Introduction and Objectives Biologic efficacy for severe asthma varies depending on age of asthma onset and inflammatory phenotype. This post hoc analysis assessed the effect of tezepelumab in patients with severe, uncontrolled asthma with childhood-onset and current allergic or non-eosinophilic disease and adult-onset and current eosinophilic or non-allergic disease using pooled data from the phase 2b PATHWAY and phase 3 NAVIGATOR studies. Methods PATHWAY (NCT02054130) and NAVIGATOR (NCT03347279) were multicentre, randomized, placebo-controlled studies with similar designs. Included patients (12–80 years old) received tezepelumab 210 mg or placebo subcutaneously every 4 weeks for up to 52 weeks. Annualized asthma exacerbation rates (AAERs) over 52 weeks were assessed in patients grouped by age of asthma onset (childhood-onset, <18 years; adult-onset, ≥18 years) in combination with inflammatory phenotype (non-eosinophilic, baseline blood eosinophil count [BEC] <150 cells/μL; eosinophilic, baseline BEC ≥150 cells/μL; or allergy to a perennial aeroallergen [positive/negative serum specific immunoglobulin E test]). Subgroups were not mutually exclusive. Results Tezepelumab reduced the AAER over 52 weeks versus placebo by 53% (95% CI: 34–67) in patients with childhood-onset, allergic asthma (tezepelumab, n=181; placebo, n=180); 49% (95% CI: 9–72) in patients with childhood-onset, non-eosinophilic asthma (tezepelumab, n=58; placebo, n=57); 67% (95% CI: 57–74) in patients with adult-onset, eosinophilic asthma (tezepelumab, n=330; placebo, n=332); and 54% (95% CI: 37–67) in patients with adult-onset, non-allergic asthma (tezepelumab, n=198; placebo, n=204). Conclusions Tezepelumab reduced exacerbations versus placebo in a broad population of patients with severe, uncontrolled asthma grouped by age of onset and inflammatory phenotype. Please refer to page A287 for declarations of interest related to this abstract.

SummaryBackgroundChildren with risk alleles at the 17q21 genetic locus who wheeze during rhinovirus illnesses have a greatly increased likelihood of developing childhood asthma. In mice, overexpression of the 17q21 gene ORMDL3 leads to airway remodelling and hyperresponsiveness. However, the mechanisms by which ORMDL3 predisposes to asthma are unclear. Previous studies have suggested that ORMDL3 induces endoplasmic reticulum (ER) stress and production of the type I interferon (IFN)‐regulated chemokine CXCL10.ObjectiveThe purpose of this study was to determine the relationship between ORMDL3 and rhinovirus‐induced ER stress and type I IFN in human leucocytes.MethodsER stress was monitored by measuring HSPA5, CHOP and spliced XBP1 gene expression, and type I IFN by measuring IFNB1 (IFN‐β) and CXCL10 expression in human cell lines and primary leucocytes following treatment with rhinovirus. Requirements for cell contact and specific cell type in ORMDL3 induction were examined by transwell assay and depletion experiments, respectively. Finally, the effects of 17q21 genotype on the expression of ORMDL3, IFNB1 and ER stress genes were assessed.ResultsTHP‐1 monocytes overexpressing ORMDL3 responded to rhinovirus with increased IFNB1 and HSPA5. Rhinovirus‐induced ORMDL3 expression in primary leucocytes required cell–cell contact, and induction was suppressed by plasmacytoid dendritic cell depletion. The degree of rhinovirus‐induced ORMDL3, HSPA5 and IFNB1 expression varied by leucocyte type and 17q21 genotype, with the highest expression of these genes in the asthma‐associated genotype.Conclusions and Clinical RelevanceMultiple lines of evidence support an association between higher ORMDL3 and increased rhinovirus‐induced HSPA5 and type I IFN gene expression. These associations with ORMDL3 are cell type specific, with the most significant 17q21 genotype effects on ORMDL3 expression and HSPA5 induction evident in B cells. Together, these findings have implications for how the interaction of increased ORMDL3 and rhinovirus may predispose to asthma.

