Non-atopic Controls Research Articles

Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis

BackgroundAtopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear.ObjectiveWe sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin.MethodsWe applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set.ResultsTwo thousand four hundred thirty differentially expressed genes (false discovery rate, P < .05) were identified, of which 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for “extracellular space” and “defense response” were enriched, whereas “lipid metabolic processes” were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferon–mediated stress response.ConclusionThese analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development.

Atopy patch test with Aleuroglyphus ovatus antigen in patients with atopic dermatitis

Epicutaneous test made with dust mite antigens. Evaluation of the response of the epicutaneous test withAleuroglyphus ovatus antigen in atopic patients. We patch tested 119 individuals, 48 with atopic dermatitis, 50 with respiratory allergy and 21 healthy controls. We compare the positive response frequency to a closed patch test usingAleuroglyphus ovatusantigen in different concentrations and 48 and 96h reading times among those individuals. Six patients with atopic dermatitis (12.5%) and 4 with respiratory atopy (8.0%) had positive reactions. None of the non-atopic controls had a positive response. As the antigen concentration raised, the number of positive reactions to epicutaneous test raised as well. Our data suggest a positive relation between Atopy Patch Test positive responses andAleuroglyphus ovatusantigen concentration, no matter the kind of the atopic clinical expression.

OX40 ligand: a potential costimulatory molecule in atopic asthma

Objective: We aimed to assess the percentage of peripheral blood B-lymphocytes expressing OX40 ligand (OX40L) in adult atopic and non-atopic asthmatic patients, and in healthy controls. Methods: This case–control study included 15 atopic asthmatic patients, 15 non-atopic asthmatic patients, and 15 healthy controls. Atopic status was determined by skin prick test reaction to the most common locally-encountered allergens. For all subjects, pulmonary function tests and measurement of total serum immunoglobulin E (IgE) levels by ELISA were performed. In addition, the percentage of B-lymphocytes expressing OX40L was assessed by flow cytometry in all three groups. Results: OX40L expression was significantly higher in atopic asthmatics than in non-atopic asthmatics and controls, but did not differ significantly between non-atopic asthmatics or controls. Among atopic asthmatics, OX40L expression correlated positively with total serum IgE levels, but not with age, disease duration, or values of forced expiratory volume in the first second. Conclusion: The over-expression of OX40L in atopic asthmatic patients appears to be linked to markers of the atopic status as total serum IgE, and signifies the vital role of OX40L in the atopic mechanism. Further large-scale studies are needed to investigate the role of OX40L in other atopic diseases and its relation to disease activity and severity.

Potential non‐T cells source of interleukin‐4 in food allergy

Recently, a study from the Consortium of Food Allergy Research (CoFAR) showed that allergen-induced IL-4 expression in CD25(+) mononuclear cells was increased in allergic patients. However, they did not find the expected increase in GATA-3 expression, suggesting that allergen-induced IL-4 might not be of T-cell origin. We sought to determine whether other cell types were responsible for the increased IL-4 expression in the CD25(+) cell population. Comparing six allergic patients and six healthy controls, we analyzed the CD25(+) isolated population from PBMC for the presence of potential IL-4-expressing non-T cells. We also compared spontaneous expression levels of surface markers (CD203c, CD63, CD25, and HLA-DR) on basophils from whole blood of 42 peanut-allergic patients and from 12 non-atopic controls. Expression of these markers was also evaluated following basophil activation in eight peanut-allergic patients selected from the previous cohort. In addition to CD4(+) T cells, a substantial proportion of non-T cells were found in the CD25(+) -isolated cell population: basophils, NK, and NK-T cells with a mean percentage ± s.e.m. of 5.24 ± 0.63%, 6.65 ± 1.01%, and 6.01 ± 1.04%, respectively. The majority of these cells exhibited positive intracytoplasmic staining for IL-4. Expression of CD63 and CD25 was significantly higher in allergic patients compared with controls (p < 0.05). Interestingly, we found a significantly higher proportion of activated basophils expressing HLA-DR, compared with non-activated basophils (p < 0.05). Our results support the suggested key role of non-T cells secreting IL-4 in food allergy, particularly basophils, which may also play a central role in antigen presentation.

