Enhancing T cell responses and tumour immunity by vaccination with peptides conjugated to a weak NKT cell agonist.

Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.

Effects of treatment changes on asthma phenotype prevalence and airway neutrophil function.

BACKGROUND: Asthma inflammatory phenotypes are often defined by relative cell counts of airway eosinophils/neutrophils. However, the importance of neutrophilia remains unclear, as does the effect of ICS treatment on asthma phenotypes and airway neutrophil function. The purpose of this study was to assess asthma phenotype prevalence/characteristics in a community setting, and, in a nested preliminary study, determine how treatment changes affect phenotype stability and inflammation, with particular focus on airway neutrophils. METHODS: Fifty adult asthmatics and 39 non-asthmatics were assessed using questionnaires, skin prick tests, spirometry, exhaled nitric oxide (FENO) measurement, and sputum induction. Twenty-one asthmatics underwent further assessment following treatment optimisation (n = 11) or sub-optimisation (n = 10). RESULTS: Forty percent (20/50) had eosinophilic asthma (EA) and 8% had neutrophilic asthma. EA was associated with increased FENO, bronchodilator reversibility (BDR) and reduced lung function (p < 0.05). Following optimisation/sub-optimisation, the EA/NEA (non-eosinophilic asthma) phenotype changed in 11/21 (52%) asthmatics. In particular, fewer subjects had EA post treatment optimisation, but this was not statistically significant. However, a significant (p < 0.05) reduction in FENO, ACQ7 score, and BDR was observed after treatment optimisation, as well as an increase in FEV1-% predicted (p < 0.05). It was also associated with reduced eosinophils (p < 0.05) and enhanced neutrophil phagocytosis (p < 0.05) in EA only, and enhanced neutrophil oxidative burst in both EA and NEA (p < 0.05). CONCLUSIONS: In this community based population, non-eosinophilic asthma was common, less severe than EA, and at baseline most asthmatics showed no evidence of inflammation. In the nested change in treatment study, treatment optimisation was associated with reduced sputum eosinophils, improved symptoms and lung function, and enhanced neutrophil function, but a significant reduction in EA could not be demonstrated. TRIAL REGISTRATION: The nested change in treatment component of this study is registered at the Australia and New Zealand Clinical Trial Registry ( www.ANZCTR.org.au ) ACTRN12617001356358 . Registration date 27/09/2017. Retrospectively registered.

Absence of airway inflammation in a large proportion of adolescents with asthma.

BACKGROUND AND OBJECTIVE: Neutrophilic inflammation has been implicated in non-eosinophilic asthma (NEA) in adults, but little is known about NEA in children/adolescents. We assessed clinical and inflammatory characteristics of NEA in adolescent asthma. METHODS: Airway inflammation, sputum endotoxin, airway hyper-reactivity, atopy and lung function were assessed in 77 adolescents with asthma and 68 without asthma (12-17 years). Asthma was identified on the basis of wheeze and asthma history. RESULTS: The proportion of NEA (sputum eosinophils <2.5%) was 54%. In this group, atopy, sputum neutrophil, eosinophil, eosinophil cationic protein (ECP), endotoxin, neutrophil elastase and IL-8 levels were not different from those without asthma. In contrast, eosinophilic asthma (EA) was associated with atopy and sputum ECP and IL-8. The majority of NEA had no evidence of inflammation; only 14% had neutrophilia (≥61% neutrophils), compared with 11% of EA, and 15% of those without asthma. Small differences in FEV1 (NS) were found between EA and NEA, but symptom prevalence and severity was not different (63% of EA and 52% of NEA were classified moderate to severe). CONCLUSION: NEA is common in adolescent asthma and has similar clinical characteristics as EA. Neutrophils do not appear to play a role in NEA in adolescents, and underlying mechanisms may not involve airway inflammation.

Metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses.

Published studies have shown that workers in animal slaughterhouses are at a higher risk of lung cancers as compared to the general population. No specific causal agents have been identified, and exposures to several chemicals have been examined and found to be unrelated. Evidence suggests a biological aetiology as the risk is highest for workers who are exposed to live animals or to biological material containing animal faeces, urine or blood. To investigate possible biological exposures in animal slaughterhouses, we used a metagenomic approach to characterise the profile of organisms present within an aerosol sample. An assessment of aerosol exposures for individual workers was achieved by the collection of personal samples that represent the inhalable fraction of dust/bioaerosol in workplace air in both cattle and sheep slaughterhouses. Two sets of nine personal aerosol samples were pooled for the cattle processing and sheep processing areas respectively, with a total of 332,677,346 sequence reads and 250,144,492 sequence reads of 85 bp in length produced for each. Eukaryotic genome sequence was found in both sampling locations, and bovine, ovine and human sequences were common. Sequences from WU polyomavirus and human papillomavirus 120 were detected in the metagenomic dataset from the cattle processing area, and these sequences were confirmed as being present in the original personal aerosol samples. This study presents the first metagenomic description of personal aerosol exposure and this methodology could be applied to a variety of environments. Also, the detection of two candidate viruses warrants further investigation in the setting of occupational exposures in animal slaughterhouses.