SummaryBackgroundAsthma exacerbations contribute to significant morbidity, mortality and healthcare utilization. Furthermore, viral infections are associated with asthma exacerbations by mechanisms that are not fully understood.ObjectiveThe aim of this analysis was to determine whether cytokine patterns in patients with colds could identify risks for subsequent asthma exacerbations.MethodsWe analysed cytokine levels in nasal lavage fluid (NLF) in 59 subjects (46 with asthma) with acute upper respiratory symptoms and after symptomatic resolution. Analyte choice was based on potential relevance to asthma exacerbations: antiviral (IFN‐α, IFN‐β, IFN‐γ, IFN‐λ1, IP‐10, TRAIL), cell recruiting (G‐CSF, IL‐1β, IL‐8, MCP‐1, MCP‐3, TNF‐α), polarizing (CXCL13, IL‐10, IL‐13, IL‐17, TSLP), and injury remodelling (fibronectin, IL‐33, MMP‐9, VEGF).ResultsThe overall cytokine response induced during viral infections was not different between asthmatic and non‐asthmatic individuals for a wide array of cytokines. However, mean levels of VEGF, TNF‐α and IL‐1β were 1.7‐, 5.1‐ and 4.7‐fold higher in samples from asthma subjects who exacerbated in the first 3 weeks of the cold compared with those who did not exacerbate (P = 0.006, 0.01, 0.048, respectively). Using receiver operating characteristic curve‐defined thresholds, high VEGF and TNF‐α levels predicted a shorter time‐to‐exacerbation after NLF sampling (25% exacerbation rate: 3 vs. 45 days, and 3 vs. 26 days; P = 0.03, 0.04, respectively).Conclusion and Clinical RelevanceAlthough they produce similar cytokine responses to viral infection as non‐asthmatics, asthmatics with higher levels of VEGF and TNF‐α in NLF obtained during acute cold phases predicted subsequent asthma exacerbations in this cohort of patients with mild‐to‐moderate disease. In the future, stratifying the risk of an asthma exacerbation by cytokine profile may aid the targeting of personalized treatment and intervention strategies.

SummaryBackgroundThe majority of asthma exacerbations are related to viral respiratory infections. Some, but not all, previous studies have reported that low interferon responses in patients with asthma increase the risk for virus‐induced exacerbations.ObjectiveWe sought to determine the relationship between lower airway inflammatory biomarkers, specifically interferon gene expression, and the severity or presence of an exacerbation in asthmatics experiencing a naturally occurring viral infection.MethodsSputum samples were analysed from subjects in an asthma exacerbation study who experienced a confirmed viral infection. Subjects were monitored for daily symptoms, medication use and peak expiratory flow rate until baseline. Sputum samples were assessed for cell counts and gene expression.ResultsInterferon gamma expression was significantly greater in patients with asthma exacerbations compared to non‐exacerbating patients (P = 0.002). IFN‐α1, IFN‐β1 and IFN‐γ mRNA levels correlated with the peak Asthma Index (r = 0.58, P < 0.001; r = 0.57, P = 0.001; and r = 0.51, P = 0.004, respectively). Additionally, IL‐13, IL‐10 and eosinophil major basic protein mRNA levels were greater in patients with asthma exacerbations compared to non‐exacerbating patients (P = 0.03, P = 0.06 and P = 0.02, respectively), and IL‐13 mRNA correlated with the peak Asthma Index (P = 0.006).ConclusionsOur findings indicate that asthma exacerbations are associated with increased rather than decreased expression of interferons early in the course of infection. These findings raise the possibility that excessive virus‐induced interferon production during acute infections can contribute to airway inflammation and exacerbations of asthma.

Chronic idiopathic urticaria (CIU) is increasingly recognized to have an autoimmune mechanism. We present characterization of the prevalence of autoimmune markers in CIU, their patterns of expression, and implications for disease course. We retrospectively evaluated 195 patients with a diagnosis of CIU over a 2-yr span for the presence of autoimmunity including antinuclear antibody (ANA), anti-microsomal antibody (ATPO), anti-thyroglobulin antibody (ATG), and chronic urticaria (CU) index. The patients were categorized into controlled and refractory subgroups based on their response to the use of antihistamines with or without the use of a leukotriene receptor antagonist. Among those tested for the respective autoimmune markers in our CIU cohort, the prevalence of a positive test for ANA (titer>1:160), ATPO, ATG, and CU Index were 29%, 6%, 26%, and 38% respectively. In comparing controlled and refractory subgroups, the percent of ANA positive (22.6 vs 40.4; p=0.04) and CU index positive (15.0 vs 52.0; p=0.01) patients differed significantly; however, a similar relationship was not observed for ATPO or ATG antibodies. Furthermore, in those patients with positive CU indices or ANA, the percent of patients categorized as refractory was higher. Logistic regression analysis of individual autoimmune markers and combination of markers in CIU suggests that the CU index independently offers the best characteristics to help predict clinical course. Our data indicates the CU index independently is a better predictor of disease severity than any combination of additional autoimmune markers (ANA, ATG, or ATPO).

