CD137−/− mice were provided by Professor Dr Robert Mittler from Emory University (Atlanta, GA, USA). C57BL/6J control mice

were purchased from Charles River (Sulzfeld, Germany). The animal protocols were constructed according to institutional and state guidelines and approved by the local animal welfare committee. Eight to 10-week-old age- and sex-matched mice were immunized with ovalbumin [OVA, lipopolysaccharide (LPS) content-reduced <10 EU/mg protein, as described previously [28]] using two protocols (Fig. 1), as follows. Allergy protocol.  Mice were sensitized twice intraperitoneally (i.p.) with OVA (20 µg in 200 µl 0·9% NaCl) in adjuvant (Imject Pexidartinib order Alum®, PerbioScience, Bonn, Germany) followed by six challenges in which 20 µg OVA in 40 µl of 0·9% NaCl was given intranasally (i.n.) (allergy protocol). Control mice (Alum) received injections and challenges of 0·9% NaCl. Mice were killed 24 h after the last local allergen provocation. Tolerance protocol.  To induce respiratory tolerance, MI-503 cell line WT and CD137−/− mice were pretreated twice i.n. with 500 µg OVA in 40 µl of 0·9% NaCl. Thereafter, mice underwent allergy protocol as described above. BALF from each individual mouse was obtained by flushing the lungs with PBS/2 mM ethylenediamine tetraacetic acid (EDTA); the total number and

differentiation of BALF cells were then determined as described previously [28]. Lung histological sections and computer-based quantification of the degree of pulmonary inflammation [haematoxylin and eosin (H&E)] and mucus production [periodic acid-Schiff (PAS)] were performed as described previously

[28]. Serum levels of OVA-specific IgE, IgG1 and IgG2a were measured by enzyme-linked immunosorbent assay (ELISA), according to standard protocol. IgE concentrations PD184352 (CI-1040) (pg/ml) were calculated using a standard curve for mouse anti-OVA IgE (AbD Serotec, Oxford, UK). Data for IgG1 and IgG2a are presented as titre values derived from analysis of optical density (OD) values versus factors of serum dilution series using a logarithmic curve-fitting model. Spleen and bronchial lymph node (bLN) isolated cells were restimulated in vitro with OVA (200 µg/ml) in RPMI-1640 containing 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cytokines (IL-4, IL-5, IL-13, IFN-γ) were measured in supernatants after 3 days using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Cell cultures were pulsed with 3[H]-thymidine and incorporated activity was measured in a Betaplate scintillation counter. Single-cell suspensions from spleen, lung and bLN were incubated with fluorescently labelled antibodies for 20 min at 4°C in phosphate-buffered saline (PBS)/0·5% bovine serum albumin (BSA). Intracellular staining of forkhead box protein 3 (FoxP3) was performed using the eBioscience kit, according to the manufacturer’s instructions. Briefly, cells were surface-stained, fixed and incubated with antibody to FoxP3 for 30 min at 4°C.