Efficient depletion of chronic lymphocytic leukemia B cells using serial rounds of immunomagnetic depletion.

Functional studies of cellular immunity in patients with leukemia often require separation of leukemic cells from other peripheral blood mononuclear cells (PBMCs). This can pose a challenge when the number of leukemic cells is very high, such as in untreated patients with chronic lymphocytic leukemia (CLL). We found that when leukemia cell frequency was very high, anti-CD19 coated immunomagnetic beads did not thoroughly deplete B cells when used according to manufacturer's instructions. In this study, we depleted leukemic B cells using a modified protocol comprising serial rounds of depletion using immunomagnetic beads at reduced bead to cell ratios. This resulted in more effective B cell depletion with the use of fewer immunomagnetic beads, and without affecting viability or yield of non-B cells. CD19- PBMC subsets were retained, and serial depletion rounds did not activate T cells and monocytes. The positively isolated CLL cells were of high purity and were available for downstream analysis. This is a convenient and cost-effective method to enable in vitro analysis of immunocompetent cells from patients with leukemia.

Synergism of tapasin and human leukocyte antigens in resolving hepatitis C virus infection.

UNLABELLED: CD8+ T-cell responses to hepatitis C virus (HCV) are important in generating a successful immune response and spontaneously clearing infection. Human leukocyte antigen (HLA) class I presents viral peptides to CD8+ T cells to permit detection of infected cells, and tapasin is an important component of the peptide loading complex for HLA class I. We sought to determine if tapasin polymorphisms affected the outcome of HCV infection. Patients with resolved or chronic HCV infection were genotyped for the known G/C coding polymorphism in exon 4 of the tapasin gene. In a European, but not a US, Caucasian population, the tapasin G allele was significantly associated with the outcome of HCV infection, being found in 82.5% of resolvers versus 71.3% of persistently infected individuals (P = 0.02, odds ratio [OR] = 1.90 95% confidence interval [CI] = 1.11-3.23). This was more marked at the HLA-B locus at which heterozygosity of both tapasin and HLA-B was protective (P < 0.03). Individuals with an HLA-B allele with an aspartate at residue 114 and the tapasin G allele were more likely to spontaneously resolve HCV infection (P < 0.00003, OR = 3.2 95% CI = 1.6-6.6). Additionally, individuals with chronic HCV and the combination of an HLA-B allele with an aspartate at residue 114 and the tapasin G allele also had stronger CD8+ T-cell responses (P = 0.02, OR = 2.58, 95% CI-1.05-6.5). CONCLUSION: Tapasin alleles contribute to the outcome of HCV infection by synergizing with polymorphisms at HLA-B in a population-specific manner. This polymorphism may be relevant for peptide vaccination strategies against HCV infection.

Relationship between airway neutrophilia and ageing in asthmatics and non-asthmatics.

BACKGROUND AND OBJECTIVE: Increased sputum neutrophilia has been observed in asthma, but also during normal ageing in asthmatics and non-asthmatics. It remains unclear what constitutes 'normal' neutrophil levels in different age groups. METHODS: We assessed the relationship between age and airway neutrophils of 194 asthmatics and 243 non-asthmatics (age range: 6-80 years). Regression analyses were used to assess this relationship adjusted for confounders including asthma status, atopy, gender, smoking and current use of inhaled corticosteroids (ICS). Age-corrected reference values for different age groups were determined using the 95th percentile of non-asthmatic participants. RESULTS: Age was positively associated with sputum neutrophils in both asthmatic and non-asthmatic adults (0.46% neutrophil increase/year (95% confidence interval (CI) 0.18, 0.73) and 0.44%/year (0.25, 0.64, respectively), but no association was found in the <20-year age category. Individuals with high sputum neutrophil counts (>95th percentile of non-asthmatic counts for any given age group) were significantly more likely to be asthmatic (odds ratio = 2.5; 95% CI: 1.3, 5.0), with the greatest effect observed in the older age group. Other factors that independently associated with increased sputum neutrophil levels included atopy in non-asthmatic adults, male gender and current use of ICS in asthmatic adults. Age-specific reference values for neutrophil percentage were under 20 years-76%, 20-40 years-62%, 40-60 years-63% and over 60 years-67%. CONCLUSIONS: Airway neutrophilia is related to age in adults, with a neutrophilic asthma phenotype present in older adults. The use of appropriate age-specific reference values is recommended for future studies aimed at elucidating the role of neutrophils in asthma.

