NK Cells Kill Stem Cells Figure 7. Decreased lysis of DPSCs by NK cells were seen when monocytes, T cells and B cells were sorted out from co-cultures of DPSCs with these cells before the addition of NK cells. Purified total population of monocytes or those depleted of CD16+ subsets (Fig. 7A), purified T cells (Fig. 7B) and purified B cells (Fig. 7C) were each co-cultured with 16106 DPSCs per plate at 1:1 ratios for 24–48 hours, before the subsets of monocytes or T cells or B cells were sorted out and removed from DPSCs. Sorted DPSCs from monocytes, T cells and B cells were then labeled with 51Cr, washed 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. After 4 hours of incubation the supernatants were removed and the released radioactivity counted by a c counter. % cytotoxicity was determined at different E:T ratio, and LU30/106 cells were calculated using the inverse of the number of effectors needed to lyse 30% of the DPSCs X100. doi:10.1371/journal.pone.0009874.g007 augmented when monocytes were added to MSCs and NK cells when compared to those in the absence of monocytes (p,0.05) (Figs. 5A and 5B). However, the levels of IL-6 and TNF-a reach plateau when monocytes were added to untreated or anti-CD16 mAb and/or IL-2 treated NK cells and MSCs (Figs. 5A and 5B). Secretion of VEGF was only observed in samples containing MSCs, and monocytes further increased secreted VEGF by MSCs (p,0.05) (Fig. 5C). Finally, as expected IL-2 treated NK cells secreted moderate amounts of IFN-c which were synergistically increased when co-cultured in the presence of MSCs (p,0.05) (Fig. 5D). The addition of anti-CD16 mAb in combination with IL-2 to NK cells in the absence of MSCs increased secretion of IFN-c when compared to IL-2 alone treated NK cells in the absence of MSCs. IFN-c secreted levels remained similar between IL-2 alone and IL-2 and anti-CD16 mAb treated NK cells cultured with MSCs (Fig. 5D). Monocytes added to IL-2 or IL-2 and anti-CD16 antibody treated NK cells in the absence of MSCs or those in the presence of MSCs, synergistically increased the levels of secreted IFN-c (p,0.05) (Fig. 5D). However, the highest increase in IFN-c release was seen when monocytes were added to IL-2 or IL-2 and anti-CD16 mAb treated NK cells with MSCs (Fig. 5D). These results indicated that monocytes increased all of the above-mentioned cytokines in co-cultures with MSCs, and further synergized with IL-2 or IL-2 and anti-CD16 mAb treated NK samples to increase the release of cytokines in the co-cultures of NKs and MSCs. Similar results were obtained when NK cells were co-cultured with monocytes and DPSCs (Fig. 6). Total populations of monocytes or those depleted of CD16+ subset trigger significant release of IFN-c by NK cells in a three way interaction with MSCs or DPSCs Purified irradiated total monocytes or those depleted of CD16+ subsets were each co-cultured with MSCs or DPSCs for 24– 48 hours before untreated or IL-2 and/or anti-CD16 mAb pre- treated NK cells were added. The incubation was then continued for another 24–48 hours before the supernatants were removed and subjected to specific ELISAs for IFN-c (Fig. 6). The differences in secretion of IFN-c between the two monocyte samples were either moderate or very slight when NK, monocytes and MSCs were all present in the co-cultures (Figs. 6A and 6B). Monocytes cultured with or without MSCs (Fig. 6A) and DPSCs (Fig. 6B) increased secretion of IFN-c substantially when added to IL-2 or IL-2 and anti-CD16 antibody treated NK cells. Since monocytes alone or monocytes cultured with MSCs or DPSCs did not have any effect on IFN-c secretion in all the experiments tested, we did not include them in Fig. 6 (data not shown). Protection of DPSCs from NK cell mediated cytotoxicity could still be seen when monocytes were sorted out from DPSCs before they were exposed to NK cells Purified total population of monocytes or those depleted of CD16+ subsets were each co-cultured with DPSCs for 24– 48 hours, before they were sorted out and removed from DPSCs. DPSCs were then labeled with 51Cr and cultured with untreated or IL-2 and/or anti-CD16 mAb pre-treated NK cells. As shown in Fig. 7A, protection from NK cell mediated lysis could still be seen when monocytes were removed from DPSCs before their exposure to NK cells. However, this effect was not specific to monocytes alone since the addition of either T cells (Fig. 7B) or B cells (Fig. 7C) to DPSCs and their removal after 24–48 hours were also protective against NK cell mediated lysis of DPSCs. Monocytes and T cells are targets for NK cell mediated lysis Since significant IFN-c secretion could also be obtained in the co-cultures of IL-2 treated NK cells with monocytes we determined whether they were also targets of NK cell mediated lysis. As shown in Fig. 8 both monocytes and T cells were targets of Figure 8. Monocytes and T cells are targets for NK cell mediated lysis. 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 DPSCs, or 51Cr labeled monocyts or 51Cr labeled T cells at different effector to target (E:T) ratios. After 4 hours of incubation the supernatants were removed and the released radioactivity counted by a c counter. % cytotoxicity was determined at different E:T ratio, and LU30/ 106 cells was calculated using the inverse of the number of effectors needed to lyse 30% of either DPSCs, monocytes or T cells X100. One of three representative experiments is shown in this figure. doi:10.1371/journal.pone.0009874.g008 PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9874