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

Reference Values and Determinants of Fractional Concentration of Exhaled Nitric Oxide in Healthy Children

PurposeMeasurement of the fractional concentration of exhaled nitric oxide (FeNO) is a quantitative, noninvasive, simple, safe method of assessing airway inflammation. While FeNO measurement has been standardized, reference values for elementary school children are scarce. The aim of this study was to establish reference values for FeNO in children.MethodsFeNO was measured in elementary school children at 6-12 years of age in Seoul, Korea, following American Thoracic Society guidelines and using a chemiluminescence analyzer (NIOX Exhaled Nitric Oxide Monitoring System, Aerocrine, Sweden). A total of 1,252 children completed a modified International Study of Asthma and Allergy in Children (ISAAC) questionnaire; FeNO was measured in 1,063 children according to the protocol and in 808 children defined as healthy controls.ResultsMean FeNO were 10.32 ppb, 16.58 ppb, and 12.36 ppb in non-atopic, atopic, and all 808 healthy controls, respectively. FeNO was not associated with age and gender. The FeNO reference equations were determined by multiple linear regression analysis, taking into account the variables of age, height, weight, total IgE, eosinophil percent, and bronchial hyper-responsiveness (methacholine PC20). FeNO=0.776+0.003×total IgE+0.340×eosinophil percent; coefficient of determination (R2)=0.084 in the 501 healthy non-atopic controls. FeNO=-18.365+1.536×eosinophil percent, R2=0.183 in the 307 healthy atopic controls; and FeNO=-7.888+0.130×Height+0.004×total IgE+1.233×eosinophil percent, R2=0.209 in the 808 all healthy controls. Eosinophil percent was correlated with FeNO in all healthy controls. FeNO was not associated with BMI.ConclusionThis study provides reference values for FeNO that can be used to evaluate airway inflammation in elementary school children. Determinants that could most accurately predict FeNO in healthy school-age children were assessed.

Serial Changes in Serum Eosinophil-associated Mediators between Atopic and Non-atopic Children after Mycoplasma pneumoniae pneumonia.

PurposeMycoplasma pneumoniae pneumonia (MP) is associated with the exacerbation, timing, and onset of asthma. The goal of this study was to elucidate the impact of MP on eosinophil-related hyper-reactive amplification in atopic children.MethodsWe studied 48 patients with MP (26 atopic, 22 non-atopic), between 3 and 12 years of age. Serial changes in blood eosinophil counts, serum interleukin-5 (IL-5), and serum eosinophil cationic protein (ECP) levels were measured in atopic and non-atopic children with MP upon admission, recovery, and at 2 months post-recovery. Serum IL-5 and ECP levels were measured by enzyme-linked immunosorbent assays; eosinophil counts were measured using an autoanalyzer.ResultsSerial changes in serum IL-5, ECP, and total eosinophil counts were significantly higher in atopic patients, relative to non-atopic controls (P≤0.001). Serum IL-5 and ECP levels were significantly higher in atopic patients at all three time points tested, while eosinophil counts were higher in the clinical recovery and follow-up phases, but not in the acute phase. Furthermore, among atopic patients, serum ECP levels were significantly higher in the recovery and follow-up phases than in the acute phase.ConclusionsThe present study demonstrated significant differences in eosinophil counts, serum IL-5, and serum ECP levels between atopic and non-atopic children with MP at admission, recovery, and 2 months after clinical recovery. These outcomes are suggestive of eosinophil-related hyperreactivity in atopic children, with this status maintained for at least 2 months after MP.

Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

BackgroundAirway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.ObjectiveTo investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.MethodsHuman bronchial fibroblasts and CD4+T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4+T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.ResultsCo-culture of CD4+T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17+/CCR6+ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4+T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.ConclusionInteraction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.

Elevated levels of exhaled nitric oxide in patients with anorexia nervosa

BackgroundNitric oxide (NO) is involved in eating behavior and inflammatory response. Moreover, there is evidence that NO production is altered in patients with anorexia nervosa (AN).AimTo assess whether the overproduction of NO in AN can affect NO level in exhaled air.Materials and methodsExhaled NO level was studied in 23 girls with AN and compared with that of healthy age- and gender-matched nonatopic controls.ResultsExhaled NO levels were significantly higher in girls with AN compared with healthy age-matched controls.ConclusionsIt appears that anorexia nervosa was accompanied by a higher level of exhaled NO, likely resulting from a systemic increase in NO production because of the severe catabolic state.