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry.

BACKGROUND: Airway inflammation is commonly assessed by sputum induction followed by a differential cell count (DCC) using light microscopy. This method is prone to intercounter variability and poor reproducibility. We aimed to develop a more objective method using flow cytometry (FCM). METHODS: Fifty-six sputum inductions were conducted in 41 adults (23 asthmatics). Sputum was processed, a cytospin prepared for DCC, and the remainder immunolabeled for FCM using CD45, CD14, and CD16-specific antibodies to distinguish major leukocyte populations. Aliquots of 15 samples were frozen at -80°C to assess the effects of cryostorage. DCC and FCM were compared, and viability of individual cell populations was determined by FCM. RESULTS: FCM and DCC, and fresh and frozen samples, were significantly correlated, R = 0.54-0.87; all P < 0.0001, and R = 0.57 to 1; P < 0.005, respectively. There was a significant neutrophil loss after cryostorage (from median 30.5-17.4% of total leukocytes; P < 0.0001). Cell viability was higher for lymphocytes compared to granulocytes or macrophages (P < 0.001). With the exception of the expected higher levels of eosinophils (P < 0.005), no significant difference in cell differentials or viability was observed between asthmatics and nonasthmatics using either DCC or FCM. CONCLUSIONS: FCM is a suitable means of assessing leukocyte populations in induced sputum. Sample storage at -80°C prior to FCM is feasible, but may be detrimental to neutrophils, although good correlations were still observed between fresh and frozen samples. Large differences in viability were found between individual cell populations suggesting that viability dye use may be necessary.

Functional invariant natural killer T-cell and CD1d axis in chronic lymphocytic leukemia: implications for immunotherapy.

Invariant natural killer T cells recognize glycolipid antigens such as α-galactosylceramide presented by CD1d. In preclinical models of B-cell malignancies, α-galactosylceramide is an adjuvant to tumor vaccination, enhancing tumor-specific T-cell responses and prolonging survival. However, numerical and functional invariant natural killer T-cell defects exist in patients with some cancers. Our aim was to assess this axis in patients with chronic lymphocytic leukemia. The numbers of circulating invariant natural killer T cells and the expression of CD1d on antigen-presenting cells were evaluated in patients with chronic lymphocytic leukemia and age-matched controls. Cytokine profile and in vitro proliferative capacity were determined. Patient- and control-derived invariant natural killer T-cell lines were generated and characterized, and allogeneic and autologous responses to α-galactosylce-ramide-treated leukemia cells were assessed. Absolute numbers and phenotype of invariant natural killer T cells were normal in patients with untreated chronic lymphocytic leukemia, and cytokine profile and proliferative capacity were intact. Chemotherapy-treated patients had reduced numbers of invariant natural killer T cells and myeloid dendritic cells, but α-galactosylceramide-induced proliferation was preserved. Invariant natural killer T-cell lines from patients lysed CD1d-expressing targets. Irradiated α-galactosylceramide-treated leukemic cells elicited allogeneic and autologous invariant natural killer T-cell proliferation, and α-galactosylceramide treatment led to increased proliferation of conventional T cells in response to tumor. In conclusion, the invariant natural killer T-cell and CD1d axis is fundamentally intact in patients with early-stage chronic lymphocytic leukemia and, despite reduced circulating numbers, function is retained in fludarabine-treated patients. Immunotherapies exploiting the adjuvant effect of α-galactosylceramide may be feasible.

Measurement of ß-(1,3)-glucan in household dust samples using Limulus amebocyte assay and enzyme immunoassays: an inter-laboratory comparison.

