All mice were housed in pathogen-free conditions in the animal center of The Medical College of Shanghai Jiao Tong University (Shanghai, China). Animal care and use were in compliance with institutional guidelines. Mouse forestomach carcinoma (MFC), a mouse gastric cancer cell line, and EVP4593 clinical trial B16F10, a melanoma cell line of B6 (H-2b) mouse origin were purchased from the Shanghai Cell Biology Institutes, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI (Roswell Park Memorial Institute) medium 1640 (GIBCO, USA) containing 12.5% fetal calf serum (FCS), penicillin G learn more (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a

humidified incubator with a 5% CO2 atmosphere. Major reagents Human recombinant CCL3 and CCL20 expressed in Brevibacillus choshinensis and purified to homogeneity was provided by Dr. Shiro Kanegasaki (Effector Cell Institute, Tokyo, Japan). Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNFα), interleukin 4 (IL-4), IL-2, and IL-7 were purchased from Becton Dickinson (New Jersey, USA). Biotinylated anti-F4/80 mAb, Cy-chrome-conjugated streptavidin, phycoerythrin (PE)-labeled anti-B220 mAb, fluorescein isothiocyanate (FITC)-labeled anti-CD11c mAb, rat anti-DEC-205 mAb, FITC-labeled goat anti-rat IgG (Fab)2 antibodies, FITC-labeled mAb against CD40, F4/80,

CD11b, or CD80, and PE-labeled mAb against Ia, CD8α, or CD86 were provided by Pharmigen mTOR tumor (CA, USA). Mitomycin C (MMC) was purchased from Jingmei Biothe (Shenzhen, China). Cell preparation B6 mice were injected via the tail vein with 1 mg MycoClean Mycoplasma Removal Kit CCL3 and CCL20 in 100 μl phosphate-buffered saline (PBS) or with the same dose PBS (control). Peripheral blood (0.8 ml per mouse) was obtained by cardiac puncture from anesthetized mice at the indicated time intervals (0 h, 8 h, 16 h, 24 h, 48 h, 72 h, 120 h) after CCL3 and CCL20 injection. Peripheral blood mononuclear cells (PBMCs) were prepared from peripheral blood by density separation with Ficoll. PBMCs were stained with biotinylated anti-F4/80 mAb followed with Cy-chrome-conjugated

streptavidin, PE-labeled anti-B220 mAb, and FITC-labeled anti-CD11c mAb for fluorescence-activated cell sorter (FACScan, Becton Dickinson) analysis and sorting of F4/80-B220-CD11c+ cells. Reanalysis by FACS showed that the purity of these sorted F4/80-B220-CD11c+ cells was greater than 98%. DC development DCs were generated as described previously [6, 13]. Briefly, purified peripheral blood-derived F4/80-B220-CD11c+ cells from mice injected with CCL3 and CCL20 were cultured at a concentration of 3 × 105 cells/ml in RPMI 1640 medium containing 10% FCS, GM-CSF (4 ng/ml), and IL-4 (10 ng/ml) for 5 d to induce their differentiation into immature DCs. These were cultured further in GM-CSF and TNFα (5 ng/ml) for 3 to 4 d to induce their maturation.