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distinct cytokine expression patterns between viral and nonviral asthma exacerbations. Asthma exacerbations triggered by a nonviral factor, most commonly an allergen, were characterized with increased frequency of IL-4–producing CD31CD41 T cells (Fig 2, A); however, the frequency of that population did not correlate with FEV1% (see Fig E5, A, in this article’s Online Repository at www.jacionline.org). IL-4 in combination with other TH2-related cytokines, such as IL-5 and IL-13, are involved in the development of pathophysiological features of asthma and allergic inflammation.1,7 In addition, increased numbers of IL-10– producing CD31CD41 T cells were detected in those with nonvirus asthma exacerbations than in healthy controls and subjects with stable asthma (Fig 2, B). Interestingly, a negative correlation was found between IL-102producing CD31CD41 T cells and FEV1% (Fig E5, B). IL-17A expression in CD41 T cells represented a peripheral marker of asthma exacerbation. Both viral and nonviral acute asthma episodes were strongly associated with increased percentage of IL-17A–producing CD31CD41 T cells (Fig 2, C), which showed a negative correlation with FEV1% (Fig E5, C). IL-17A induces the production of proinflammatory cytokines and chemokines by airway epithelial cells, fibroblasts, and other cell types, which plays a role in the influx of neutrophils and macrophages to the site of inflammation, and their excessive activation leading to tissue damage.1,7,8 The induction of IL-22–producing CD31CD41 T cells was also identified in asthma exacerbations triggered by a nonviral factor (Fig 2, D), without showing any correlation with FEV1% (Fig E5, D). The percentage of IFNg–producing T cells was similar in all groups (Fig E5, E) and did not correlate with FEV1% (Fig E5, E). In addition to single cytokine expression, our data demonstrate increased frequency of CD41 T cells with a coexpression phenotype of IL-17A1IL-101, IL-41IL-101, IL-41IL-17A1, IL-41IL-221, IL-17A1IL-221, and IL-221IL-101 in nonviral asthma exacerbations, suggesting a general activation of effector cytokines in CD41 T cells (see Fig E6 in this article’s Online Repository at www.jacionline.org). In contrast to IL-17A, IL-22 exhibits proinflammatory, anti-inflammatory properties as well as tissue regenerative features.7,8 It might be assumed that the simultaneous production of IL-22 and IL-17A is responsible for overcoming the tissue damage resulting from the ongoing inflammatory process. IL-22 is an IL-10 family cytokine member and plays an important role in mucosal immunity. The role of IL-222producing T cells in bronchial asthma in humans is unknown.8 Interestingly, increased levels of IL-22 in the serum of subjects with asthma have been reported.8 Furthermore, studies in animal models have demonstrated an involvement of IL-22 in regulation of the infiltration of eosinophils to the airways.9 In addition, it may be involved in the stabilization of lung epithelial integrity and homeostasis. However, further studies are required to clarify the role of TH22 cells in asthma and their exacerbations. Individuals treated with systemic corticosteroids showed heterogeneity in the populations of effector and regulatory T-cell subsets in the periphery, and it requires further studies for in-depth analyses (see Fig E7 in this article’s Online Repository at www.jacionline.org). In conclusion, our data demonstrate that multicolor flow cytometry represents a relatively rapid tool to investigate peripheral blood lineage-specific CD41 T-cell characterization that may open a new window for the identification of the etiology of asthma exacerbations. Distinct immune activation and immune regulation pathways were demonstrated in exacerbations due to viral or nonviral etiology. Longitudinal studies are required for

