RPMI medium. Cells were activated with 2 µg/ml (eBioscience) or 0.5 µg/ml (BD) of plate-bound anti-CD3 (145-2C11) and 1 µg/ml (eBioscience) or 4 µg/ml (BD) of plate-bound anti-CD28 (37–51). CD4+ T cells were iso- lated using positive or negative selection kits (Miltenyi Biotec). Naive CD4+CD25CD44low T cells were preenriched using CD4 negative selection kit and sorted to >99% purity with a FACSAria II (BD) using anti-CD4 (BD), anti-CD25 (eBioscience), and anti-CD44 (BD) antibodies. Naive cells were polarized toward Th2 with 5 µg/ml of plate-bound anti-CD3 and 5 µg/ml of plate-bound anti-CD28 in the presence of 20 ng/ml of recombi- nant IL-4 (R&D Systems) and 20 µg/ml of blocking IFN- (R&D Systems) as previously described (Watford et al., 2010). Th17 differentiation was driven by stimulating CD4+CD25 T cells with 2 µg/ml of plate-bound anti-CD3 and 1 µg/ml of plate-bound anti-CD28 antibodies in the presence of 1 ng/ml of recombinant human (rh) TGF- (R&D Systems), 200 U/ml rhIL-6 (R&D Systems), and 10 µg/ml of blocking IL-4 and IFN- anti- bodies (R&D Systems) in complete IMDM medium (Invitrogen). Immunological analysis. For real-time PCR, mRNA was extracted with the RNeasy kit (QIAGEN) and converted as first-strand cDNA with the MMLV reverse transcription (Invitrogen). Each PCR was performed on an MX3000P qPCR machine (Agilent Technologies) from 50 ng cDNA using SYBR green technology (QIAGEN), and genes were amplified with the fol- lowing primers (Invitrogen): IL-4 (forward, 5-ACAGGAGAAGGGACGC- CAT-3; reverse, 5-GAAGCCCTACAGACGAGCTCA-3), IL-5 (forward, 5-AGCACAGTGGTGAAAGAGACCTT-3; reverse, 5-TTCAATGCA- TAGCTGGTGATTT-3), IL-13 (forward, 5-AGACCAGACTCCCCTGT- GCA-3; reverse, 5-TGGGTCCTGTAGATGGCATTG-3), GATA3 (forward, 5-AGGCAAGATGAGAAAGAGTGCCTC-3; reverse, 5-CTC- GACTTACATCCGAACCCGGTA-3), RORt (forward, 5-CCGCTGA- GAGGGCTTCAC-3; reverse, 5-TGCAGGAGTAGGCCACATTACA-3), SOCS1 (forward, 5-ACCTTCTTGGTGCGCGAC-3; reverse, 5-AAGC- CATCTTCACGCTGAGC-3), SOCS2 (forward, 5-GGTTGCCGGAGG- AACAGTC-3; reverse, 5-GAGCCTCTTTTAATTTCTCTTTGGC-3), and SOCS3 (forward, 5-CCTTCAGCTCCAAAAGCGAG-3; reverse, 5-GCTCTCCTGCAGCTTGCG-3). Cytokine secretion in cell culture supernatants was analyzed by ELISA (BD or R&D Systems). Total and antigen-specific serum IgE were measured by ELISA, as described previously (Mangan et al., 2007). Flow cytometry. Surface marker expression was assessed by flow cytometry using anti-CD3 (BD), anti-CD4 (BD), or anti-CD8 (eBioscience). For cyto- kine staining, cells were restimulated in complete medium at 37°C for 4 h with 50 ng/ml PMA (Sigma-Aldrich), 500 ng/ml ionomycin (Sigma-Aldrich), and GolgiPlug (BD) or for 6 h with 2.5 µg/ml Concanavalin A (Sigma-Aldrich) and 10 µg/ml Brefeldin A (Sigma-Aldrich). Cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD), followed by intracellular anti–IL-4, anti–IFN-, or anti–IL-17 (BD or eBioscience) staining. For phospho-STAT analysis, the cells were fixed after stimulation, permeabilized with the Perm III buffer (BD), and stained with antibodies specific for phospho-STAT5, phospho- STAT3, and phospho-STAT6 according to the Phosflow (BD) instructions. Data were collected on a CyAn flow cytometer (Beckman Coulter) or on BD FACSCanto II (BD) and analyzed using FlowJo software (Tree Star). Helminth egg injections. Eggs from S. mansoni–infected mice were ob- tained as described previously (Smith et al., 2009). SEA at <1 endotoxin unit/mg (Chromogenic LAL; BioWhittaker) was prepared as described pre- viously (Smith et al., 2009). Mice were given an i.p injection of either 10,000 S. mansoni eggs or PBS. For the adoptive transfer experiments, RAG-1/ mice were reconstituted with CD4+ sorted from WT or SOCS2/ mice be- fore injection. In separate experiments, 5,000 S. mansoni eggs or PBS were injected into the footpad of mice to examine local cellular responses to SEA. Mice were culled and spleens or popliteal LNs removed 10 d after injection. AD. Calcipotriol (MC903; Sigma-Aldrich) was resuspended in ethanol at 50 µM. A total of 1 nM was applied daily to the outer and inner surfaces JEM Vol. 208, No. 7 A r t i c l e of the right ear (20 µl/ear) as described previously (Li et al., 2006; Elentner et al., 2009). Ethanol (20 µl/ear) was applied as a vehicle control. Ear thick- ness was monitored daily using digital calipers (Kroeplin). Spleens and au- ricular draining LNs of the MC903 or vehicle-treated mice were extracted at day 5 and at the end of the study. The cells were restimulated with anti- CD3 and anti-CD28 and the supernatants harvested after 48 h. MC903 or vehicle-treated ear samples were formalin fixed, embedded in paraffin, and stained with hematoxylin and eosin. 