follow-up with an average percentage decline of 5.8% per year (p = 0.005), while the frequencies of Ag-specific IL-2-secreting T cells increased during treatment (p = 0.02). These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-γ- and IL-2-secreting cells over 28 mo. Simultaneous measurement of IFN-γ and IL-2 secretion at the single-cell level revealed a codominance of IFN-γ-only secreting and IFN-γ/IL-2 dual secreting CD4+ T cells in active disease that shifted to dominance of IFN-γ/IL-2-secreting CD4+ T cells and newly detectable IL-2-only secreting CD4+ T cells during and after treatment. These distinct T cell functional signatures before and after treatment suggest a novel immunological marker of mycobacterial load and clinical status in tuberculosis that now requires validation in larger prospective studies.

Incomplete understanding of mechanisms and clinicopathobiological heterogeneity in asthma hinders research progress. Pathogenic roles for T-helper-type 17 (Th17) cells and invariant T cells implied by murine data have yet to be assessed in man. We aimed to investigate the role of Th17 and mucosal associated invariant T (MAIT) cells in airway inflammation; to characterise associations between diverse clinical and immunological features of asthma; and to identify novel multidimensional asthma endotypes.In this single-centre, cross-sectional observational study in the UK, we assessed volunteers with mild-to-severe asthma and healthy non-atopic controls using clinical and physiological assessment and immunological sampling of blood, induced sputum, endobronchial biopsy, and bronchoalveolar lavage for flow cytometry and multiplex-electrochemiluminescence assays. Primary outcomes were changes in frequencies of Th17 and MAIT cells between health and asthma using Mann-Whitney U tests and the Jonckheere-Terpstra test (linear trend across ranked groups). The study had 80% power to detect 60% differences in T-cell frequencies at p0·05. Bayesian Network Analysis (BNA) was used to explore associations between parameters. Topological Data Analysis (TDA) was used to identify multidimensional endotypes. The study had local research ethics approval. All participants provided informed consent.Participants were 84 male and female volunteers (60 with mild-to-severe asthma and 24 healthy, non-atopic controls) aged 18-70 years recruited from clinics and research cohorts. Th17 cells and γδ17 cells were not associated with asthma, even in severe neutrophilic forms. MAIT-cell frequencies were strikingly reduced in asthma compared with health (median frequency in blood 0·9% of CD3+ cells [IQR 0·3-1·8] in asthma vs 1·6 [1·2-2·6] in health, p=0·005; in sputum 1·1 [0·7-2·0] vs 1·8 [1·6-2·3], p=0·002; and in biopsy samples 1·3 [0·7-2·3] vs 3·9% [1·3-5·3%], p=0·02), especially in severe asthma where BAL regulatory T cells were also reduced compared with those in health (4·4, 3·1-6·1, vs 8·1, 5·6-10; p=0·02). BNA and TDA identified six novel clinicopathobiological clusters of underlying disease mechanisms, with elevated mast cell mediators tryptase (p0·0001), chymase (p=0·02), and carboxypeptidase A3 (p=0·02) in severe asthma.This study suggests that Th17 cells do not have a major pathogenic role in human asthma. We describe a novel deficiency of MAIT cells in severe asthma. We also provide proof of concept for application of TDA to identification of multidimensional clinicopathobiological endotypes. Endotypes will require validation in further cohorts.Wellcome Trust.

T-cell responses during tuberculosis (TB) help contain Mycobacterium tuberculosis in vivo but also cause collateral damage to host tissues. Immune regulatory mechanisms may limit this immunopathology, and suppressed cellular immune responses in patients with TB suggest the presence of regulatory activity. CD4+CD25(high) regulatory T cells mediate suppressed cellular immunity in several chronic infections but have not been described in TB.To determine whether regulatory T cells are increased in patients with TB and whether they suppress cellular immune responses.We compared the frequency of circulating regulatory T cells in 27 untreated patients with TB and 23 healthy control subjects using two specific markers: cell-surface CD25 expression and FoxP3 mRNA expression in peripheral blood mononuclear cells.We detected a threefold increase in the frequency of CD4 + CD25(high) T cells (p0.001) and a 2.2-fold increase in FoxP3 expression (p = 0.006) in patients with TB, and there was a positive correlation between these markers (r = 0.58, p0.001). Increased expression of interleukin-10 and transforming growth factor-beta1 mRNA was also detected in patients with TB but did not correlate with regulatory T-cell markers. Ex vivo depletion of CD4 + CD25(high) cells from peripheral blood mononuclear cells resulted in increased numbers of M. tuberculosis antigen-specific IFN-gamma-producing T cells in seven of eight patients with TB (p = 0.005). Finally, FoxP3 expression was increased 2.3-fold in patients with extrapulmonary TB compared with patients with purely pulmonary TB (p = 0.01) and was amplified 2.6-fold at disease sites relative to blood (p = 0.043).Regulatory T cells are expanded in patients with TB and may contribute to suppression of Th1-type immune responses.

Background Background rates of latent tuberculosis infection in low prevalence regions of Britain are unknown. These would be valuable data for interpreting positive IGRA results, and guiding cost-benefit analyses. The management of a large outbreak of tuberculosis occurring in a rural district hospital provided an opportunity to determine the background rates and epidemiology of IGRA-positivity amongst unselected hospital patients in a low-prevalence region of U.K. Methods As part of a public health surveillance project we identified 445 individuals exposed to the index cases for clinical assessment and testing by a TB-specific interferon-γ release assay (IGRA): T-Spot.TB. Uniquely, an additional comparator group of 191 age-matched individuals without specific recent exposure, but with a similar age distribution and demographic, were recruited from the same wards where exposure had previously occurred, to undergo assessment by questionnaire and IGRA. Results Rates of IGRA positivity were 8.7% (95%CI, 4.2-13, n=149) amongst unexposed patients, 9.5%(3.0-22, n=21) amongst unexposed staff, 22%(14–29, n=130) amongst exposed patients, 11%(6.1-16, n=142) amongst exposed staff. Amongst the individuals without history of recent exposure to the outbreak, IGRA-positivity was associated with prior TB treatment (OR11, P.04) and corticosteroid use (OR5.9, P.02). Background age-specific prevalences of IGRA-positivity amongst unexposed individuals were: age Conclusions Background rates of IGRA-positivity remain high amongst unselected white-Caucasian hospital inpatients in U.K. These data will aid interpretation of future outbreak studies. As rates peak in the 5th and 6th decade, given an ageing population and increasing iatrogenic immunosuppression, reactivation of LTBI may be a persistent hazard in this population for several decades to come.