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better understanding of immune response associated with asthma exacerbations. The knowledge of the dynamics of changes in peripheral blood T cells may allow the development of rapid tests for cellular diagnosis of asthma exacerbations. Agnieszka S. Wegrzyn, PhDa,b Bogdan Jakiela, PhDc Beate R€ uckert, Sci Teca,b Marek Jutel, MDd M€ ubeccel Akdis, MD, PhDa,b Marek Sanak, MD, PhDc Cezmi A. Akdis, MDa,b From athe Swiss Institute of Allergy and Asthma Research, University of Zurich, Zurich, Switzerland; bthe Christine K€uhne-Center for Allergy Research and Education, Davos, Switzerland; cthe Department of Internal Medicine, Jagiellonian University, Krakow, Poland; and dthe Department of Clinical Immunology, Wroclaw Medical University, Wroclaw, Poland. E-mail: [email protected]. Or: akdisac@siaf. uzh.ch. The authors’ laboratories are supported by European Commission’s Seventh Framework Programme under Mechanisms of the Development of Allergy (grant agreement no. 261357) and Post-infectious immune reprogramming and its association with persistence and chronicity of respiratory allergic diseases (grant agreement no. 260895), Swiss Polish Research Programme (grant no. PSPB-072/2010), and the Swiss National Science Foundation (grant no. 310030_156823). Disclosure of potential conflict of interest: M. Jutel has received consultancy fees from Allergopharma, Biomay AG, Anergis, and the Polish National Science Centre, from which he has also received or has grants pending; he has received payment for delivering lectures from Stallergenes, GlaxoSmithKline, and Allergopharma as well as for the development of educational presentations from the Polish Society of Allergoplogy. C. A. Akdis has institutional ties to the Christine K€uhne-Center for Allergy Research and Education and the European Academy of Allergy and Clinical Immunology. The rest of the authors declare that they have no relevant conflicts of interest. REFERENCES 1. Lloyd CM, Saglani S. T cells in asthma: influences of genetics, environment, and T-cell plasticity. J Allergy Clin Immunol 2013;131:1267-74; quiz 75. 2. Jackson DJ, Sykes A, Mallia P, Johnston SL. Asthma exacerbations: origin, effect, and prevention. J Allergy Clin Immunol 2011;128:1165-74. 3. Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy: multiple suppressor factors at work in immune tolerance to allergens. J Allergy Clin Immunol 2014;133:621-31. 4. Karagiannidis C, Akdis M, Holopainen P, Woolley NJ, Hense G, Ruckert B, et al. Glucocorticoids upregulate FOXP3 expression and regulatory T cells in asthma. J Allergy Clin Immunol 2004;114:1425-33. 5. Purwar R, Campbell J, Murphy G, Richards WG, Clark RA, Kupper TS. Resident memory T cells (T(RM)) are abundant in human lung: diversity, function, and antigen specificity. PLoS One 2011;6:e16245. 6. Grzanka J, Leveson-Gower D, Golab K, Wang XJ, Marek-Trzonkowska N, Krzystyniak A, et al. FoxP3, Helios, and SATB1: roles and relationships in regulatory T cells. Int Immunopharmacol 2013;16:343-7. 7. Akdis M, Burgler S, Crameri R, Eiwegger T, Fujita H, Gomez E, et al. Interleukins, from 1 to 37, and interferon-gamma: receptors, functions, and roles in diseases. J Allergy Clin Immunol 2011;127:701-21, e1-70. 8. Akdis M, Palomares O, van de Veen W, van Splunter M, Akdis CA. TH17 and TH22 cells: a confusion of antimicrobial response with tissue inflammation versus protection. J Allergy Clin Immunol 2012;129:1438-49; quiz 50-1. 9. Takahashi K, Hirose K, Kawashima S, Niwa Y, Wakashin H, Iwata A, et al. IL-22 attenuates IL-25 production by lung epithelial cells and inhibits antigen-induced eosinophilic airway inflammation. J Allergy Clin Immunol 2011;128:1067-76; e1-6. Available online January 17, 2015. http://dx.doi.org/10.1016/j.jaci.2014.12.1866

Prevention of allergic asthma through Der p 2 peptide vaccination To the Editor: House dust mites (HDMs) are a major source of allergens that affect more than 50% of allergic patients.1-3 The

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FIG 1. Derp 2.1 exhibits therapeutic potential in a mouse model of Dermatophago€ıdes pteronyssinus– induced asthma. A, Schematic representation of the HDM antigen–allergic model. B, Penh values (n 5 8-12 mice per group). C, Total BAL cell numbers (n 5 8-12 mice per group). D, BAL eosinophil values (n 5 8-12 mice per group). E, BAL neutrophil values (n 5 8-12 mice per group). ***P < .001, **P < .01, and *P < .05.

