Strategies to Rescue Mesenchymal Stem Cells (MSCs) and Dental Pulp Stem Cells (DPSCs) from NK Cell Mediated Cytotoxicity

BACKGROUND: The aim of this paper is to study the function of allogeneic and autologous NK cells against Dental Pulp Stem Cells (DPSCs) and Mesenchymal Stem Cells (MSCs) and to determine the function of NK cells in a three way interaction with monocytes and stem cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that freshly isolated untreated or IL-2 treated NK cells are potent inducers of cell death in DPSCs and MSCs, and that anti-CD16 antibody which induces functional split anergy and apoptosis in NK cells inhibits NK cell mediated lysis of DPSCs and MSCs. Monocytes co-cultured with either DPSCs or MSCs decrease lysis of stem cells by untreated or IL-2 treated NK cells. Monocytes also prevent NK cell apoptosis thereby raising the overall survival and function of NK cells, DPSCs or MSCs. Both total population of monocytes and those depleted of CD16(+) subsets were able to prevent NK cell mediated lysis of MSCs and DPSCs, and to trigger an increased secretion of IFN-gamma by IL-2 treated NK cells. Protection of stem cells from NK cell mediated lysis was also seen when monocytes were sorted out from stem cells before they were added to NK cells. However, this effect was not specific to monocytes since the addition of T and B cells to stem cells also protected stem cells from NK cell mediated lysis. NK cells were found to lyse monocytes, as well as T and B cells. CONCLUSION/SIGNIFICANCE: By increasing the release of IFN-gamma and decreasing the cytotoxic function of NK cells monocytes are able to shield stem cells from killing by the NK cells, resulting in an increased protection and differentiation of stem cells. More importantly studies reported in this paper indicate that anti-CD16 antibody can be used to prevent NK cell induced rejection of stem cells

Increased Lysis of Stem Cells but Not Their Differentiated Cells by Natural Killer Cells; De-Differentiation or Reprogramming Activates NK Cells

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-γ were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-γ. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells

Total monocytes or CD16⁻ subsets of monocytes synergize with NK cells and MSCs or DPSCs to induce IFN-γ secretion.

<p>MSCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g006" target="_blank"><b>Fig. 6A</b></a>) or DPSCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g006" target="_blank"><b>Fig. 6B</b></a>) at 1×10⁵ cells/well were co-cultured with highly purified and irradiated total monocytes or CD16⁻ subsets of monocytes at 1∶1 MSCs or DPSCs to monocyte ratios for 24–48 hours before untreated or IL-2 (1000 u/ml) pre-treated or anti-CD16mAb (3 µg/ml) pre-treated, or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 µg/ml) pre-treated NK cells at 1∶1 MSCs or DPSCs to NK cells were added. NK cells were pre-treated as indicated for 24–48 hours before they were added to the co-cultures of monocytes and MSCs or DPSCs. After 24–48 hours of the addition of NK cells the supernatants were removed and subjected to specific ELISA for IFN-γ. One of four representative experiments is shown in this figure.</p

Monocytes are potent inducers of NFκB activity in HEp2 cells.

<p>HEp2 cells were transfected with NFκB 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 NFκB activity in the samples was determined over the control HEp2 cells in the absence of immune effectors.</p

Decreased killing of MSCs by NK cells when MSCs were co-cultured with monocytes prior to the addition of NK cells.

<p>Monocytes were purified from PBMCs and irradiated as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#s2" target="_blank">Material and Methods</a> section. MSCs (1×10⁶ 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g002" target="_blank"><b>Fig. 2A</b></a>) or treated with anti-CD16 mAb (3 µg/ml) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g002" target="_blank"><b>Fig. 2B</b></a>), IL-2 (1000 u/ml) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g002" target="_blank"><b>Fig. 2C</b></a>), or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 µg/ml) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g002" target="_blank"><b>Fig. 2D</b></a>) 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 γ counter. % cytotoxicity was determined as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#s2" target="_blank">Material and Methods</a> section. One of three representative experiments is shown in this figure.</p

Monocytes and T cells are targets for NK cell mediated lysis.

<p>Highly purified NK cells were left untreated or treated with anti-CD16 mAb (3 µg/ml), IL-2 (1000 u/ml), or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 µg/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 γ 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.</p

Total monocytes or those depleted of CD16⁺ subsets of monocytes protect MSCs and DPSCs against NK cell mediated cytotoxicity.

<p>MSCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g003" target="_blank"><b>Fig. 3A</b></a>) or DPSCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g003" target="_blank"><b>Fig. 3B</b></a>) each at 1×10⁶ cells/plate were co-cultured with highly purified and irradiated total monocytes or CD16⁻ subsets of monocytes at 1∶1 MSCs to monocyte ratio for 24–48 hours before they were detached, washed and labeled with ⁵¹Cr and added to untreated or IL-2 (1000 u/ml) pre-treated or anti-CD16mAb (3 µg/ml) pre-treated, or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 µg/ml) pre-treated NK cells at different E:T ratios. NK cells were pre-treated as indicated for 24–48 hours before they were added to the co-cultures of monocytes with MSCs. After 4 hours of incubation the supernatants were removed and the released radioactivity counted by a γ 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 MSCs or DPSCs X100. Both autologous and allogeneic DPSCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g003" target="_blank"><b>Fig. 3C</b></a>) at 1×10⁶ cells/plate were cultured with highly purified and irradiated total monocytes or CD16- subsets of monocytes at 1∶1 DPSCs to monocytes for 24–48 hours before they were detached, washed and labeled with ⁵¹Cr and added to untreated or IL-2 (1000 u/ml) pre-treated or anti-CD16mAb (3 µg/ml) pre-treated, or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 µg/ml) pre-treated NK cells at different E:T ratios. NK cells were pre-treated as indicated for 24–48 hours before they were added to the co-cultures of monocytes with DPSCs. After 4 hours of incubation the supernatants were removed and the released radioactivity counted by a γ counter. % cytotoxicity was determined, and LU₃₀/10⁶ cells were calculated using the inverse of the number of effectors needed to lyse 30% of DPSCs X100.</p

Monocytes prevent NK cell death induced by anti-CD16mAb.

<p>NK cells were labeled with FITC before they were either left untreated or treated with anti-CD16 mAb (3 µg/ml), IL-2 (1000 u/ml), or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 µg/ml) and immediately added to monocytes at 1∶1 (NK: monocyte) ratios. After an overnight incubation the levels of cell death in NK cells were determined by flow cytometric analysis of Propidium Iodide stained cells. FITC labeled NK cells were gated to determine the levels of PI stained NK cells. 10,000 events were analyzed for each sample. One of three representative experiments is shown in this figure.</p

Monocytes synergize with NK cells and MSCs to induce IL-6, TNF-α, VEGF and IFN-γ.

<p>MSCs (1×10⁵ 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 µg/ml) pre-treated, or a combination of IL-2 (1000 u/ml) and anti-CD16 mAb (3 µg/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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g005" target="_blank"><b>Fig. 5A</b></a>), TNF-α (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g005" target="_blank"><b>Fig. 5B</b></a>), VEGF (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g005" target="_blank"><b>Fig. 5C</b></a>), and IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009874#pone-0009874-g005" target="_blank"><b>Fig. 5D</b></a>) 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.</p