RATIONALE: Eosinophilic asthma and nasal polyps are characterized by prominent eosinophilic inflammation. In a study of the humanized antibody to IL-5, reslizumab, in patients with eosinophilic asthma, we evaluated a subgroup with nasal polyps. METHODS: Patients with poorly controlled asthma on high-dose inhaled corticosteroids and ≥3% sputum eosinophils were randomized to reslizumab 3.0mg/kg or placebo at weeks 0, 4, 8, and 12. A pre-planned analysis was performed on data from patients with a history of nasal polyps. Asthma Control Questionnaire (ACQ) score, clinical asthma exacerbations (CAEs), and FEV1 were assessed at week 15. RESULTS: 106 patients were enrolled; 38 had nasal polyps (age: 48 years; sputum eosinophils: 17.7%; ACQ: 2.6; FEV1: 2.1L/s). In this subgroup, ACQ score improved significantly following reslizumab versus placebo (-0.94 ± 0.35, p=0.01). No patients in the reslizumab group had a CAE versus 19% in the placebo group (p=0.04). In patients without nasal polyps (age: 44 years; sputum eosinophils: 7.5%; ACQ: 2.7; FEV1: 2.2L/s) who received reslizumab, there were no significant changes in ACQ score or incidence of CAEs. Changes in FEV1 in the reslizumab versus placebo groups were 0.20 ± 0.10L/s (p=0.062) in patients with polyps and 0.25 ± 0.11L/s (p=0.026) in patients without polyps. Changes in responses to the individual symptom questions led to the improved ACQ scores in the polyp subgroup. CONCLUSIONS: In patients with poorly controlled eosinophilic asthma who received reslizumab, FEV1 improved regardless of the presence of nasal polyps. Asthma symptom control and exacerbations improved in the subgroup with nasal polyps who received reslizumab, but not in those without nasal polyps.

