NK Cells Kill Stem Cells total monocytes or CD16- subsets of monocytes at 1:1 DPSCs to monocytes for 24–48 hours before they were detached, washed and labeled with 51Cr and added to untreated or IL-2 (1000 u/ml) pre-treated or anti-CD16mAb (3 mg/ml) pre-treated, or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 mg/ml) pre-treated NK cells at different E:T ratios. NK cells were pre-treated as indicated for 24–48 hours before they were added to the co- cultures of monocytes with DPSCs. After 4 hours of incubation the supernatants were removed and the released radioactivity counted by a c counter. % cytotoxicity was determined, and LU30/106 cells were calculated using the inverse of the number of effectors needed to lyse 30% of DPSCs X100. doi:10.1371/journal.pone.0009874.g003 cytotoxicity was calculated as follows: % Cytotoxicity ~ Experimental cpm { spontaneous cpm Total cpm { spontaneous cpm LU 30/106cells were calculated using the inverse of the number of effector cells needed to lyse 30% of tumor target cells X100. Surface and DNA Staining and apoptosis assay Staining was performed by labeling the cells with antibodies or propidium iodide and Annexin V as described previously [19] [16,27]. Luciferase reporter assay Transfections were performed using NFkB Luciferase reporter vector [28] and Lipofectamine 2000 reagent (Invitrogen, CA) in Opti-MEM media (Invitrogen, CA) for 18 hours after which they were adhered to the plate overnight before different immune effectors at 1:1 Effector to target ratios were added. The cells were then lysed with lysis buffer and the relative Luciferase activity was measured using the Luciferase assay reagent kit obtained from Promega (Madison, WI). Statistical analysis An unpaired, two-tailed student t- test was performed for the statistical analysis. One way ANOVA with a Bonferroni post test was used to compare the different groups. Results MSCs and DPSCs were selected based on their phenotypic and functional properties MSCs were CD166+CD105+CD99+CD342CD452and CD142 based on the flow cytometric analysis (data not shown). In addition, MSCs were capable of differentiating to osteogenic, chondrogenic and adipogenic lineages (data not shown, www.clonetics.com). DPSCs were isolated as described previously [26] and is shown by us and by others [10] to have osteogenic, chondrogenic and adipogenic properties. IL-2 activated NK cells are potent inducers of cell death in MSCs Highly purified human NK cells were treated with and without IL- 2 for 8–12 hours before they were added to 51Cr labeled MSCs (Fig. 1A) or DPSCs (Fig. 1B) or NK sensitive K562s (Fig. 1C). The addition of freshly isolated untreated NK cells had lower cytotoxic activity against MSCs (Fig. 1A), DPSCs (Fig. 1B) and K562s (Fig. 1C). However, activation with IL-2 increased cytotoxicity substantially and resulted in a significant lysis of stem cells (p,0.05) (Fig. 1). Anti-CD16 antibody induces death in a subset of untreated and IL-2 treated NK cells and inhibits NK cell mediated lysis of MSCs and DPSCs NK cells were left untreated or treated with anti-CD16 antibody and/or IL-2 for 8-12 hours before they were added to 51Cr labeled MSCs or DPSCs. As shown in a number of previous studies [18,19,20] and below, anti-CD16 mAb treatment induced death in a subset of NK cells and inhibited NK cell cytotoxicity against MSCs and DPSCs (p,0.05) (Fig. 1). The addition of the combination of IL-2 and anti-CD16 mAb treatment also induced death in a subset of NK cells and inhibited NK cell cytotoxicity against MSCs and DPSCs when compared to IL-2 activated NK cells (p,0.05) (Fig. 1). Figure 4. Monocytes prevent NK cell death induced by anti- CD16mAb. NK cells were labeled with FITC before they were either left untreated or treated with anti-CD16 mAb (3 mg/ml), IL-2 (1000 u/ml), or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 mg/ml) and immediately added to monocytes at 1:1 (NK: monocyte) ratios. After an overnight incubation the levels of cell death in NK cells were determined by flow cytometric analysis of Propidium Iodide stained cells. FITC labeled NK cells were gated to determine the levels of PI stained NK cells. 10,000 events were analyzed for each sample. One of three representative experiments is shown in this figure. doi:10.1371/journal.pone.0009874.g004 PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9874