Flow cytometry data were acquired on a LSRFortessa

(Becton Dickinson) and analyzed with FlowJo software (version 8.8.6, Tree Star). Female (BALB/c×C57BL/6) F1 mice were irradiated at 600+600 Rad with an interval of 3 h and received 107 BM cells from IL-10-GFP C57BL/6 female mice 22, provided by Giorgio Trinchieri (NCI, Frederick). After 8 wk correct reconstitution was checked by flow cytometry, after staining peripheral blood cells with PE anti-Kd, PE-Cy7 anti-CD4 and allophycocyanin anti-Foxp3. Transplanted mice were inoculated with CT26 subcutaneously and treated with OX86 or PBS. After 24 h, tumors were collected and GFP fluorescence was GDC-0449 evaluated in CD4+CD25high cells without any restimulation. In this experiment we could not identify Treg cells by Foxp3 staining because the fixation/permeabilization step induced the loss of GFP expression. BALB/c and CD40−/− tumor-bearing mice were intratumorally injected with OX86 or rat IgG plus 4×107 FITC-conjugated latex micro-spheres of 1 μm diameter (Polysciences). After 24 h, dLNs were mechanically and enzymatically disaggregated (by incubation for 30 min at 37°C with 400 U/mL of collagenase

D). The absolute counts of FITC+ CD11c PE-Cy7+ cells were done for each sample. BMs were collected from femurs and tibias of BALB/c and CD40−/− mice. Cells were cultured for 10 days in IMDM with 10% FBS supplemented with conditioned medium from a murine fibroblast cell line engineered Akt inhibitor to express mouse GM-CSF (corresponding to 20 ng/mL of recombinant GM-CSF). The differentiation state of cells was checked by flow cytometry. TILs were enriched by ficoll gradient from single-cell suspensions of mechanically disaggregated tumors 24 h after OX86 or rat IgG treatment. CD4+CD44highCD62Llow Tem cells were sorted using a FACSAria (Becton Dickinson) from TILs pooled from different mice

and cultured with BMDCs at 1:1 ratio. After 24 h, BMDC activation was analyzed by flow cytometry. Treg cells pooled from splenocytes from different Foxp3-GFP mice were sorted using a FACSaria (Becton Dickinson) as CD4+GFP+CD8−B220−CD11b− cells. Sclareol Purity after sorting was assessed around 98%. Sorted Treg cells were activated overnight with coated anti-CD3 (1 μg/mL) plus OX86 or rat IgG (10 μg/mL). RNA was purified using mirVana Kit (Ambion), and checked for integrity and purity by Agilent Bioanalyzer. Each sample was analyzed in duplicate. RNA (0.2 μg) was reverse transcribed, labeled with biotin and amplified using the Illumina RNA TotalPrep amplification kit (Ambion). Biotinylated sample (1 μg) was hybridized at 58°C overnight to an expression Bead Chip MouseRef_8_v2.0 array (Illumina). Array chips were washed, stained with 1 μg/mL Cy3-streptavidin (GE Healthcare Europe GmbH) and scanned with an Illumina BeadArray Reader (Illumina).