RATIONALE: Airway eosinophilia is a risk factor for virus-induced wheezing and exacerbations of asthma. We sought to determine whether eosinophils inhibit the epithelial cell antiviral response to human rhinovirus (HRV) infection.METHODS: We utilized an in vitro cell culture model consisting of BEAS-2B epithelial cell line, primary human eosinophils, and poly-IC or HRV-1A. BEAS-2B cells were cultured for 24 - 48 hours and poly-IC (25 mcg/mL) or HRV-1A (20 MOI) was added with or without eosinophils (106 per mL) for an additional 24 hours. Transwell plates were used to separate the eosinophils and BEAS-2B cells. A pharmacological inhibitor (SB 431542) of the activin receptor-like kinase (ALK5) was added to the culture to inhibit TGF-beta signaling. Supernatants were collected for ELISA and RNA was prepared from BEAS-2B cells for quantitative PCR of IFN-beta1 and IFN-lambda1.RESULTS: Both poly-IC and HRV-1A induced BEAS-2B expression of IFN-beta1 and IFN-lambda1 mRNA. The addition of eosinophils resulted in a 74% suppression of poly-IC induced IFN-lambda1 (p=0.023) and a tendency for 30% suppression of IFN-beta1 (p=0.11). Eosinophils also suppressed HRV-1A induced IFN-beta1 (83%, p=0.002) and IFN-lambda1 (80%, p=0.002). Separation of eosinophils from the BEAS-2B cells in transwell plates did not affect the suppression. TGF-beta1 protein concentrations were elevated in the cultures containing eosinophils. Inhibition of TGF-beta signaling abrogated eosinophil mediated suppression of IFN-beta1 and IFN-lambda1.CONCLUSIONS: Eosinophil release of TGF-beta suppresses HRV-induced epithelial cell expression of IFN-beta1 and IFN-lambda1. Since these interferons have antiviral effects, this may represent a mechanism by which airway eosinophilia promotes virus-induced exacerbations of asthma. RATIONALE: Airway eosinophilia is a risk factor for virus-induced wheezing and exacerbations of asthma. We sought to determine whether eosinophils inhibit the epithelial cell antiviral response to human rhinovirus (HRV) infection. METHODS: We utilized an in vitro cell culture model consisting of BEAS-2B epithelial cell line, primary human eosinophils, and poly-IC or HRV-1A. BEAS-2B cells were cultured for 24 - 48 hours and poly-IC (25 mcg/mL) or HRV-1A (20 MOI) was added with or without eosinophils (106 per mL) for an additional 24 hours. Transwell plates were used to separate the eosinophils and BEAS-2B cells. A pharmacological inhibitor (SB 431542) of the activin receptor-like kinase (ALK5) was added to the culture to inhibit TGF-beta signaling. Supernatants were collected for ELISA and RNA was prepared from BEAS-2B cells for quantitative PCR of IFN-beta1 and IFN-lambda1. RESULTS: Both poly-IC and HRV-1A induced BEAS-2B expression of IFN-beta1 and IFN-lambda1 mRNA. The addition of eosinophils resulted in a 74% suppression of poly-IC induced IFN-lambda1 (p=0.023) and a tendency for 30% suppression of IFN-beta1 (p=0.11). Eosinophils also suppressed HRV-1A induced IFN-beta1 (83%, p=0.002) and IFN-lambda1 (80%, p=0.002). Separation of eosinophils from the BEAS-2B cells in transwell plates did not affect the suppression. TGF-beta1 protein concentrations were elevated in the cultures containing eosinophils. Inhibition of TGF-beta signaling abrogated eosinophil mediated suppression of IFN-beta1 and IFN-lambda1. CONCLUSIONS: Eosinophil release of TGF-beta suppresses HRV-induced epithelial cell expression of IFN-beta1 and IFN-lambda1. Since these interferons have antiviral effects, this may represent a mechanism by which airway eosinophilia promotes virus-induced exacerbations of asthma.

RATIONALE: The Chronic Urticaria (CU) Index serum test has been available to help identify patients with autoimmune urticaria. We sought to determine the clinical utility of the CU Index level, specifically with respect to disease severity and responsiveness to medications. METHODS: In an IRB approved retrospective study of patients seen at the University of Wisconsin Allergy/Immunology clinic over a period of two years, patients with a diagnosis of chronic urticaria with a CU Index measurement were reviewed. The clinical history of these patients was examined, specifically capturing the value of their CU Index as well as the therapeutic management utilized and whether subjective symptom control was achieved. RESULTS: We examined 62 patients. 42 patients remained symptomatic despite the use of antihistamines with or without leukotriene inhibitors (refractory patients), and 20 were responsive (controlled patients). The prevalence of a positive CU Index (defined as a CU Index greater than or equal to 10) in refractory patients was significantly greater than in controlled patients (52% vs. 15%, p=0.006). Furthermore, the median CU Index for refractory patients was significantly greater than in controlled patients (10.8 vs. 5.05, p=0.009). CONCLUSIONS: A positive or elevated Chronic Urticaria Index value suggests an increased severity of chronic urticaria requiring a more aggressive level of medication use to achieve control.