Dermatophagoides pteronyssinus allergen, Der p 2, is one of the most representative HDM allergens and is recognized by more than 90% of HDM-allergic patients.1-3 Continuous exposure to this allergen leads to the development of asthma, a pathology characterized by bronchoconstriction and airway inflammation. Genetic engineering led to the development of new therapeutic recombinant hypoallergenic derivatives with lower IgE and T-cell reactivity.4 Two recombinant hypoallergenic peptides derived from Der p 2 were recently described to exhibit less in vivo allergenicity than the Der p 2 allergen while preserving immunogenicity.5,6 Thus, hypoallergenic peptides may represent candidates for specific immunotherapy, especially in HDM-allergic asthma. To investigate whether Der p 2–derived peptides can modulate asthma in mice, we tested the prophylactic effect of 3 recombinant Der p 2 hypoallergenic derivatives in a mouse model of HDMinduced asthma (see the Methods section in this article’s Online Repository at www.jacionline.org): Der p 2.1 (amino acids 153), Der p 2.2 (amino acids 54-129), and the Der p 2.212.1 hybrid molecule. Mice were vaccinated with 1 of these 3 peptides or an irrelevant peptide as control (major birch pollen allergen, Bet v 1) 10 days before the first sensitization to the HDM extract and 1

day before the fourth sensitization (Fig 1, A). All 3 recombinant peptides decreased airway hyperresponsiveness in response to methacholine, suggesting that these peptides may influence the course of the disease. Interestingly the Der p 2.1 peptide exhibited a stronger effect than did the other hypoallergenic derivatives (Fig 1, B). In the next step, we analyzed airway inflammation and observed a dramatic decrease in the cellular infiltrate in the bronchoalveolar lavage (BAL) of Der p 2.1–vaccinated mice in conjunction with a significant drop in the eosinophil and neutrophil counts (Fig 1, C-E). Together, these results validate the prophylactic effect of the hypoallergenic Der p 2.1 peptide and suggest its ability to prevent the development of HDM-allergic asthma by inhibiting airway inflammation and hyperresponsiveness. To assess this hypothesis, we focused on the Der p 2.1 peptide in our next experiments. As shown in Fig 2, Der p 2.1 prevents the alteration of lung function in mice. Indeed, invasive measures of lung function revealed a significantly lower lung resistance and elastance in mice treated with the Der p 2.1 derivate when compared with mice treated with the control peptide (Fig 2, A and B). Given that Der p 2.1 vaccination dampened immune cell recruitment in the airways (Fig 1, C-E), we analyzed the levels of inflammatory

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FIG 2. Vaccination with Der p 2.1 normalizes lung function and influences airway inflammation. A and B, Resistance and elastance values obtained by invasive measurements (n 5 8-12 mice per group). C, BAL levels of chemokines after vaccination (n 5 4-6 mice per group). D, T-cell responses in lung mucosa (n 5 5-8 mice per group). E, Serum levels of Der p 2–specific IgE, IgG1, and IgG2a after Der p 2.1 vaccination (n 5 5 mice per group). F, Penh values obtained after vaccination with Der p 2.1 in mouse model of Dermatophago€ıdes farinae–induced asthma (n 5 8-12 mice per group). KC, Keratinocyte derived chemokine; MCP-1, monocyte chemoattractant protein-1; ***P < .001, **P < .01, and *P < .05.