Environmental levels of ß-(1,3)-glucan, an inflammatory fungal cell wall component, have been suggested to be related to respiratory symptoms. However there is currently little data comparing ß-(1,3)-glucan detection methods and/or results obtained in different laboratories. The aim of this study was to compare levels of ß-(1,3)-glucans detected in household dust samples (n = 40) using different extraction/detection methods (Limulus amebocyte assay (LAL), inhibition enzyme immunoassay (EIA) and sandwich EIA) in five different laboratories. Dust sample aliquots were sent to participating centres, extracted and analysed for ß-(1,3)-glucan according to standard in-house procedures. Significant differences in the levels of ß-(1,3)-glucan were observed between all laboratories (geometric mean levels ranging from 15.4 µg g (-1) to 4754 µg g(-1) dust; p < 0.0001) with the exception of those using a similar LAL method. The inhibition EIA used in laboratory D produced mean ß-(1,3)-glucan measurements 80-100 times higher than the LAL assays, 4 times higher than the sandwich EIA in the same lab, 17.6 times those obtained with the EIA in lab E and 363 times those obtained in the EIA in laboratory C. Pearson's correlations generally showed significant associations between methods and laboratories, particularly those using similar methodology (R ranging from 0.5 to 0.8; p < 0.001), although some poor and even inverse correlations were observed. Bland-Altman analyses showed moderate to good agreement between most assays, although clear absolute differences were observed. In conclusion, although results obtained with different methods were often significantly correlated and therefore comparable in relative terms, direct comparison of results between laboratories and assays may be inappropriate.

Measurement of exhaled nitric oxide in a general population sample: a comparison of the Medisoft HypAir FE(NO) and Aerocrine NIOX analyzers.

BACKGROUND AND OBJECTIVE: Measuring the fraction of nitric oxide in exhaled breath (FE(NO)) is increasingly utilized to assess airway inflammation in asthma. The primary aim of this study was to compare exhaled nitric oxide measurements obtained using two devices from different manufacturers, that is, the recently marketed portable and electrochemical-based Medisoft HypAir FE(NO) and the well-established chemiluminescence-based Aerocrine NIOX analyzer, in an unselected population. METHODS: FE(NO) measurements were conducted in 106 subjects (86 healthy; 20 asthmatic; 56.6% atopic). Atopy and health status were assessed by skin prick tests and questionnaire, respectively. RESULTS: The two instruments showed strong correlation over a wide range of FE(NO) measurements (8-261.3 ppb with the HypAir, 5.6-156.8 ppb with the NIOX; r = 0.98; p < .0001). This correlation was observed in the population as a whole, as well as in healthy non-atopics, healthy atopics, and atopic asthmatics when considered separately. The measurements on the HypAir FE(NO) were consistently 1.6 times (95% CI 1.11-2.05) higher than those obtained with the NIOX. CONCLUSIONS: FE(NO) measurements obtained with the HypAir FE(NO) correlated well with the NIOX, but were approximately 1.6 times higher. Therefore, a conversion factor is required if results are to be compared with the NIOX instrument.

The inhibitory receptor NKG2A determines lysis of vaccinia virus-infected autologous targets by NK cells.

Signals transduced by inhibitory receptors that recognize self-MHC class I molecules prevent NK cells from being activated by autologous healthy target cells. In order for NK cells to be activated upon contact with an infected cell, the balance between the activating and inhibitory signals that regulate NK cell function must be altered in favor of activation. By studying liver-derived NK cells, we show that only a subpopulation of NK cells expressing high levels of the inhibitory receptor NKG2A are able to lyse autologous vaccinia-infected targets, and that this is due to selective down-regulation of HLA-E. These data demonstrate that release from an inhibitory receptor:ligand interaction is one mechanism that permits NK cell recognition of a virally infected target, and that the variegated expression of inhibitory receptors in humans generates a repertoire of NK cells with different antiviral potentials.

HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection.

Natural killer (NK) cells provide a central defense against viral infection by using inhibitory and activation receptors for major histocompatibility complex class I molecules as a means of controlling their activity. We show that genes encoding the inhibitory NK cell receptor KIR2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand directly influence resolution of hepatitis C virus (HCV) infection. This effect was observed in Caucasians and African Americans with expected low infectious doses of HCV but not in those with high-dose exposure, in whom the innate immune response is likely overwhelmed. The data strongly suggest that inhibitory NK cell interactions are important in determining antiviral immunity and that diminished inhibitory responses confer protection against HCV.

MHC class I receptors on natural killer cells: on with the old and in with the new.

The functions of natural killer (NK) cells are controlled by an abundance of activating and inhibitory receptors. Many of these interact with MHC class I molecules. The MHC system also interacts with cytotoxic T-lymphocytes and has been shown to comprise a rapidly evolving family of molecules. This challenges the functional relationship of NK cell receptors with their ligands. Although individual receptors have become subject to species-specific expansions over evolutionary time, the main themes of the NK cell interaction with MHC class I have been preserved. This review details the interaction of NK cell receptors with MHC class I and discusses their unexpectedly rapid evolution.