Mast cells, Mast/Stem Cell Growth Factor receptor (c-kit/cd117) and IgE may be integral to the pathogenesis of endemic pemphigus foliaceus

Introduction: Pemphigus foliaceus (PF) is endemic in some South American countries, especially in Colombia and Brazil; in Brazil, it is also known as fogo selvagem (FS). We aimed to study the presence of mast cells and the expression of the mast/stem cell growth factor receptor (c-kit/CD117) in PF skin biopsies, as well as the role of IgE in the disease pathogenesis. Methods: Forty-four skin biopsies from patients affected by endemic PF (EPF) (30 patients from El Bagre, Colombia, and 14 from the northeastern region of Sao Paulo State, Brazil), 48 control biopsies from Colombian and Brazilian endemic areas, and additional control biopsies from none endemic areas in Colombia and the USA non were studied. Immunohistochemistry (IHC) was performed to evaluate skin biopsies with anti-mast cell tryptase (MCT), anti-c-kit and anti-IgE antibodies. We also searched for serum IgE in 30 EPF and 30 non-atopic controls from the El Bagre region via ELISA. In our El Bagre patients and controls, we also searched for IgE in skin samples by direct immunofluorescence. Results: In 100% of the EPF biopsies, MCT, c-kit and IgE were identified with stronger expression relative to control biopsies, especially in the inflammatory infiltrates around upper dermal blood vessels and dermal eccrine glands. IgE staining was positive along the BMZ in some EPF skin samples. The DIF results confirmed a linear deposition of IgE at the BMZ. Increased IgE serum levels were also noted in PF patients relative to controls.. Conclusions: In patients with EPF, the observed increased expression of MCT, c-kit and IgE in lesional skin, associated with higher serum IgE levels may indicate possible IgE participation in the antigenic response.

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cohort, were genotyped in 451 unrelated Atopic dermatitis patients and 375 non-atopic controls by means of restriction enzyme digestion. Allele, genotype and haplotype frequencies were subsequently compared between cases and controls. Results In this analysis, none of the investigated Single nucleotide polymorphisms or haplotypes in the Claudin-1 gene showed a significant association with Atopic dermatitis. Conclusion Our results do not support a substantial role for Claudin-1 variation in Atopic dermatitis pathogenesis. Yet, additional studies in independent cohorts are needed to confirm these results.

Human Leukocyte Antigen-G and Regulatory T Cells during Specific Immunotherapy for Pollen Allergy

Background: T_H2-biased immune responses are important in allergy pathogenesis. Mechanisms of allergen-specific immunotherapy (SIT) might include the induction of regulatory T cells (Tregs) and immunoglobulin (Ig) G₄ blocking antibodies, a reduction in the number of effector cells, and skewing of the cytokine profile towards a T_H1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG₄). Methods: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG₄ were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with pollen extract in vitro and immune factors were evaluated. Results: During SIT, the main changes in the peripheral blood were an increase in CXCR3⁺CD4⁺CD25⁺CD127^low/- Tregs and a decrease in CCR4⁺CD4⁺CD25⁺CD127^low/- Tregs, an increase in allergen-specific IgG₄, and a decrease in sHLA-G during the first half of the treatment period. In the PBMC in vitro experiments, the following changes were observed upon allergen-stimulation: an increase in CD4⁺CD25⁺CD127^low/- Tregs and ILT2⁺CD4⁺CD25⁺CD127^low/- Tregs, an increase in IL-10 and IL-2 levels, and an increase in sHLA-G that was most pronounced at the start of SIT. Conclusions: The changes in CXCR3⁺CD4⁺CD25⁺CD127^low/- Treg, IgG₄, and sHLA-G levels in the peripheral blood and in ILT2⁺ Treg, IL-10, IL-2, and sHLA-G levels upon in vitro allergen stimulation suggest an upregulation in immunomodulatory factors and, to some degree, a shift towards T_H1 during SIT.

Enterovirus infections in early childhood and the risk of atopic disease – a nested case‐control study

Enterovirus infections in childhood have been associated with a reduced risk of atopy in cross-sectional studies. To study the relation between enterovirus infections in the first 2years of life and atopic disease with IgE sensitization in a prospective study setting. This was a nested case-control study among children who had been followed from birth. Neutralizing antibodies against 12 enterovirus serotypes were analysed at the age of 2years from 71 atopic children and 142 non-atopic control children. Atopy was defined as having an atopic disease and IgE antibodies against at least one aeroallergen by the age of 5years. Cumulative exposure to different enterovirus serotypes was inversely associated with atopy [odds ratio (OR) 0.73; 95% confidence interval (CI): 0.56-0.96]. The most pronounced protection was seen when echoviruses were analysed as a separate group (OR 0.63; 95%CI: 0.46-0.88). We propose that exposure to several different enteroviruses in early childhood is inversely associated with atopic diseases. Our results support the hypothesis that repeated microbial infections in early life may protect from atopic sensitization and atopic diseases.

