NK Cells Kill Stem Cells Figure 1. Potent lysis of MSCs and DPSCs by the Natural Killer cells; inhibition by anti-CD16 antibodies. Highly purified NK cells were 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) for 8– 12 hours before they were added to the 51Cr labeled MSCs (Fig. 1A) or 51Cr labeled DPSCs (Fig. 1B) or 51Cr labeled K562 (Fig. 1C) at different effector to target (E:T) ratios. After 4 hours of incubation the supernatants were removed and the released radioactivity was counted by a c counter. % cytotoxicity was determined as indicated in the Materials and Methods section. One of five representative experiments is shown in this figure. doi:10.1371/journal.pone.0009874.g001 Institutional Review Board (IRB) were obtained from the blood donors and all the procedures were approved by the UCLA-IRB. ELISA and Multiplex Cytokine Array kit Single ELISAs were performed as described previously [13]. Fluorokine MAP cytokine multiplex kits were purchased from R&D Systems (Minneapolis, MN) and the procedures were conducted as suggested by the manufacturer. To analyze and obtain the cytokine concentration, a standard curve was generated by either two or three fold dilution of recombinant cytokines provided by the manufacturer. Analysis was performed using the Star Station software. 51Cr release cytotoxicity assay: The 51Cr release assay was performed as described previously [22]. Briefly, MSCs or DPSCs were co-cultured with irradiated (10 Gy) subsets of monocytes for 24– 48 hours before they were labeled with 51Cr for 1 hour, after which they were washed and added to NK samples. In several experiments monocytes were sorted out from the stem cell co-cultures before they were labeled with 51Cr and added to the NK cells. 100% removal of monocytes from the stem cells was achieved when the sorted samples were checked either by microscopy or by flow cytometric analysis of stained cells. After a 4 hour incubation period, the supernatants were harvested and counted for released radioactivity. The percentage of Figure 2. Decreased killing of MSCs by NK cells when MSCs were co-cultured with monocytes prior to the addition of NK cells. Monocytes were purified from PBMCs and irradiated as indicated in the Material and Methods section. MSCs (16106 cells/plate) were cultured with the irradiated monocytes (monocyte: MSC ratio of 1:1) for 24–48 hours before they were removed from the plates, washed and labeled with 51Cr and used as targets in the cytotoxicity assays against NK cells. The NK samples were either left untreated (Fig. 2A) or treated with anti-CD16 mAb (3 mg/ ml) (Fig. 2B), IL-2 (1000 u/ml) (Fig. 2C), or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 mg/ml) (Fig. 2D) for 24–48 hours before they were added to 51Cr labeled MSCs at different effector to target (E:T) ratios. Supernatants were removed after 4 hours of incubation and the released radioactivity counted by a c counter. % cytotoxicity was determined as indicated in the Material and Methods section. One of three representative experiments is shown in this figure. doi:10.1371/journal.pone.0009874.g002 PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9874