tions (total population) for the prevention of NK cell mediated cytotoxicity against MSCs and DPSCs. Both total population and CD16+ depleted subsets of monocytes could significantly prevent NK cell mediated lysis of MSCs (Fig. 3A) and DPSCs (Fig. 3B and 3C). Similar results were obtained in regards to the protection of DPSCs by monocytes against NK cell mediated lysis when the function of NK cells and monocytes was assessed with both allogeneic and autologous DPSCs (Fig. 3C). Furthermore, the removal of CD16 antibody by extensive washings of NK cells before their culture with monocytes and stem cells had similar results to those which were left unwashed and used in co-cultures with monocytes and stem cells (data not shown). Monocytes prevent anti-CD16 antibody mediated NK cell death Since monocytes were capable of protecting MSCs and DPSCs from NK cell mediated lysis we reasoned that they may also protect NK cells from undergoing cell death. Therefore, to investigate the protection of NK cells by monocytes, untreated or IL-2 treated and/or anti-CD16mAb treated NK cells were co- cultured with monocytes immediately after purification and treatment, and their viability was determined after an overnight incubation. As shown in Fig. 4 monocytes prevented NK cell death induced by anti-CD16 antibody in the presence and absence of IL- NK Cells Kill Stem Cells 2 treatment (p,0.001). Therefore, monocytes are potent inducers of NK cell survival. Monocytes increase IFN-c, IL-6, TNF-a and VEGF secretion substantially when cultured with and without NK cells and MSCs or NK cells and DPSCs Irradiated monocytes were added to MSCs for 24–48 hours before the addition of untreated or IL-2 pre-treated and/or anti- CD16 antibody pre-treated NK cells. After 24–48 hours of incubation with NK cells the supernatants were removed, and the levels of IFN-c, IL-6, TNF-a and VEGF secretion were determined using both multiplex cytokine array assay and single ELISAs (Fig. 5). Monocytes increased the secretion of IL-6 and TNF-a in co-cultures with either NK cells or with MSCs (p,0.05) (Figs. 5A and 5B). However, the highest increase was obtained when monocytes were added to NK and MSCs (p,0.05) (Figs. 5A and 5B). As expected, IL-2 increased the secretion of TNF-a and IL-6 moderately in NK cells and the combination of IL-2 and anti- CD16 mAb had synergistic effect on the release of TNF-a by the NK cells (Figs. 5A and 5B). The addition of monocytes increased synergistically the secretion of IL-6 and TNF-a in all treated NK samples (p,0.05) (Figs. 5A and 5B). MSCs induced synergistically the secretion of IL-6 when cultured with NK samples (p,0.05) (Fig. 5A). Secretion of IL-6 and TNF-a were significantly Figure 6. Total monocytes or CD162 subsets of monocytes synergize with NK cells and MSCs or DPSCs to induce IFN-c secretion. MSCs (Fig. 6A) or DPSCs (Fig. 6B) at 16105 cells/well were co-cultured with highly purified and irradiated total monocytes or CD162 subsets of monocytes at 1:1 MSCs or DPSCs to monocyte ratios for 24–48 hours before 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 1:1 MSCs or DPSCs to NK cells were added. NK cells were pre-treated as indicated for 24–48 hours before they were added to the co-cultures of monocytes and MSCs or DPSCs. After 24–48 hours of the addition of NK cells the supernatants were removed and subjected to specific ELISA for IFN-c. One of four representative experiments is shown in this figure. doi:10.1371/journal.pone.0009874.g006 PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9874