Imbalance of serum IL-10 and TGF-β in patients with pollen food syndrome

BackgroundPollen food syndrome is one of the main causes of food allergies in adults. However, the intrinsic immunological mechanisms remain unclear. MethodsForty pollinosis sufferers [23 with a food allergy (PSFA) and 17 without a food allergy (PS)] and 17 non-atopic healthy controls were included in this study. The PSFA group was subdivided into an oral allergy syndrome group, a systemic reaction group, and an anaphylactic reaction group according to their symptoms after eating the suspected foods. Serum IL-10 and TGF-β levels of all participants were determined by ELISA. Clinical characteristics of the patients were also evaluated. ResultsThere were no significant differences in age, sex, pollen-associated symptoms, duration of respiratory disease, and positive parental history of atopy between the PSFA and PS groups. Compared to healthy controls, serum IL-10 levels of both the PSFA group and PS group were significantly lower (p≤0.01), but TGF β levels were significantly higher in the PSFA group (35.3±5.6ng/ml vs. 31.2±6.6ng/ml, respectively; p=0.037). Within the PSFA group, IL-10 levels in the anaphylactic reaction subgroup were significantly lower compared to oral allergy syndrome subgroup (1.87±0.47pg/ml vs. 1.40±0.30pg/ml, respectively; p=0.027). More severe food allergy symptoms were associated with lower serum IL-10 levels. In contrast, the highest serum levels of TGF-β were found in patients from the anaphylactic reaction subgroup. ConclusionsWith the exception of a defect in regulatory cells represented by the reduction of IL-10, other potential immunological mechanisms (e.g., Th17 or IL-23 together with TGF-β) may be involved in the development of pollen food syndrome.

Characterisation of CD154+ T cells following ex vivo birch allergen stimulation defines a close relationship between T cell subsets in healthy volunteers

BackgroundAllergic sensitisation has been ascribed to a dysregulated relationship between allergen-specific Th1, Th2 and regulatory T cells. We hypothesised that the relationship between these T cell subsets could be better defined using a short-term allergen stimulation system followed by direct analysis of CD154-positive T cells. Using peripheral blood samples from birch pollinosis patients and healthy non-atopic controls, we sought to explore the frequencies and phenotype of birch-stimulated CD154-positive T helper cells following ex vivo birch allergen stimulation.ResultsActivated CD154-positive Th1, Th2 and Tr1-like cells, that co-expressed IFNγ, IL-4 and IL-10 respectively, were identified in both birch-allergic and non-allergic participants. We observed a close correlation between Th1, Th2 and Tr1-like cell frequency in non-allergic volunteers, such that the three parameters increased together to maintain a low Th2: Th1 ratio. The relationship between Th1, Th2 and Tr1-like responses was dysregulated in birch-allergic patients, with abrogation of the IL-10 response and a higher Th2: Th1 ratio. A close correlation was observed between Th2 cell frequency and the absolute concentration of birch-specific IgE within the birch-allergic group, and we confirmed previous reports of a more differentiated T cell phenotype in allergic subjects.ConclusionsThe findings demonstrate an important balance between IFNγ, IL-4 and IL-10 T cell responses to birch allergen in health, where Th2 responses to allergens were frequently observed, but apparently balanced by Th1 and regulatory responses. The detection of CD154 positive T cells after short-term antigen stimulation may be a useful method for the detection of T cell responses to allergens when cost, speed and convenience are priorities.

Increased expression of CD69 antigen on human peripheral blood natural killer cells in patients with allergic rhinitis.

