tion of IFN-c, TNF-a, IL-1a and IL-1b cytokines and caused elevation in the levels of chemokines and inducible nitric oxide (iNOS) [14]. Finally, indoleamine 2,3-dioxygenase (IDO) and Prostaglandin E2 (PGE2) were also shown to be the key mediators of MSC inhibition of NK cells [6]. Overall, these studies indicated the inhibitory role of key factors induced during the interaction of MSCs with the immune effectors. We have previously coined the term of ‘‘split anergy’’ for the responses observed by NKDC (NK cells dissociated from tumor conjugates) and NKC (NK cells not dissociated from the tumor conjugate). Whereas NKDC responded to IL-2 activation for cytotoxicity, they were unresponsive to IL-2 mediated induction of proliferation or secretion of cytokines. In contrast, NKC showed an inverse response namely, they did not respond to IL-2 activation for cytotoxicity, but proliferated and secreted cytokines [15,16]. Treatment of NK cells with IL-2 and anti-CD16mAb also induced split anergy by significantly decreasing the NK cell cytotoxicity while increasing the cytokine secretion capabilities of NK cells [17,18,19,20]. Furthermore, IL-2 rescued anti-CD16 mAb mediated apoptosis induced in a subset of NK cells [19]. Loss of cytotoxicity in NK cells was exacerbated when NK cells were either treated with F(ab)’2 fragment of anti-CD16 mAb or treated with a combination of MHC-Class I and anti-CD16 mAb while the same treatments resulted in an increased secretion of cytokines [17,20]. These results suggested that increase in signaling load on NK cells is likely to result in a decrease in cytotoxicity while increasing secretion of cytokines by the NK cells. Therefore, three distinct functional outcomes could be observed in NK cells which have either interacted with sensitive tumor-target cells or treated with anti-CD16 mAb in the presence of IL-2 treatment, namely; 1- Loss of cytotoxicity, 2-gain in the ability to secrete cytokines and 3- death in a subset of NK cells. Whether these functions are carried out by the same NK cell subsets or there are distinct subsets of NK cells with distinct functional capabilities require further studies. However, similar to T cells, NK cells can also be functionally anergized or induced to undergo cell death during increased signaling load. Increased activity of NFkB in tumor cells was found to be inhibitory for the function of NK cells since blocking NFkB in tumor cells made these cells susceptible to lysis by the NK cells [21,22]. Therefore, to determine which subpopulations of immune effectors were able to protect DPSCs/MSCs against NK cell mediated lysis, we undertook studies to first identify the subset of immune effectors, which was able to increase NFkB in stem cells substantially, and to determine their role in the overall increase in the survival and function of stem cells. It is quite well established in tumor immunotherapy that certain effectors of the myeloid arm of the immune system induce resistance in tumor cells and cause suppression of NK cell mediated cytotoxicity [23]. Monocytes, an important subset of the myeloid arm of the immune effectors are known to be the major inducers of resistance in tumor cells and are shown to significantly aid in the progression of cancer in tumor-bearing hosts [23,24,25]. However, the mechanisms by which monocytes contribute to the progression of cancer have not been fully elucidated yet. Therefore, studies reported in this paper may also have the potential to shed light on this very important question. Our recent findings indicated that monocytes protect oral tumors via NFkB dependent and independent pathways (manuscript submitted). In addition, the role of monocytes in suppression of NK cell mediated cytotoxicity against MSCs/DPSCs has not been explored yet. Furthermore, whether a unique subset or all monocytes mediate suppression of NK cell mediated cytotoxicity against MSCs needs to be explored. Studies reported in this paper are significant in NK Cells Kill Stem Cells several ways. First, we demonstrate that both allogeneic and autologous NK cells are capable of lysing stem cells significantly and that lysis can be blocked by the treatment of NK cells with anti-CD16 antibody. Second, either the total populations of monocytes or those depleted of CD16+ subsets are capable of increasing survival of DPSCs/MSCs against NK cells in three way interactions, raising the overall secretion of key cytokines responsible for the resistance and differentiation of stem cells and protection against NK cell mediated cytotoxicity. Therefore, the effect of monocytes on NK cells are reminiscent of the effect of anti-CD16 mAb or target mediated induction of split anergy in NK cells [16,19]. Thus, the protection of stem cells against NK cell lysis is partly due to the direct induction of resistance of stem cells by monocytes and indirectly by serving as a shield to protect lysis of stem cells by the NK cells since they too are greatly susceptible to NK cell mediated lysis in a three way interaction. Finally, by delineating the optimal cell-cell interaction required for the protection of stem cells from NK cell mediated cytotoxicity, we can design strategies to successfully implant either allogeneic or autologous stem cells in different tissue engineering applications. Overall, our studies demonstrate that monocytes are important catalysts of survival for different cellular subsets, and protect stem cells from NK cell mediated lysis, thereby elevating the collective functions of the cells. Materials and Methods Cell Lines, Reagents, and Antibodies HEp2 cells were obtained from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. MSCs were purchased from Clonetics and cultured with the basal medium provided by the manufacturer. DPSCs were isolated as described previously [26] and they were cultured in complete DMEM supplemented with 10% FBS. Recombinant IL-2 was obtained from the NIH repository. The NK, CD16+ and CD162 monocyte purification kits were obtained from Stem Cell technologies (Stem Cell, Vancouver, Canada). The ELISA kits for IFN-c, VEGF and IL-6 were purchased from R&D Systems (Minneapolis, MN). The TNF-a ELISA was developed in our laboratory and reported previously [19]. The multiplex cytokine array kit was purchased from R&D Systems. The Fluorescein Isothiocyanate (FITC) conjugated Annexin V/Propidium Iodide (PI) kit was purchased from Coulter Immunotech (Miami, FL). Purification of NK cells, Total monocytes and CD162 Monocytes PBMCs from healthy donors were isolated as described before [16]. Briefly, peripheral blood lymphocytes were obtained after Ficoll-hypaque centrifugation and adherence to the plate for 1 hour. Purified NK cells were negatively selected by using an NK cell isolation kit. The purity of NK cell population was found to be greater than 90% based on flow cytometric analysis of anti-CD16 antibody stained cells. The levels of CD3+ T cell staining in purified population of NK cells remained low at 2.4%61%, similar to that obtained by the non-specific staining using isotype control antibody throughout the experimental procedures. The adherent subpopulation of PBMCs was detached from the tissue culture plates and the total population of monocytes and those depleted of CD16+ subsets (CD162) were purified using isolation kits obtained from Stem Cell Technologies (Vancouver, Canada). Greater than 95% purity was achieved for each subset based on flow cytometric analysis of CD14 and CD16 antibody stained monocytes. Written informed consents approved by UCLA PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9874