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Blood, 15 February 2006, Vol. 107, No. 4, pp. 1484-1490. Prepublished online as a Blood First Edition Paper on October 20, 2005; DOI 10.1182/blood-2005-07-2775.
IMMUNOBIOLOGY Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferationFrom the Istituto Giannina Gaslini, Genoa; Centro di Eccellenza per la Ricerca Biomedica and Dipartimento di Oncologia, Biologia e Genetica, Università di Genova, Genoa; Istituto Nazionale per la Ricerca sul Cancro, Genoa; and Dipartimento di Medicina Sperimentale, Università di Genova, Genoa, Italy.
In recent years, mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However, no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells, whereas they only partially affect the proliferation of activated NK cells. In addition, we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30, NKG2D, and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly, MSCs expressed the known ligands for these activating NK receptors—ULBPs, PVR, and Nectin-2. Moreover, NK-mediated lysis was inhibited when IFN-  -exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation.
Mesenchymal stem cells (MSCs) are known for their characteristic of being multipotent stem cells, capable of forming bone, cartilage, and other mesenchymal tissues.1,2 In particular, in vitro experiments demonstrated that clonal MSCs can differentiate into different lineages including not only osteoblasts, chondrocytes, and adipocytes but also muscle cells,3 cardiomyocytes,4,5 and neural precursors.6 Moreover, MSCs are a component of the bone marrow stroma that have been shown to support hemopoiesis by providing suitable cytokines and growth factors.7,8 More recently, another function has been ascribed to MSCs. Thus, as shown by different groups, MSCs can inhibit T-cell responses induced by mitogens or alloantigens.9-11 The mechanisms underlying such immunosuppressive activity are only in part understood. Recent studies suggest that soluble factors produced by MSCs represent key mediators of MSC-mediated inhibition,9,12,13 though cell contact might also be involved.14-17 Little information is available regarding the cellular interactions between natural killer (NK) cells, major effector cells of innate immunity, and MSCs. NK cells are known to display strong cytolytic activity against tumor- or virus-infected cells.18,19 Their function is regulated by a series of surface receptors transducing inhibitory or activating signals. Inhibitory receptors are represented mainly by killer immunoglobulin-like receptors (KIRs)20 that are specific for allotypic determinants shared by different HLA class I alleles and CD94/NKG2A specific for HLA-E21 (a nonclassic MHC class 1 molecule), whose levels of expression are directly proportional to HLA class I surface density.22 In normal conditions, the expression of classic HLA class I or HLA-E molecules on the surfaces of normal autologous cells prevents activation of NK cells because of the interaction with these inhibitory receptors. The down-regulation of HLA class I expression or even the loss of single HLA class I alleles at the surface of tumor- or virus-infected cells can result in a lack of inhibitory interactions and can lead to NK-cell activation.23 Under these conditions, target cells become susceptible to NK-mediated killing. Different receptors and coreceptors are responsible for NK-cell activation on interaction with target cells.24 The natural cytotoxicity receptors (NCRs) NKp46, NKp30, and NKp44 represent crucial receptors for NK-cell triggering25 and mediate cytotoxic activity and cytokine production. Thus far, the NCR ligands expressed on target cells have not been identified. Other important receptors involved in NK-cell activation are NKG2D26 and DNAM-1,27 the ligands of which are represented by MICA/B and ULBPs for NKG2D28-30 and the poliovirus receptor (PVR) and Nectin-2 for DNAM-1.31 A series of coreceptors capable of supporting NCR-mediated NK-cell triggering has been described. These include 2B4,32 NTBA,33 NKp80,34 and CD59.35 CD48 represents the known ligand of 2B4,36 whereas NTBA mediates homophilic interactions.37 In this study we analyzed the effect of the interaction between MSC and NK cells. We show that MSCs can inhibit the IL-2-induced proliferation of resting, unactivated NK cells, whereas they had only a partial inhibitory effect on proliferating NK cells. More important, activated NK cells could efficiently lyse MSCs. This information may have relevant implications in novel bone marrow (BM) transplantation approaches in which MSCs are infused to optimize engraftment or to suppress graft-versus-host disease (GVHD).38-40