Allergic rhinitis (AR) is an inflammatory disorder of the nasal mucosa with high morbidity and prevalence. Natural killer (NK) cells might have a role in AR. We aimed to evaluate the changes of the markers and receptors on NK cells in AR patients compared to the non-atopic controls.Flow cytometric analysis was used with double staining of the Peripheral Blood Mononuclear Cells (PBMCs) to examine the expression of CD25 and CD69 markers, and NKG2D and NKG2A receptors on NK cells of 20 patients with AR and 20 non-atopic controls. The serum total IgE level was measured by Enzyme-linked Immunosorbent Assay.The expression of CD69 antigen on NK cells in AR patients was significantly higher than that of healthy group (p=0.03). No significant changes were observed between CD25, NKG2D and NKG2A expression on the surface of NK cells from healthy and AR subjects. Our study also showed that there was no significant correlation between the expression of CD69, CD25, NKG2D and NKG2A and level of serum total IgE in AR patients and normal subjects.These results indicated that the expression of CD69 antigen on NK cells of AR patients was increased. The high expression of CD69 on NK cells in AR patients suggested that these cells were activated, probably due to the cytokines secreted from allergen-stimulated T cells and activated monocytes.

Transcriptional Analysis of Hair Follicle-Derived Keratinocytes from Donors with Atopic Dermatitis Reveals Enhanced Induction of IL32 Gene by IFN-γ

We cultured human hair follicle-derived keratinocytes (FDKs) from plucked hairs. To gain insight into gene expression signatures that can distinguish atopic dermatitis from non-atopic controls without skin biopsies, we undertook a comparative study of gene expression in FDKs from adult donors with atopic dermatitis and non-atopic donors. FDK primary cultures (atopic dermatitis, n = 11; non-atopic controls, n = 7) before and after interferon gamma (IFN-γ) treatment were used for microarray analysis and quantitative RT-PCR. Comparison of FDKs from atopic and non-atopic donors indicated that the former showed activated pathways with innate immunity and decreased pathways of cell growth, as indicated by increased NLRP2 expression and decreased DKK1 expression, respectively. Treatment with IFN-γ induced the enhanced expression of IL32, IL1B, IL8, and CXCL1 in the cells from atopic donors compared to that in cells from non-atopic donors at 24 h after treatment. IL1B expression in FDKs after IFN-γ treatment correlated with IL32 expression. We hypothesized that overexpression of IL32 in hair follicle keratinocytes of patients with atopic dermatitis would lead to the excessive production of pro-IL1β and that the activation of IL1β from pro-IL1β by inflammasome complex, in which NLRP2 protein might be involved, would be augmented. This is the first report to show enhanced induction of cytokine/chemokine genes by IFN-γ in atopic dermatitis using cultured FDKs.

Differences in fungi present in induced sputum samples from asthma patients and non-atopic controls: a community based case control study

BackgroundThere is emerging evidence for the presence of an extensive microbiota in human lungs. It is not known whether variations in the prevalence of species of microbiota in the lungs may have aetiological significance in respiratory conditions such as asthma. The aim of the study was to undertake semi-quantitative analysis of the differences in fungal species in pooled sputum samples from asthma patients and controls.MethodsInduced sputum samples were collected in a case control study of asthma patients and control subjects drawn from the community in Wandsworth, London. Samples from both groups were pooled and then tested for eukaryotes. DNA was amplified using standard PCR techniques, followed by pyrosequencing and comparison of reads to databases of known sequences to determine in a semi-quantitative way the percentage of DNA from known species in each of the two pooled samples.ResultsA total of 136 fungal species were identified in the induced sputum samples, with 90 species more common in asthma patients and 46 species more common in control subjects. Psathyrella candolleana, Malassezia pachydermatis, Termitomyces clypeatus and Grifola sordulenta showed a higher percentage of reads in the sputum of asthma patients and Eremothecium sinecaudum, Systenostrema alba, Cladosporium cladosporioides and Vanderwaltozyma polyspora showed a higher percentage of reads in the sputum of control subjects. A statistically significant difference in the pattern of fungi that were present in the respective samples was demonstrated using the Phylogenetic (P) test (P < 0.0001).ConclusionThis study is novel in providing evidence for the widespread nature of fungi in the sputum of healthy and asthmatic individuals. Differences in the pattern of fungi present in asthma patients and controls merit further investigation. Of particular interest was the presence of Malassezia pachydermatis, which is known to be associated with atopic dermatitis.