RATIONALE: Since airway eosinophilia is a predictor of asthma exacerbations and viral infections account for the majority of exacerbations, we hypothesize that eosinophils (EOS) alter the response to virus-induced airway inflammation. We chose to study human EOS regulatory activity in altering host responses to virus stimulation through interactions with BEAS-2B epithelial cell line.METHODS: Synthetic double-stranded RNA polyinosinic: polycytidylic acid (poly I:C) was used as a virus analogue for the co-culture of EOS with epithelial cells. Using this in vitro co-culture system, purified human peripheral blood EOS were incubated with BEAS-2B cells and stimulated by poly I:C at 25 mcg/ml for 24 hours. RT-qPCR analysis of IFN-alpha-1, IFN-beta-1 and IFN-lamba-1 (IL-29) were performed on RNA extracted from BEAS-2B cells.RESULTS: Co-culture of EOS with BEAS-2B cells stimulated with poly I:C in vitro significantly decreased IL-29 expression (p=0.023) with a trend for decreased expression of IFN-beta-1. There was no significant change in IFN-alpha-1 expression.CONCLUSIONS: The presence of EOS alters in vitro IFN expression of BEAS-2B cells. Thus, EOS may affect host response to viral infections in asthma by suppressing innate immune response of epithelial cells. RATIONALE: Since airway eosinophilia is a predictor of asthma exacerbations and viral infections account for the majority of exacerbations, we hypothesize that eosinophils (EOS) alter the response to virus-induced airway inflammation. We chose to study human EOS regulatory activity in altering host responses to virus stimulation through interactions with BEAS-2B epithelial cell line. METHODS: Synthetic double-stranded RNA polyinosinic: polycytidylic acid (poly I:C) was used as a virus analogue for the co-culture of EOS with epithelial cells. Using this in vitro co-culture system, purified human peripheral blood EOS were incubated with BEAS-2B cells and stimulated by poly I:C at 25 mcg/ml for 24 hours. RT-qPCR analysis of IFN-alpha-1, IFN-beta-1 and IFN-lamba-1 (IL-29) were performed on RNA extracted from BEAS-2B cells. RESULTS: Co-culture of EOS with BEAS-2B cells stimulated with poly I:C in vitro significantly decreased IL-29 expression (p=0.023) with a trend for decreased expression of IFN-beta-1. There was no significant change in IFN-alpha-1 expression. CONCLUSIONS: The presence of EOS alters in vitro IFN expression of BEAS-2B cells. Thus, EOS may affect host response to viral infections in asthma by suppressing innate immune response of epithelial cells.

RATIONALE: Given the presence of thymic stromal lymphopoietin (TSLP) and eosinophils as prominent components of allergic inflammation, we sought to determine whether eosinophils express the TSLP receptor a-chain (TSLPR) and respond directly to TSLP stimulation. METHODS: Eosinophil mRNA expression for TSLPR was examined by real-time quantitative PCR of purified human peripheral blood eosinophils treated with cytokines in various combinations, including IL-3, IL-5, GM-CSF, and TNFa. Immunoblotting for TSLPR protein expression was performed on eosinophil lysates with a goat anti-TSLPR antibody. Flow cytometry with a goat anti-TSLPR antibody was used to detect surface TSLPR expression. Eosinophils were stimulated with human recombinant TSLP at various concentrations for 4 hours and supernatants were analyzed by ELISA for degranulation of eosinophil derived neurotoxin (EDN). RESULTS: Expression of mRNA and cell surface TSLPR was upregulated by stimulation with TNFa and IL-5 family cytokines. TSLPR protein was also detectable by Western blotting of eosinophil lysates. Stimulation of eosinophils with TSLP resulted in phosphorylation of the STAT5 transcription factor. In addition, eosinophil stimulation with TSLP resulted in degranulation of eosinophil derived neurotoxin (EDN) at levels comparable to IL-5. Furthermore, the TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to the TSLPR. CONCLUSIONS: This study demonstrates a role for eosinophils as a target of the allergic inflammatory cytokine, TSLP. Thus, in allergic inflammation, multiple pathways can serve to activate eosinophils, perhaps in a synergistic manner.

RATIONALE: We have previously shown differences in eosinophil effector functions in vitro and sputum neutrophil levels in young and aging asthma subjects. We sought to characterize eosinophil and neutrophil inflammatory mediators involved in the pathogenesis of asthma, in young and aging asthma subjects at baseline disease. METHODS: Human subjects with mild to moderate asthma between the ages of 20-40 (younger) and 50-70 (aging) were recruited. Baseline characterizations of lung function and sputum analysis were performed. Purified eosinophils and neutrophils stimulated with calcium ionophore were evaluated by ELISA for leukotriene C4 (LTC4), eosinophil derived neurotoxin (EDN), and leukotriene B4 (LTB4) production. The sputum was analyzed for cysteinyl leukotrienes, LTB4, EDN, and neutrophil elastase (NE) levels. RESULTS: Sputum analysis revealed no difference in the percentage of eosinophils between both age groups but a trend (p = 0.08) towards a higher percentage of neutrophils in aging asthma subjects was found. In vitro LTC4, EDN, and LTB4 production in calcium ionophore stimulated eosinophils and neutrophils were comparable in both age groups. Sputum analysis revealed no differences in EDN or cysteinyl leukotriene production. However, there was a trend (p = 0.10) for higher NE levels and significantly lower levels (p = 0.05) of LTB4 in the sputum of aging asthma subjects. CONCLUSIONS: Eosinophils and neutrophils in aging asthma subjects are capable of comparable leukotriene production in vitro. Despite a higher percentage of sputum neutrophils in aging asthma subjects, a decreased level of LTB4 and increased level of NE was found in vivo suggesting that specific age-related regulation of neutrophil function is occurring in the airway.