Isolation and culture of MSCs
After obtaining ethics committee approval and informed consent, human MSCs were obtained from discarded bone tissues from 15 pediatric patients undergoing surgery to correct major scoliosis. Bone marrow cell suspensions were plated at a concentration of 1 x 106 cells/mL in 25-cm2 tissue-culture flasks in Mesencult basal medium supplemented with MSC stimulatory supplements (both from StemCell Technologies, Vancouver, BC, Canada) and incubated at 37°C in a 5% CO2 humidified atmosphere. After 24 hours, nonadherent cells were removed and fresh medium was added. Half the medium volume was replaced twice a week. When the cultures nearly reached confluence, cells were detached by treatment with trypsin/EDTA solution (BioWhittaker, Cambrex, Verviers, Belgium) and replated at 5 x 105 cells per 75-cm2 tissue-culture flask. MSCs were used in the experiments only after 2 to 3 expansion passages to ensure depletion of monocytes/macrophages. In some experiments, MSCs were cultured for 48 hours in the presence of 100 U/mL IFN- Isolation and culture of NK cells NK cells were isolated from the peripheral blood of healthy donors (some were also donors of MSCs) using the RosetteSep method (StemCell Technologies). Purified NK cells were cultured on irradiated feeder cells in the presence of 1.5 ng/mL phytohemagglutinin (Life Technologies, Paisley, Scotland) and 100 U/mL recombinant IL-2 (Proleukin; Chiron, Emeryville, CA) to obtain long-term activated polyclonal or, by limiting dilution, clonal NK cells. Alternatively, NK cells were used in cytotoxicity assays immediately after separation or on short-term activation (20 hours-7 days) with 100 U/mL IL-2 or 2 ng/mL IL-12 (PeproTech). Monoclonal antibodies and cytofluorimetric analysis The following monoclonal antibodies (mAbs), produced in our laboratory, were used in this study: JT3A (IgG2a, anti-CD3), c127 (IgG1, anti-CD16), c218 (IgG1, anti-CD56), BAB281 and KL247 (IgG1 and IgM, respectively, anti-NKp46), Z231 and KS38 (IgG1 and IgM, respectively, anti-NKp44), A76 and F252 (IgG1 and IgM, respectively, anti-NKp30), BAT221 (IgG1, anti-NKG2D), GN18 and F5 (IgG3 and IgM, respectively, anti-DNAM-1), MA127 (IgG1, anti-NTBA), GL183 (IgG1, anti-KIR2DL2/3/S2, anti-CD158b1/b2/j), EB6b (IgG1, anti-KIR2DL1/S1, anti-CD158a/h), Z27 (IgG1, anti-KIR3DL1, anti-CD158e), AZ158 (IgG2a, anti-KIR3DL1/2, anti-CD158e/k), Z270 and Z199 (IgG1 and IgG2b, respectively, anti-NKG2A), Y9 (IgM, anti-CD94), L95 (IgG1, anti-PVR), L14 (IgG2a, anti-Nectin-2), BAM195 (IgG1, anti-MICA), and A6-136 (IgM, anti-HLA class I).20,24,30,31,41
Anti-CD105-PE (N1-3A1, IgG1), anti-CD29 (4B7R, IgG1), anti-CD106 (1.G11B1, IgG1), and anti-CD166 (3A6, IgG1) were purchased from Ancell (Bayport, MN). M295 (IgG1, anti-ULBP1), M310 (IgG1, anti-ULBP2), M551 (IgG1, anti-ULBP3), and M475 (IgG1, anti-ULBP4) were kindly provided by Amgen (Seattle, WA). Anti-CD34 (8G12, IgG1), anti-CD45 and anti-CD45-PerCP (2D1, IgG1), and anti-CD14 (M To analyze the surface markers of MSCs, cell samples were first stained with the appropriate mAbs at 4°C for 30 minutes, washed, and incubated with PE-conjugated AffiniPure F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Then, after washing, cells were resuspended in RPMI 1640 medium supplemented with 5% FCS and analyzed on a FACScalibur by using the Cell Quest program (both from Becton Dickinson). To compare the surface densities of various molecules among different MSC populations, we calculated the mean ratio fluorescence intensity (MRFI)—the ratio between the mean fluorescence intensity (MFI) of cells stained with the selected mAb and that of cells stained with the second reagent only (negative