chemokines in BAL. We observed a significant decrease in the production of monocyte chemoattractant protein-1 (chemokine C-C motif ligand-2), eotaxin (chemokine C-C motif ligand-11), and keratinocyte derived chemokine (chemokine C-X-C ligand 1), thus explaining the dampened infiltrate observed in Der p 2.1–vaccinated mice (Fig 2, C). Because this hypoallergenic peptide has been described as a poor inducer of T-cell proliferation and activation,5,6

we investigated whether vaccination could modulate primary Tcell responses to inhaled allergen, primed in the lung tissue using flow cytometry. Interestingly, we observed a decreased frequency of IL-4– and IL-17–producing CD41 T cells (Fig 2, D), 2 T-cell subsets known for their deleterious effects in allergic asthma.7 No effect was observed on the frequency of IL-131 or IFN-g1 CD41 T cells (Fig 2, D). In addition, we observed decreased IgE

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reactivity in the BAL of Der p 2.1–vaccinated mice, confirming previous reports demonstrating the lack of IgE reactivity toward this recombinant hypoallergenic derivative (Fig 2, E).5,6 However, contrary to recent results, we did not observe an increase in the inhibitory Der p 2–specific IgG1 (Fig 2, E).8 Furthermore, we observed no influence of Der p 2.1 vaccination on the production of Der p 2–specific IgG2a. This result could be explained by the fact that B cells require IL-4 to produce both IgG1 and IgE. Consequently, by decreasing IL-4–producing T cells, vaccination with Der p 2.1 also reduced the production of TH2-related immunoglobulins. Given their structural homology, Der p and Der f antigens demonstrate an important IgE cross-reactivity.9 On the basis of this cross-reactivity, we investigated whether vaccination with Der p 2.1 peptide could control the development of Der f–induced airway hyperresponsiveness. Vaccination with Der p 2.1 inhibited bronchial hyperresponsiveness in Der f–induced asthma to the same extent as in the Der p–induced asthma model (Fig 2, F). Collectively, these data demonstrate the protective effect of Der p 2.1 vaccinations given before and during HDM sensitization. By inhibiting T-cell–mediated IL-4 production, Der p 2.1 vaccination is believed to dampen IgE production, leading to a global attenuation on airway inflammation and hyperresponsiveness. Lower levels of IgE lead to fewer allergen-IgE pathogenic complexes and therefore to less activation of innate cells such as eosinophils, mastocytes, and basophils. The fact that IgG1 was also decreased is intriguing. The decrease in IgG1 may be explained by the fact that IL-4 is also involved in the production of IgG1 in mice. Der p 2.1 vaccination could also promote the expansion of immunosuppressive cells, such as regulatory T cells or induced-regulatory B cells.10-12 Indeed, many reports demonstrate that specific immunotherapy promotes the emergence of IL-10–producing T and B cells in allergic patients.10-12 Another interesting finding is the fact that vaccination also dampens the frequency of TH17 cells in lungs. Given the potent role of these cells in severe asthma,13 this therapeutic strategy could represent an interesting alternative in some cases of severe allergic asthma. Most interestingly, we demonstrated that vaccination with Der p 2.1 could be effective in Der f asthmatic mice. Consequently, this vaccine may reduce IgE-mediated as well as T-cell–mediated allergic inflammation irrespective of the HDM species. Finally, the protective role provided by vaccination with a hypoallergenic peptide could be of interest clinically, especially in sensitized children in whom allergic processes are sequential from sensitization to asthma. For these individuals, peptide immunotherapy may offer an alternative to block the allergic process and prevent its progression to allergic asthma. We thank the Cytocell Cytometry Facility in Nantes for expert technical assistance. Gregory Bouchaud, PhDa,b,c,d,e* Faouzi Braza, PhDa,b,c,d* Julie Chesn e, PhDa,b,c,d David Lair, MSa,b,c,d Kuan-Wei Chen, PhDf Camille Rolland-Debord, MDa,b,c,d Dorian Hassoun, MSa,b,c,d Tiphaine Roussey-Bihou ee, MDa,b,c,d Marie-Aude Cheminant, BSa,b,c,d Sophie Brouard, PhDc,g,h Marie Bodinier, PhDe Susanne Vrtala, PhDf Antoine Magnan, MDa,b,c,d,i

