Briefly, a mouse was placed into the main chamber of the plethysmograph. The mouse was exposed to nebulized PBS and methacholine (Sigma-Aldrich) in PBS using an ultrasonic nebulizer. As an index of in vivo airway obstruction, buy BYL719 enhanced pause (Penh) values were measured and expressed as relative values compared to baseline Penh values following PBS exposure for each methacholine concentration (1–25 mg/ml). Levels of plasma OVA-specific IgE

(OVA-IgE) in challenged mice were measured by enzyme-linked immunosorbent assay (ELISA), as described previously [16]. Th1 and Th2 cytokine levels (IL-4, IL-5, IL-13, IFN-γ) were measured in BALF by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. To estimate OVA-specific T cell proliferation in vivo, we used OTII CD4+ cells labelled with CFSE; Molecular Probes, Eugene, OR, USA). Single-cell spleen suspensions from OTII mice were depleted of dendritic cells (DCs) using CD11c microbead and automatic magnetic-activated cell sorting (autoMACS) system

(Miltenyi Biotech, Auburn, CA, USA). The purity of CD4+ cells was estimated to be over 90% using a flow cytometer. Cells were incubated with 5 µM CFSE, according to the manufacturer’s instructions. CFSE-labelled OTII cells (5 × 106 cells) were transferred intravenously into each IgG or PBS-administered wild-type mouse. After injection, mice were challenged with OVA for 30 min a day for 2 days. Seventy-two hours after the OTII cell transfer, mononuclear cells from the thoracic lymph nodes were stained with anti-CD4-magnetic-activated PDK4 cell sorting Selleckchem Stem Cell Compound Library (BD Biosciences, Franklin Lakes, NJ, USA) to analyse transferred CD4+ OTII cell proliferation using a flow cytometer. Data were analysed using Cellquest (BD Biosciences) and FlowJo

software (Treestar, Ashland, OR, USA). To analyse the function of lung CD11c+ antigen-presenting cells (APCs), they were collected 24 h after the mice were administered with 1 mg of IgG or PBS, as described previously [17]. Briefly, mouse lungs were minced and then incubated in the digestion medium consisting of RPMI-1640 (Sigma-Aldrich), 5% fetal bovine serum (Sigma-Aldrich), 1 mg/ml collagenase type 4 (Roche Diagnostics, Indianapolis, IN, USA) and deoxyribonuclease I (bovine pancreas; Wako). Lung CD11c+ APCs were isolated using the CD11c microbeads and autoMACS system according to the manufacturer’s instructions. The purity of CD11+ cells was estimated to be over 80% using a flow cytometer. OTII CD4+ cells were isolated from OTII mouse spleens using the MACS system. OTII CD4+ cells (2·5 × 105 cells/well) were co-cultured in a 96-well plate in complete medium with lung CD11c+ APCs (2·5 × 104 cells/well) from naive WT mice after PBS or IgG administration. Cultures were stimulated in vitro with an OVA323–339 peptide (5 µg/ml; GenWay Biotech, San Diego, CA, USA) or medium for 6 h.