control). Long-term activated polyclonal and clonal NK cells were analyzed for the expression of activating and inhibitory receptors by single- and double-fluorescence analyses. Briefly, cells were incubated with appropriate mAbs followed by FITC- or PE-conjugated isotype-specific second reagents (Southern Biotechnology Associates, Birmingham, AL). Proliferation assays by CFSE dilution and 3H-thymidine uptake IL-2-induced proliferation of resting NK cells was evaluated by the 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution method. Briefly, freshly isolated NK cells were labeled with 5 µM CFSE (Molecular Probes, Eugene, OR) in serum-free RPMI 1640 for 7 minutes at 37°C. Labeled cells were washed twice with RPMI 1640 containing 10% FCS and then cultured in the presence of 100 U/mL IL-2 with or without irradiated MSCs (at different NK/MSC ratios ranging from 1:1 to 8:1). In some experiments, resting NK cells were induced to proliferate in the presence of 100 ng/mL IL-15 (PeproTech). Short-term activated NK cells were harvested at day 7 or 10 of culture and were stained with anti-CD45-PerCP and anti-CD56-PE mAbs. NK-cell proliferation was evaluated as CFSE fluorescence by triple-fluorescence cytofluorimetric analysis of CD45+CD56+ lymphocytes. Proliferation of long-term activated NK cells was evaluated by 3H-thymidine uptake. After culture for 3 or 7 days in the presence of IL-2 (100 U/mL) with or without irradiated MSCs (at different NK/MSC ratios ranging from 1:1 to 16:1), cells were pulsed for 18 hours with 3H-thymidine at 1 µCi (0.037 MBq)/well (Amersham Biosciences, Buckinghamshire, United Kingdom) and then harvested, and 3H-thymidine incorporation was measured using the Plate Chameleon Multilabel Counter (Hidex, Turku, Finland). Cytolytic assays Polyclonal (either short-term or long-term activated) and clonal NK cells were tested for cytolytic activity in a 4-hour chromium 51 (51Cr) release assay, as previously described.41 Autologous or allogeneic MSCs were used as targets. Briefly, MSCs were detached with trypsin/EDTA (ethylenedi-aminetetraacetic acid) solution, labeled with 100 µg 51Cr/106 cells, and plated at 5000 cells/well in 96-well V-bottom microplates. To test the lytic potential of NK cells against MSCs, effectors were plated at different effector-to-target (E/T) ratios. For mAb-mediated blocking experiments, NK cells were preincubated with mAbs specific to the various activating or inhibitory receptors (of IgM isotype, if available) at the concentration of 10 µg/mL and, after washing, were used in the cytolytic assay at a 10:1 or 5:1 E/T ratio. For masking of HLA class I molecules, MSCs were preincubated with saturating amounts of the anti-HLA class I A6136 mAb. To characterize the signaling pattern of activating and inhibitory receptors of polyclonal and clonal NK cells, redirected-killing assays were performed. To this purpose, the murine mastocytoma cell line P815 was used as target in the presence of mAbs of IgG isotype at the concentration of 0.5 µg/mL. Cytokine production
To evaluate IFN-
MSCs prevent the IL-2-induced proliferation of resting NK cells but have only a partial inhibitory effect on proliferating NK cells MSCs have been shown to sharply suppress T-cell proliferation of resting and activated T cells.9,42 To assess whether MSCs could exert a similar inhibitory effect on NK cells, we cultured allogeneic MSCs with resting, unactivated NK cells in the presence of exogenous IL-2 (100 U/mL) or with proliferating NK cells that had been cultured in IL-2 for more than 7 days. As shown in Figure 1A, MSCs prevented the proliferation of resting NK cells. It is of note that the inhibition of NK-cell proliferation occurred at all NK/MSC ratios tested (ranging from 1:1 to 8:1; data not shown). On the other hand, as shown in Figure 1B, only partial inhibition of proliferation of activated NK cells could be detected, and the extent of the inhibitory effect was dependent on the NK/MSC ratio. We further evaluated whether MSCs could also inhibit NK-cell proliferation induced by IL-15. Although not shown, MSCs had a similar suppressive effect on fresh NK cells when cultured in the presence of 100 ng/mL IL-15.