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From aINSERM, UMR1087, l’institut du thorax, bCNRS, UMR 6291, cUniversite de Nantes, dDHU2020 medecine personnalisee des maladies chroniques, and eINRA, UR1268 BIA, Nantes, France; fthe Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Vienna, Austria; gInstitut National de la Sante Et de la Recherche Medicale INSERM U1064, hInstitut de Transplantation Urologie Nephrologie du Centre Hospitalier Universitaire H^otel Dieu, and iCHU de Nantes, l’institut du thorax, Service de Pneumologie, Nantes, France. E-mail: [email protected]. *These authors contributed equally to this work. This study was supported by the Societe Franc¸aise d’Allergologie, the Fonds de recherche en sante respiratoire, the Fondation Recherche Medicale, and the Genavie Fundation. G.B. was supported by The People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under Research Executive Agency (REA) (grant agreement no. 624910). S.V. was supported by the Austrian Science Fund Fonds zur F€orderung der wissenschaftlichen Forschung (FWF) (grant no. F4602). The Region Pays de La Loire also funded this work in the frame of the REAL2 project. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest. REFERENCES 1. Bousquet PJ, Chinn S, Janson C, Kogevinas M, Burney P, Jarvis D, et al. Geographical variation in the prevalence of positive skin tests to environmental aeroallergens in the European Community Respiratory Health Survey I. Allergy 2007;62:301-9. 2. Meyer CH, Bond JF, Chen MS, Kasaian MT. Comparison of the levels of the major allergens Der p I and Der p II in standardized extracts of the house dust mite, Dermatophagoides pteronyssinus. Clin Exp Allergy 1994;24:1041-8. 3. van der Zee JS, van Swieten P, Jansen HM, Aalberse RC. Skin tests and histamine release with P1-depleted Dermatophagoides pteronyssinus body extracts and purified P1. J Allergy Clin Immunol 1988;81:884-96. 4. Valenta R, Ferreira F, Focke-Tejkl M, Linhart B, Niederberger V, Swoboda I, et al. From allergen genes to allergy vaccines. Annu Rev Immunol 2010;28:211-41. 5. Chen KW, Blatt K, Thomas WR, Swoboda I, Valent P, Valenta R, et al. Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy. J Allergy Clin Immunol 2012;130:435-43.e4. 6. Chen KW, Focke-Tejkl M, Blatt K, Kneidinger M, Gieras A, Dall’Antonia F, et al. Carrier-bound nonallergenic Der p 2 peptides induce IgG antibodies blocking allergen-induced basophil activation in allergic patients. Allergy 2012;67:609-21. 7. Botturi K, Langelot M, Lair D, Pipet A, Pain M, Chesne J, et al. Preventing asthma exacerbations: what are the targets? Pharmacol Ther 2011;131:114-29. 8. Linhart B, Narayanan M, Focke-Tejkl M, Wrba F, Vrtala S, Valenta R. Prophylactic and therapeutic vaccination with carrier-bound Bet v 1 peptides lacking allergenspecific T cell epitopes reduces Bet v 1-specific T cell responses via blocking antibodies in a murine model for birch pollen allergy. Clin Exp Allergy 2014;44:278-87. 9. Heymann PW, Chapman MD, Aalberse RC, Fox JW, Platts-Mills TA. Antigenic and structural analysis of group II allergens (Der f II and Der p II) from house dust mites (Dermatophagoides spp). J Allergy Clin Immunol 1989;83:1055-67. 10. Braza F, Chesne J, Castagnet S, Magnan A, Brouard S. Regulatory functions of B cells in allergic diseases. Allergy 2014;69:1454-63. 11. Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy: multiple suppressor factors at work in immune tolerance to allergens. J Allergy Clin Immunol 2014;133:621-31. 12. van de Veen W, Stanic B, Yaman G, Wawrzyniak M, Sollner S, Akdis DG, et al. IgG4 production is confined to human IL-10-producing regulatory B cells that suppress antigen-specific immune responses. J Allergy Clin Immunol 2013;131:1204-12. 13. Chesne J, Braza F, Mahay G, Brouard S, Aronica M, Magnan A. IL-17 in severe asthma: where do we stand? Am J Respir Crit Care Med 2014;190:1094-101. Available online February 11, 2015. http://dx.doi.org/10.1016/j.jaci.2014.12.1938