10 fields were randomly defined through- out the tissue and counted blindly by two people using a graticule at 400×.The number of eosinophils per square millimeter of tissue was then calculated. Experimental allergic airway inflammation. Mice were sensitized with OVA to induce allergic airway inflammation in a protocol optimized for mice on C57BL/6J strain background, as described previously (Fallon et al., 2009). Mice were administered 50 µg OVA in alum (Thermo Fisher Scientific) i.p. on days 0 and 14 and subjected to airway exposure with 1% OVA in PBS for 20 min on days 27–31. 24 h after the last OVA aerosol challenge, lung function was analyzed by an invasive method where mice were tracheosto- mized and ventilated using a whole-body plethysmography with a pneumo- tachograph linked to a transducer (EMMS) to determine changes in RL in response to increasing doses of inhaled methacholine (10, 30, 60, and 100 mg/ml; Sigma-Aldrich). Lungs from allergen-sensitized and challenged mice were taken and fixed in 10% formaldehyde-saline for histology (Mangan et al., 2007). Sections of lungs were stained with hematoxylin and eosin for assessment of lung inflammation and Periodic acid-Schiff stained for the enumeration of goblet cells and mucus production. OT/II CD4+ cell transfer model of allergic airway inflammation. Spleen and LNs were harvested from OT/II and SOCS2/xOT/II mice. CD4+ T cells were enriched from single cell suspension by negative selection using the EasySep mouse CD4+ T cell enrichment kit (STEMCELL Technol- ogies). Enriched CD4+ T cells were subsequently sorted to >99% purity with a MoFlo (Beckman Coulter) using anti-CD4 (BD) antibodies. A total of 1.5 × 107 purified CD4+ cells isolated from OT/II or SOCS2/xOT/II mice were injected i.v. into RAG-1/ recipient mice. Control mice received PBS. RAG-1/ mice were administered 50 µg OVA adsorbed onto alum (Thermo Fisher Scientific) i.p. on days 1 and 8 and were subjected to airway exposure with 1% OVA aerosol in PBS for 20 min on days 9–13. 24 h after the last OVA aerosol challenge, lung function and immunological analysis was performed as described in the previous section. Lavage was performed and cytokines were detected in BAL fluid as in the previous section. Lungs were homogenized and cytokines determined (expressed as picogram of cytokine per milligram of lung protein) as previously described (Mangan et al., 2007). Immunoblotting and immunoprecipitation. CD4+ cells were prestimu- lated with anti-CD3 and anti-CD28 for 48 h before activation with rIL-4 (R&D Systems), rIL-12 (PeproTech), or rIL-6 (PeproTech) as indicated. Cells lysates were analyzed byWestern blotting as previously described (Tannahill et al., 2005). Phosphorylated STAT proteins were detected using the following anti- bodies: phospho-STAT6 (Imgenex), phospho-STAT3 (Abcam), and phospho- STAT4 (Invitrogen). Blots were subsequently reprobed with total STAT6, 3, or 4 (Santa Cruz Biotechnology, Inc.).To analyze SOCS2 and SOCS3 expression, total splenocytes from WT or SOCS2/ mice were prestimulated with anti- CD3,anti-CD28,and rIL-2 (R&D Systems) as shown.Cell lysates were immuno- blotted with antibodies against SOCS3 (IBL International), SOCS2 (Cell Signaling Technology), and -tubulin (Sigma-Aldrich). For SOCS3 analysis in CD4+ cells, equal amounts of total protein were immunoprecipitated with anti- SOCS3 (IBL International) that had been precoupled to protein A Sepharose beads. SOCS3 protein was subsequently analyzed by Western blotting as previ- ously described (Tannahill et al., 2005). Densitometry was performed using ImageJ software (National Institutes of Health). Statistical analysis. Data were analyzed with Prism4 (GraphPad Software). Statistical differences were determined by two-tailed unpaired or paired Stu- dent’s t test as appropriate. 1529