In Vivo DC Editing by NK Cells Figure 4. DC editing by NK cells generates a more protective immune response during cancer cell vaccination. Mice were vaccinated with immunogenic tumor cells alone (TS/A), tumor cells mixed with MHC-negative ells (TS/A + YAC-1) or MHC-negative cells alone (YAC-1) (five mice per group). In a group of mice, anti-asialo GM1 mAbs were administered i.p. 48 h before administration of cell vaccines to deplete NK cells. After 21 days, mice were challenged with a lethal dose of tumor cells and monitored for tumor growth twice a week for two months. Mice vaccinated with tumor cells mixed with MHC-negative cells (TS/A + YAC-1) displayed a delay in tumor growth and a striking survival to TS/A cancer cell challenge. This protective effect was abrogated when mice were depleted of NK cells (TS/A + YAC-1 + anti-asialo GM1). doi:10.1371/journal.pone.0039170.g004 Antibodies and Flow Cytometry Animals undergoing in vivo NK cell depletion received two i.p. injections with anti-asilao GM1 at day –2/0 (anti-NK rabbit serum, 200 ml/mouse of 1:10 diluted stock solution, Wako Neuss, Germany). After 36 h from injection, inguinal LNs were harvested and the cell suspensions obtained were labeled with anti-CD11c, anti-DX5, anti-CD40, anti-CD80, anti-CD86 and anti-MHC class II (all from eBioscience, San Diego, CA ) and analyzed by flow cytometry (Facs Canto II, BD). Proliferation Assay For the proliferation assay, spleen cells from B6 mice were labeled with 5 mM carboxyfluorescein succinimidyl ester (CFSE) in PBS plus 0.1% BSA for 10 min at 37uC. After extensive washing with PBS plus 0.1% BSA, cells were incubated with allogeneic CD11c DCs sorted from draining LN of mice injected with YAC-1 cells that had or had not been subjected to NK cell depletion. The DCs were extensively washed in PBS before culture with T cells. After 6 days, CFSE fluorescence was evaluated on CD3+ cells by flow cytometry. Enzyme-linked Immunospot Assays The frequencies of IFN-c-producing spleen cells from vaccinated animals were determined after three weeks by an enzyme-linked immunospot (ELISPOT) assay performed on splenocytes. Multi- screen-IP plates (Millipore, Bedford, MA and BD Pharmingen, San Jose, CA, USA) were coated overnight with 10 mg/ml of anti-IFN-c mAb in PBS (endogen, Woburn, MA and BD Pharmingen, San Jose, CA, USA). Plates were then washed with RPMI 1640 and blocked for 3 h with PBS-2% bovine serum albumin. Splenocytes were counted in complete RPMI 1640 and then seeded at a 2-fold serial dilution, starting from 46105 cells per well, in duplicate, in the presence or absence of: (i) irradiated (20,000 rads) TS/A tumor cells; (ii) YAC-1 cells (both at 10:1 effector/stimulator cell ratio); (iii) endogenous retroviral gp70-derived AH1 peptide, the 9-amino acid H-2Ld- restricted peptide (SPSYVYHQF, synthesized by INBIOS S.r.l., Napoli, Italy) at final concentration of 10 mg/ml. AH1 is the immunodominant CD8 antigen expressed on surface of TS/A cancer cell lines [37]. Where indicated, cell stimulation was also obtained by PMA and ionomycin at final concentration of 50 ng/ml and 500 ng/ml, respectively (SIGMA). After 40 h of incubation, plates were washed with PBS-0.05% Tween 20 and incubated with 1 mg/ml biotinylated secondary mAb to IFN-c (Endogen and BD Pharmingen, San Jose, CA, USA) in PBS-1% bovine serum albumin for 3 h at room temperature. Horseradish peroxidase-conjugated streptavidin (1:5,000) was then added for 2 h at room temperature. After washing, the plates were stained with AEC staining kit (Sigma and BD Pharmingen, San Jose, CA, USA) and spots were counted using a stereomicroscope. A .2-fold increase in number of spots over the control (splenocytes cultured with no stimulus) was considered as a positive response. Data were expressed as number of spot-forming cells per million of spleen cells. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39170