In Vivo DC Editing by NK Cells Figure 3. DC editing by NK cells promotes antigen specific CTL expansion. A: Mice were inoculated with immunogenic TS/A cells, TS/A cells mixed with MHC-devoid cells (YAC-1) or YAC-1 cells alone. 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, splenocytes were restimulated with either TS/A cells, the TS/A MHC class I-restricted immunodominant peptide AH1 or with YAC-1 cells and the frequencies of IFNc-producing cells were determined by ELISPOT assay. A significant increase of antigen-specific IFNc-producing cells was detectable in mice vaccinated with TS/A mixed with YAC-1 cells (TS/A + YAC-1). The increase in antigen-specific CTL was abrogated when mice had been depleted of NK cells before vaccination (TS/A + YAC-1 + anti asialo GM1). B: Similar experiments were performed in perforin KO mice (pfn 2/2) and in parallel with wild type (WT) mice. A significant lower number of TS/A tumor-specific CTL was induced by vaccination in perforin KO mice when compared to wild type mice. Bars represent mean values and SEM of results obtained in three independent experiments (three mice per group). ** = p , 0,001; * = p , 0,005. doi:10.1371/journal.pone.0039170.g003 week-old female wild type BALB/c (H-2Kd) mice and wild-type C57BL/6 (H-2b) mice were purchased from Harlan (Udine, Italy). Perforin2/2 mice (CByJ.B6-Prf1tm1Sdz/J) were purchased from The Jackson Laboratory (Bar Harbour, ME). Mice were subcutaneously (s.c.) injected with 2 x 106 irradiated (20,000 rads) YAC-1 cells or, where indicated, with either irradiated TS/A tumor cells alone (5 x 105) or irradiated TS/A tumor cells (5 x 105) admixed with irradiated YAC-1 cells (2 x 106). Animals undergoing in vivo NK cell depletion received three intraperitoneal (i.p.) injections with anti-asialo-GM1 antibodies (anti-NK rabbit serum, 200 ml/mouse of 1:10 diluted stock solution, Wako) at days 22/0/+1 as previously described [43] (day 0 was the day of cell vaccination. Tumor challenge was done at three weeks after vaccination with a tumorigenic dose 5 x 104 of TS/A tumor cells. Tumor growth and size was measured twice weekly using a caliper. Injection of YAC-1 into BALB/c represents an allogenic system, but the absence of both MHC class I and class II expression in YAC-1 cells minimizes the potential alloresponse. The animals were housed in pathogen-free colony and experiments were performed accord- ing to the National Regulation on Animal Research Resources and approved by the Review Board of the Istituto Nazionale per la Ricerca sul Cancro, Genoa. Lymphoid Organ Cell Isolation Inguinal lymph nodes (LNs) were surgically excised and collected in RPMI 1640/10% FCS. They were cut into small fragments using razor blades and digested in 2 mg/ml collagenase D and 30 mg/ml DNase I (Roche, Mannheim, Germany) at 37uC for 30 min. Single cell suspensions were prepared by filtration through a 70 mm cell strainer (BD Labware, San Jose, CA, USA). CD11c+ cells were isolated by positive selection using anti-CD11c microbeads and a magnetic separator (Miltenyi Biotec, Bergisch Gladbach, Germany). Spleen cells were isolated by mechanical dissociation and filtrate through a 70 mm cell strainer (BD Labware). Erythrocytes in the spleenic preparations were removed by hypotonic lysis with NH4Cl/ KHCO3 /EDTA. PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39170