Figure 10. Monocytes are potent inducers of NFkB activity in HEp2 cells. HEp2 cells were transfected with NFkB luciferase reporter vector before they were co-cultured with monocytes, purified NK cells, peripheral blood lymphocytes (PBLs), polymorphonuclear (PMNs) and peripheral blood mononuclear cells (PBMCs) at 1:1 ratio. After 4 hours of incubation the fold induction of NFkB activity in the samples was determined over the control HEp2 cells in the absence of immune effectors. doi:10.1371/journal.pone.0009874.g010 As expected, IL-2 treated NK cells lysed a much higher percentage of MSCs and DPSCs when compared to untreated NK cells, although a range of 20% to 70% cytotoxicity could be observed by untreated NK cells depending on the donor NK cells used to lyse stem cells (data not shown). In addition, anti CD16 antibody treated NK cells lost their cytotoxic function against MSCs and DPSCs since this treatment resulted in anergy as well as the death of a subset of NK cells and prevention of cytotoxicity against MSCs and DPSCs. Moreover, significant induction of IFN-c secretion could be observed in supernatants removed from the co-cultures of either IL-2 or IL-2 and anti-CD16 mAb treated NK cells with MSCs and DPSCs. When MSCs and DPSCs were cultured with either viable or irradiated monocytes before they were exposed to IL-2 treated NK cells a significant decrease in NK cell mediated cytotoxicity could be observed. Interestingly, significant lysis of MSCs and DPSCs by untreated NK cells was also significantly and reproducibly blocked by the addition of monocytes (Fig. 2). To determine whether decreased lysis of stem cells by NK cells was due to competitive lysis of monocytes by NK cells we performed several experiments. Indeed, we confirmed that monocytes were also lysed by NK cells significantly. In addition, we found not only monocytes but also T and B cells to be significant targets of NK cell mediated killing. Furthermore, when we co-cultured stem cells with monocyte subsets and sorted to remove the monocytes from the stem cells we could still observe significant inhibition of NK cell mediated lysis, arguing against the protection of stem cell lysis by NK cells being solely on the bases of competitive lysis of monocytes. Therefore, NK Cells Kill Stem Cells even though lysis of monocytes by the NK cells may in part contribute to the prevention of NK cell lysis of stem cells, interaction of monocytes with stem cells can also provide resistance of stem cells against NK cell cytotoxicity. However, this function was not specific to monocytes since the addition of T and B cells to stem cells and their removal by sorting before their culture with NK cells could also provide resistance of stem cells against NK cell mediated cytotoxicity. Therefore, it is likely that interaction of NK cells with stem cells in the presence of other immune effectors is complex and it is governed by not only the ability of NK cells to lyse other immune effectors, but also due to increased resistance of stem cells to NK cell lysis after interaction with subsets of immune effectors. Decrease in NK cell lysis of MSCs and DPSCs was paralleled with a significant induction of IFN-c in a stepwise manner. Indeed, when MSCs or DPSCs were cultured with IL-2 treated NK cells alone we could observe significant induction of IFN-c secretion. However, the highest increase was seen when NK cells were cultured with MSCs or DPSCs in the presence of monocytes. Therefore, although decreased killing of stem cells by the NK cells could be observed in the presence of monocytes, synergistic secretion of IFN-c by the NK cells in the presence of monocytes and stem cells could be observed, indicating a discordant relationship between cytotoxicity and IFN-c secretion. This was similar to the profiles which we had seen when NK cells were treated with IL-2 and anti-CD16 antibody in which significant decrease in cytotoxicity of NK cells could be observed in parallel with increased secretion of IFN-c (Fig. 5, 6 and 9). We have previously shown that IL-2 rescues NK cells from anti-CD16 mAb mediated apoptosis [19], therefore, it is possible that monocytes also contribute to rescue from apoptosis and increase in secretion of IFN-c by the NK cells. Indeed, monocytes prevent anti-CD16 mediated NK cell apoptosis (Fig. 4). Rescue of NK cells from apoptosis could be an important function of monocytes since 1- monocytes are known to increase NFkB activity in the cells, and 2- lysis of monocytes by the NK cells could result in activation and increased resistance of NK cells to cell death. In this respect T and B cells can also provide increased survival of NK cells since they too are targets of NK cell killing, but they are not capable of inducing NFkB in epithelial cells to the levels which were induced by monocytes. In addition, even though T and B cells could cause resistance of stem cells to NK cell mediated cytotoxicity, they could not raise the levels of IFN-c secretion by the IL-2 and anti- CD16 mAb treated NK cells to the levels which were observed when these cells were cultured with monocytes and stem cells (Fig. 9). These observations prompted us to speculate regarding the significance of interaction of monocytes with NK cells and stem cells. It is plausible that monocytes serve as not only effectors which provide survival for NK cells and stem cells but also they may serve as shields against NK cell lysis of stem cells. Similar to anti-CD16 antibody mediated effect on IL-2 treated NK cells, monocytes too can shield stem cells from killing by the NK cells by increasing the total IFN-c release by the NK cells while decreasing the cytotoxic function of NK cells (split anergy), resulting in an increased protection and differentiation of stem cells. Indeed, monocytes also increased TNF-a, IL-6 and VEGF secretion in the co-cultures of stem cells with NK cells which could further augment induction of NFkB and increased differentiation of stem cells. The shielding effect of monocytes could be a more generalized function of immune effectors since NK cells can also target T and B cells. This may have significant implications regarding the role of NK cells in not only limiting inflammation, but also the significance of other immune effectors PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9874