Childhood allergic asthma is associated with increased IL-13 and FOXP3 histone acetylation To the Editor: Efficient maturation of the immune system in early childhood is essential for protection against allergy and asthma later in life. In addition to genetic predisposition, epigenetic modifications can influence the risk for development of chronic inflammatory diseases such as asthma. Epigenetic mechanisms including DNA methylation and histone acetylation play a major role in

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METHODS Animal procedures Balb/c female mice aged 6 to 8 weeks were purchased from Charles River Breeding Laboratories and used for all experiments. The animals were housed in UTE IRS-UN platform (Nantes, France). The mice were maintained under specific pathogen-free, temperature-controlled conditions under a strict 12-hour light-dark cycle and were given free access to food and water. All protocols were approved by the Regional Ethical Committee for Animal Experiments of Pays de la Loire (ceea.2012.77). The mice were sensitized on days 0, 7, 14, and 21 by percutaneous application of 500 mg of total extract of Dermatophago€ıdes pteronyssinus (Der p) or Dermatophago€ıdes farinae (Der f) (Stallergenes, Antony, France) in 20 mL of dimethylsulfoxide (Sigma-Aldrich, St Louis, Mo) on the ears without any synthetic adjuvant. The mice were challenged intranasally with 250 mg of Der p in 40 mL of sterile PBS on day 27 to induce asthma and again on day 34 to induce asthma exacerbation. Animals were anesthetized with 100 mL of xylazine (ROMPUN 2%, Bayer, Lyon, France, 15 mg/kg) intraperitoneally and 100 mL of ketamine (IMALGENE1000, Merial, Lyon, France, 80 mg/kg) for both sensitizations and challenges. Mice were sacrificed by sublethal injection of dolethal (vetoquinol) on day 35 (Fig 1). For dynamic lung resistance measurements, mice were anesthetized with 200 mL of a ketamine-xylazine mix and paralyzed with 100 mL of rocuronium bromide (ESMERON, Organon, Kloosterstraat, The Netherlands, 10 mg/mL) intraperitoneally.

Derivative peptide injection Derivative (Der p 2.1) and control (Bet v1 peptide 1.2) peptides were purified as previously described by Chen et al.E1 Peptides were injected 10 days before the first sensitization and 20 days later. Der p 2.1 was solubilized with 10 mM NaH2PO4, pH 7, solution to a final concentration of 400 mg/mL. Bet v 1.2 was used as control and was solubilized with PBS to a final concentration of 150 mg/mL. Approximately 200 uL of a solution of Alu-gel (AluGel-S, SERVA Electrophoresis) containing 5 mg of the peptide was injected subcutaneously in the neck of the mice.

BAL preparation One milliliter of sterile PBS was administered intratracheally to mice through a flexible catheter. Cells and supernatants from the removed fluid were separated by centrifugation. The total cell number was determined by using optical microscopy, and the BAL cell composition was analyzed by using flow cytometry (see below). Supernatants were stored at 2808C for cytokine assays.

Flow cytometry For analysis of BAL cells, the following antibodies were used: CD3-APC, CD19 PE-Cy7, CD11c PE-Cy7, F4/80 FITC, Ly6G PerCP-Cy5.5, and CD45R PerCP-Cy5.5 (purchased from eBioscience, Paris, France); CD8 APC-H7 and CD19 APC-H7 (purchased from BD Biosciences, Paris, France); and CCR3 PE (purchased from R&D Systems, Minneapolis, Minn). Dead cells were excluded using 49-6-diamidino-2-phenylindole, dihydrochloride. Stained cells resuspended in PBS were acquired on a BD LSR II (BD Biosciences) and were analyzed on BD FACSDiva software (BD Biosciences). To study lung T-cell responses, the lungs were removed, disrupted, and digested at 378C for 1 hour in 10 mL of PBS containing DNAse I (Roche, Boulogne-Billancourt, France) and collagenase II (Invitrogen, Saint Aubin, France). Lung cells were passed through a 40-mm cell strainer with a syringe plunger, and the strainer was washed with 10 mL of RPMI 1640 media supplemented with 10% FBS, 200 mg/mL penicillin, 200 U/mL streptomycin, 4 mM L-glutamine, and 0.05 mM b-mercaptoethanol. After red blood cell lysis, 5 3 105 lung cells were

