NK Cells Kill Stem Cells Monocytes prevent NK cell mediated lysis of MSCs and DPSCs Monocytes were purified from PBMCs and irradiated as indicated in the Material and Methods section. MSCs and DPSCs were co-cultured with allogeneic irradiated monocytes for 24– 48 hours before they were labeled with 51Cr and used in the cytotoxicity assays against NK cells. NK cells were left untreated or pre-treated with anti-CD16 antibody and/or IL-2 for 24– 48 hours before they were used in the cytotoxicity assays against MSCs and DPSCs. The addition of monocytes to MSCs significantly protected the MSCs (Fig. 2 and 3A) and DPSCs (Fig. 3B) from NK cell mediated cytotoxicity (p,0.05). Significant inhibition of NK cell cytotoxicity by monocytes could be observed against untreated and IL-2 treated NK samples (p,0.05) (Figs. 2 and 3). Monocytes also increased the levels of alkaline phosphatase staining in MSCs and prevented decrease in alkaline phosphatase expression induced by IL-2 activated NK cells (data not shown). Untreated or anti-CD16 antibody treated live or irradiated monocytes were not able to mediate cytotoxicity against MSCs, DPSCs or K562 cells (data not shown). Overall, these experiments indicated that monocytes protect MSCs and DPSCs against NK cell mediated lysis. The reason cytotoxicity was shown both as percentage of killing over different E:T ratios (Figs. 1 and 2) and as Lu30/106 cells (Fig. 3) is to demonstrate the clear relationship between cytotoxicity and calculated lytic units which will be used from here on after. Both the total populations of monocytes and those depleted of CD16+ subsets prevented NK cell mediated cytotoxicity against MSCs and DPSCs Since unbound anti-CD16 antibody or shed antibody from the NK cells could affect the small subpopulation of CD16+ monocytes by modulating their function, we opted to sort out this small subset of monocytes from the total population of monocytes (162) and compared their function to the activity of monocytes containing both the CD16+ and CD162 subpopula- Figure 5. Monocytes synergize with NK cells and MSCs to induce IL-6, TNF-a, VEGF and IFN-c. MSCs (16105 cells/well) were co-cultured with and without irradiated Monocytes at 1:1 MSCs to monocytes 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 NK: monocyte ratios were added to the MSC: moncyte co-cultures. NK cells were pre-treated as indicated for 24–48 hours before they were added to the co-cultures of monocytes and MSCs. NK samples were also cultured in the absence of monocytes and MSCs. After 24–48 hours of the addition of NK cells the supernatants were removed from the cultures and the levels of IL-6 (Fig. 5A), TNF-a (Fig. 5B), VEGF (Fig. 5C), and IFN-c (Fig. 5D) were determined using multiplex cytokine array kit. The results were also confirmed for each individual cytokine using single ELISAs (data not shown). One of three representative experiments is shown in this figure. doi:10.1371/journal.pone.0009874.g005 PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9874