RATIONALE: Airway eosinophilia is a predictor of asthma exacerbations and a marker of asthma severity. Viral infections, including rhinovirus, account for the majority of asthma exacerbations. The role of eosinophils (EOS) in virus-induced inflammation remains to be defined. We chose to study human EOS regulatory activity in altering host responses to virus stimulation through interactions with peripheral blood mononuclear cells (PBMCs), monocytes, and epithelial cells (ECs). We have previously shown significant increases in IL-13 and IFN-gamma in co-culture experiments of EOS with CD4 + T-cells. The synthetic double-stranded RNA polyinosinic: polycytidylic acid (poly I: C) was used as a virus analogue for the co-culture of eosinophils with PBMCs and monocytes. METHODS: Using an in vitro co-culture system, purified human peripheral blood EOS were incubated with primary human PBMCs or monocytes and stimulated by poly I: C at 25 mcg/ml for 48 hours. EOS were also incubated with primary ECs and BEAS-2B cells for 24 hours. Supernatants from the co-cultures were collected and analyses of cytokine production were performed by ELISA. RESULTS: Co-culture of EOS with PBMCs stimulated with poly I: C in vitro significantly increased IL-13 expression (p = 0.010.) Co-culture of EOS with monocytes stimulated by poly I: C significantly increased IL-1beta expression (p = 0.008). Preliminary experiments suggest that incubation of EOS with primary ECs decreases IL-8 expression. CONCLUSIONS: The presence of EOS alters in vitro cytokine expression of PBMCs, monocytes and ECs. Thus, EOS may affect host response to viral infection in asthma by altering cytokine production of inflammatory cells as well as epithelial cells.

EOS are a prominent feature of airway inflammation in human asthma. EOS have been previously described to enhance CD4+ T-lymphocyte cytokine secretion in vitro in the presence of Staphylococcal enterotoxin B (SEB) superantigen. Since SEB is not recognized as a stimulus for asthma exacerbations, we sought to evaluate an alternate mechanism of T-lymphocyte activation relevant to human asthma. Viral infections are recognized as a common trigger for asthma exacerbations. We chose to use poly I:C as a surrogate for virus-induced stimulation to study whether EOS also enhance T-lymphocyte cytokine secretion with a stimulus modeling virus infection. Using an in vitro co-culture system, isolated purified human peripheral blood EOS and CD4+ T-lymphocytes were incubated using 100 mcg/ml poly I:C as a virus analogue. After 48 hours of co-culture, supernatants were collected and analyzed by ELISA for secretion of Th1 and Th2 cytokines, IFN-gamma and IL-13. Statistical analysis was performed using SigmaStat software. Co-culture of CD4+ T-lymphocytes and EOS with 100 mcg/ml poly I:C for 48 hours significantly enhanced secretion of Th1 and Th2 cytokines compared to CD4+ T-lymphocytes with poly I:C alone: IFN-gamma (33 ng/ml vs 17 ng/ml, n = 11, p = 0.006) and IL-13 (408 pg/ml vs 238 pg/ml, n = 11, p < 0.001.) EOS interact with CD4+ T-lymphocytes to enhance cytokine production with poly I:C stimulation in vitro. Thus, EOS may regulate T-lymphocyte responses and enhance virus-induced airway inflammation in human asthma.

