Bone marrow cells were harvested from the femur and tibiae of D011.10 mice. Subsequently, the erythrocytes were lysed. After washing with 1% FCS supplemented RPMI 1640 medium, T and B cells were depleted using mouse pans T and B dynabeads (Invitrogen). T- and B-depleted cells were incubated at 37°C. After 4 h, nonadherent cells were harvested and cultured at 5 × 106 /mL in 24-well plate in complete medium (RPMI 1640 supplemented with 8% FCS, 2 mM L-glutamin, 5 × 10−5 M β-mercaptoethanol, streptomycin, nonessential amino Pim inhibitor acids (Gebco) and 1 mM sodium pyruvate (Sigma-Aldrich)) with 1000 IU/mL of

rmGM-CSF (R&D systems), and 1000 IU/mL of rmIL-4 (R&D systems). The medium was refreshed every Tanespimycin other day for 1 week. After 1 week culturing, bone marrow-derived DCs were harvested and cultured with DX5+CD4+, DX5−CD4+ T cells or their supernatants or medium for 3 days. LPS (0.01 μg/mL; Sigma-Aldrich) was added after 1 day. The DCs obtained were cultured at 0.4 × 106 /mL with OVA323-339 peptide and OVA-specific CD4+ T cells at 1 × 106 /mL in total volume of 150 μL for 3 days. After 3 days, cytokine production was determined by flow cyto-metry. IL-12

(20 ng/mL) that was added to the co-cultures of CD4+ T cells and DCs were purchased from eBioscience. The concentrations of anti-IL-4 and anti-IL-10 antibodies used for blocking studies were chosen on the 17-DMAG (Alvespimycin) HCl basis of titration experiments where known concentrations of cytokine were effectively inhibited in a bioassay [45]. Cytokine levels in DCs cell culture supernatants were measured by ELISA using IL-12p70 kit ELISA Ready-set-Go (eBioscience) according to the manufacturer’s instructions. Matched pairs of antibodies to measure IL-12p40 were purchased from BD. The expression of the surface molecules was examined

using fluorescence-labeled antibodies against B7-H1 (MIH5) and B7-DC (TY25) from eBioscience and CD80 (16-10A-1), CD86 (GL-1), CD40 (3/23), and MHC class II from BD. CD4+ T cells were visualized by staining with anti-CD4-PerCP-Cy5.5 (L3T4/RM4-5; BD Pharmingen). KJ1-26-PE (Invitrogen) was used to detect OVA-specific T cells. Anti-IFN-γ-FITC (XMG1.2; BD Pharmingen) was used to detect IFN-γ-producing cells. The staining reactions were performed according to manufacturer’s protocol. In brief, the cells were first washed in the staining buffer (PBS containing 0.5% BSA); subsequently, the cells were incubated with antibodies for surface markers for 20 min at 4°C. For intracellular cytokine staining, Brefeldin A (10 μg/mL; Sigma-Aldrich) was added to co-culture of CD4+ T cells and DCs for 4 h. After washing, the cells were fixed using Cytofix/Cytoperm (BD Bioscience) followed by washing with Perm/wash (BD Bioscience). For determination of cytokine production, the cells were stained for intracellular cytokines in Perm/wash for 20 min.