Antigen-Specific TH Cell Monitoring by CD154 Expression in Japanese Cedar-Specific SCIT

M O N D A Y 721 Local Nasal 'Protective' Immunoglobulin G4 (IgG4) Responses in Nasal Fluid Following Grass Pollen Sublingual Immunotherapy Nausheen Saleem, BSc(Hons), Adam Chaker, MD, Ulrich Zissler, Carsten B. Schmidt-Weber, PhD, Stephen R. Durham, MA, MD, FRCP, Mohamed H. Shamji, BSc, MSc, PhD; Imperial College London, Universitatsklinkum Dusseldorf, Dusseldorf, Germany, ENT department, TU Munich, M€unchen, Germany, ENT department, TU Munich, Imperial College, London, United Kingdom, Medical Research Council and Asthma UK Centre for Allergic Mechanisms of Asthma, UK, MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, Imperial College London, United Kingdom. RATIONALE: Grass pollen sublingual immunotherapy (SLIT) is associated with induction of serum IgG4-associated blocking antibodies that prevent IgE-facilitated allergen binding to B cells (IgE-FAB). We hypothesised that local nasal inhibitory IgG4 antibodies are also induced following SLIT. METHODS: Nasal fluids were obtained from SLIT-treated patients (n57), untreated allergics (n57) and non-atopic controls (n58). Specific IgE and IgG4 to Phleum pratense (Phl p) components were measured by ISAC microarray technology. Inhibitory activity of nasal fluid and serum was measured using the IgE-FAB assay. RESULTS: Untreated allergics had elevated levels of rPhl p 1 (p50.01) and rPhl p 5-specific IgE in nasal fluid compared to non-atopic controls. SLIT-treated patients showed a trend for an increase in rPhl p 1 (mean+/SE, 6.062.7 ISU) and rPhl p 5-specific IgE (2.162.6 ISU) compared to untreated allergics (p50.12). Recombinant Phl p 5-specific IgG4 levels were increased in SLIT-treated patients compared to untreated allergics (p50.02) and non-atopic controls (p50.003). Inhibitory activity in nasal fluid for co-operative binding of allergen-IgE complexes to B cells in SLITtreated patients was significantly reduced (p50.004) compared to untreated allergics. These decreases in local nasal inhibitory activity paralleled reductions in serum inhibitory activity after SLIT (p50.03). Interestingly, the magnitude of this suppression was more profound with local antibodies (45%) compared to the peripheral antibody responses (35%). CONCLUSIONS: For the first time, IgG4 antibodies with inhibitory activity for IgE-FAB were measured in the nasal fluid of SLIT treated patients. Local IgG4 antibodies may potentially be used as a surrogate/ predictive biomarker for monitoring SLIT.

IL-35+ Regulatory T Cells Suppress Grass Pollen-Driven Th2 Responses and Are Induced Following Grass Pollen-Specific Sublingual Immunotherapy

Inducible IL-35 producing T cells are a novel subset of regulatory T cells (iTR35). We hypothesized that i) iTR35 suppress grass pollen-specific Th2 responses following ex-vivo allergen stimulation in an IL-35-dependent manner ii) iTR35 are induced following grass pollen-specific sublingual immunotherapy T effector cells from grass pollen allergics (n=9) were co-cultured with irradiated APCs and 3mcg/mL of Phleum pratenseextract in the presence/absence of iTR35 cells. Th2 cytokines and proliferative responses were measured by Luminex MagPix assay and 3H-thymidine incorporation, respectively. We quantified iTR35 cells, IL-10 and Foxp3+ Tregs in PBMCs obtained from grass pollen sublingual immunotherapy (SLIT)-treated patients, untreated allergic subjects and non-atopic controls. iTR35 cells significantly suppressed Th2 cytokines (IL-4, p=0.002; IL-5, p=0.001 and IL-13, p<0.001) and grass pollen-driven proliferative responses (p=0.006). This suppression was IL-35 (p=0.004), partially IL-10 (p=0.02) but not TGF-b-dependent. In untreated allergics, iTR35 cells (p=0.03) and IL-10+Tregs (p=0.03) were lower compared to non-atopic controls. Interestingly, the SLIT-treated group had higher proportions of iTR35 cells and IL-10+ Tregs compared to untreated allergic controls. In the SLIT-treated group, mRNA expression for IL-12p35 (p=0.01), EBI3 (p=0.04) and IL-10 (p=0.02) in allergen stimulated PBMCs was increased compared to untreated allergic controls. Foxp3+ Tregs were lower in untreated allergics (p=0.03) and the SLIT-treated group (p=0.03) compared to non-allergic controls, whereas ‘activated’ GARP+Foxp3+ T regs were increased in the SLIT-treated group (p=0.03) compared to controls. iTR35 cells suppress grass pollen-driven memory Th2 cell responses and are induced following SLIT. Whether iTR35 cells contribute to long-term tolerance remains to be tested.