RationaleCD4+ T cells are the main effector cell driving inflammation in chronic allergic conjunctivitis which is characterized by an activated conjunctival epithelium. Recent studies suggest that the conjunctival epithelium can be activated by Staphylococcus aureus (SA) present on the conjunctiva and lids in the majority of patients with chronic allergic conjunctivitis. The goal of this study was to examine the role of conjunctival epithelial cell mediators in regulation of cytokine release from activated CD4+ T cells.MethodsPeripheral blood CD4+ T cells were activated with anti-CD3/CD28 coated beads in buffer or supernates from a human conjunctival epithelial cell line (IOBA-NHC, Valladolid, Spain) stimulated with a either SA cell wall extract (CWE) or a combination of TNFα/IL-1β (positive control) or media containing either SA-CWE or a combination of TNFα/IL-1β (direct effect of SA-CWE and cytokines). T cell release of IL-13 and IFNγ were measured by ELISA.ResultsT cells activated in supernates from SA-CWE stimulated IOBA-NHC cells released markedly less IFNγ and IL-13, compared to T cells activated in buffer. Interestingly, the direct effect of SA-CWE on IFNγ release from CD3/CD28-activated T cells was the opposite, that is, SA-CWE enhanced IFNγ release. T cells activated in supernates from TNFα/IL-1β stimulated IOBA-NHC cells also released less IFNγ and IL-13.ConclusionsMediators released from conjunctival epithelial cells may play an important T cell regulatory role in chronic allergic conjunctivitis. RationaleCD4+ T cells are the main effector cell driving inflammation in chronic allergic conjunctivitis which is characterized by an activated conjunctival epithelium. Recent studies suggest that the conjunctival epithelium can be activated by Staphylococcus aureus (SA) present on the conjunctiva and lids in the majority of patients with chronic allergic conjunctivitis. The goal of this study was to examine the role of conjunctival epithelial cell mediators in regulation of cytokine release from activated CD4+ T cells. CD4+ T cells are the main effector cell driving inflammation in chronic allergic conjunctivitis which is characterized by an activated conjunctival epithelium. Recent studies suggest that the conjunctival epithelium can be activated by Staphylococcus aureus (SA) present on the conjunctiva and lids in the majority of patients with chronic allergic conjunctivitis. The goal of this study was to examine the role of conjunctival epithelial cell mediators in regulation of cytokine release from activated CD4+ T cells. MethodsPeripheral blood CD4+ T cells were activated with anti-CD3/CD28 coated beads in buffer or supernates from a human conjunctival epithelial cell line (IOBA-NHC, Valladolid, Spain) stimulated with a either SA cell wall extract (CWE) or a combination of TNFα/IL-1β (positive control) or media containing either SA-CWE or a combination of TNFα/IL-1β (direct effect of SA-CWE and cytokines). T cell release of IL-13 and IFNγ were measured by ELISA. Peripheral blood CD4+ T cells were activated with anti-CD3/CD28 coated beads in buffer or supernates from a human conjunctival epithelial cell line (IOBA-NHC, Valladolid, Spain) stimulated with a either SA cell wall extract (CWE) or a combination of TNFα/IL-1β (positive control) or media containing either SA-CWE or a combination of TNFα/IL-1β (direct effect of SA-CWE and cytokines). T cell release of IL-13 and IFNγ were measured by ELISA. ResultsT cells activated in supernates from SA-CWE stimulated IOBA-NHC cells released markedly less IFNγ and IL-13, compared to T cells activated in buffer. Interestingly, the direct effect of SA-CWE on IFNγ release from CD3/CD28-activated T cells was the opposite, that is, SA-CWE enhanced IFNγ release. T cells activated in supernates from TNFα/IL-1β stimulated IOBA-NHC cells also released less IFNγ and IL-13. T cells activated in supernates from SA-CWE stimulated IOBA-NHC cells released markedly less IFNγ and IL-13, compared to T cells activated in buffer. Interestingly, the direct effect of SA-CWE on IFNγ release from CD3/CD28-activated T cells was the opposite, that is, SA-CWE enhanced IFNγ release. T cells activated in supernates from TNFα/IL-1β stimulated IOBA-NHC cells also released less IFNγ and IL-13. ConclusionsMediators released from conjunctival epithelial cells may play an important T cell regulatory role in chronic allergic conjunctivitis. Mediators released from conjunctival epithelial cells may play an important T cell regulatory role in chronic allergic conjunctivitis.