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transferred to a 96-well round-bottom plate and were stimulated for 5 hours with a mix containing phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (500 ng/mL; Sigma-Aldrich) together with either monensin for TH17 analysis (2 mg/mL; BD Biosciences) or brefeldin A for TH2 assessment (1 mg/mL; BD Biosciences). For cytokine detection, FcRs were blocked with mouse CD16/CD32 mAbs (eBioscience). Before surface-specific staining, the cells were stained with Fixable Viability Dye 450 (BD Biosciences) to exclude dead cells. The following antibodies were used for surface staining: CD3 PeCy7 (145-2C11; BD Biosciences) and CD8 APC-H7 (53-6.7; BD Biosciences). The cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) and stained intracellularly with anti–IL-4 (11B11; eBioscience), anti–IL-13 (eBio13A; eBioscience), and anti–IL-17A (TC1118H10; BD Biosciences).

Airway hyperresponsiveness measurement Unrestrained mice were exposed to increasing doses of methacholine aerosol generated through a nebulizer containing 0 to 40 mg/mL methacholine in PBS. Enhanced pause (Penh) was measured by whole body plethysmography (Emka Technologies, Paris, France). Dynamic lung resistances were measured using a flexiVent (SCIREQ). Mice were anesthetized and connected via an endotracheal cannula to a flexiVent system. After initiating mechanical ventilation, the mouse was paralyzed with an intraperitoneal injection of 0.1 mL of a 10 mg/mL solution of rocuronium bromide. The animal was ventilated at a respiratory rate of 150 breaths/min and a tidal volume of 10 mL/kg against a positive end expiratory pressure of 3 cmH2O. Respiratory mechanics were assessed using a 1.2-second, 2.5-Hz single-frequency forced oscillation maneuver and a 3-second, broadband low-frequency forced oscillation maneuver containing 13 mutually prime frequencies between 1 and 20.5 Hz. The settings of both perturbations were configured to ensure that onset transients were omitted and that the oscillations had reached steady state in the analyzed portions of the maneuvers. Respiratory system resistance (R) was calculated using the flexiVent software by fitting the equation of motion of the linear singlecompartment model of lung mechanics using multiple linear regressions. Both maneuvers were executed every 15 seconds in alternation after each methacholine aerosol challenge to capture the time course and detailed response of the methacholine-induced bronchoconstriction.

Cytokines quantification Cytokines’ concentrations in BAL or spleen cell culture supernatants were quantified by Luminex technology (BioPlex 200 system, Bio-Rad Laboratories, Munich, Germany), using the Pro Mouse Cytokine 23-plex kit (BioRad Laboratories). Assays were performed according to the manufacturer’s specifications. Data analysis was performed using the Bio-Plex Manager Software version 4.0.

Statistical analysis All statistical analyses were conducted using GraphPad Prism 6.0 (GraphPad Software, Inc, La Jolla, Calif). All data are expressed as the mean 6 SEM. Unless otherwise specified, differences in the airway response between groups were tested for statistical significance using ANOVA. The Mann-Whitney test was used for other analyses. A P value of less than .05 was considered statistically significant.

REFERENCE E1. Chen KW, Fuchs G, Sonneck K, Gieras A, Swoboda I, Douladiris N, et al. Reduction of the in vivo allergenicity of Der p 2, the major house-dust mite allergen, by genetic engineering. Mol Immunol 2008;45:2486-98.