Surface expression on MSCs of ligands recognized by different activating NK receptors To analyze possible interactions between NK cells and MSCs, we first assessed MSCs for the expression of ligands recognized by activating NK receptors. We performed cytofluorimetric analysis using mAbs specific for known ligands of activating receptors and checked, in parallel, the presence of typical markers of MSCs. Table 1 shows the level of expression of the ligands of DNAM-1 and NKG2D triggering receptors and of HLA class I molecules in the 15 MSC populations analyzed in this study. Remarkably, MSCs expressed PVR and Nectin-2 (both ligands of DNAM-1) as well as ULBPs and MICA (both ligands of NKG2D). In particular, among the ligands of NKG2D, ULBP3 was expressed in all 15 MSC populations analyzed. Moreover, in no instance were CD48 (ligand of 2B4) and NTBA (mediating homophilic interactions) molecules expressed by MSCs. In agreement with previous reports,1,2 MSCs did not express CD34, CD45, or HLA-DR (data not shown), whereas they did express low/intermediate levels of HLA class I molecules. Figure 2 shows the cytofluorimetric analysis of MSCs derived from a representative donor (donor 2). Figure 2A shows the expression profiles of CD105, CD166, and CD29 (typical MSC markers). Figure 2B shows the surface expression of ULBP3, Nectin-2, and PVR molecules.
IL-2-activated, but not resting, NK cells lyse MSCs The finding that MSCs expressed ligands for activating NK receptors, together with the low expression of HLA class I molecules, suggested the possibility that MSCs could be susceptible to NK cell-mediated lysis. To test this hypothesis, we analyzed freshly isolated NK cells and NK-cell populations that had been exposed to IL-2 for a short (20-hour) or a long (7-day) interval for their ability to kill allogeneic MSC target cells. In agreement with a previous report,43 freshly isolated NK cells did not lyse MSCs, even when used at high E/T ratios (Figure 3A-C). However, the same NK cells, when exposed to IL-2 for 20 hours only, displayed strong cytolytic activity. For example, approximately 50% specific lysis was achieved at a 6:1 NK/MSC ratio. NK cells cultured in IL-2 for 7 days displayed even higher cytolytic activity. Although not shown, short-term culture of freshly isolated NK cells in the presence of IL-12 did not induce any significant cytolysis of MSCs, whereas low cytolytic activity could be detected only after 72 hours of culture. Having demonstrated the susceptibility of MSCs to lysis of allogeneic effector NK cells, we asked whether lysis also occurred in an autologous setting. To this aim, we performed a series of cytotoxicity experiments in which different MSC populations were used as target and autologous or allogeneic NK cells were used as effector. It is of note that these experiments were performed using long-term IL-2-activated NK cells because of the low numbers of NK cells isolated from the blood samples of pediatric donors. As shown in Figure 3D-F, MSCs were susceptible to NK-mediated lysis independently of whether autologous or allogeneic NK cells were used.
Analysis of the activating or inhibitory molecular interactions involved in the lysis of autologous and allogeneic MSCs To define which receptor-ligand interactions were involved in the NK-mediated killing of MSCs, we used the mAb-mediated masking strategy in cytotoxicity assays. Figure 4 shows the results of 2 representative experiments. In Figure 4A, NK cells derived from an allogeneic donor were used as effector cells against 51Cr-labeled MSCs. mAb-mediated masking of NKp30, NKG2D, and DNAM-1 resulted in the inhibition of lysis. On the other hand, blocking of NKp46 resulted in low inhibition, whereas NKp44 did not appear to exert any substantial role. Consistent results were obtained in 10 independent experiments using different MSCs and different allogeneic NK-cell populations. Figure 4B shows a cytotoxicity assay in which NK cells were tested against autologous MSCs. In addition, in this autologous setting, the major activating NK receptors involved were NKp30, NKG2D, and DNAM-1. Similar results were obtained in 5 independent experiments using autologous combinations. Notably, in allogeneic and autologous NK/MSC combinations, the simultaneous masking of these receptors virtually abrogated cell lysis. Remarkably, autologous MSCs are as susceptible to lysis as allogeneic MSCs. These data suggest that interactions involving HLA class I-specific inhibitory receptors, possibly occurring between NK cells and MSCs, were not sufficient to protect MSCs from lysis. In fact, mAb-mediated masking of HLA class I molecules on target cells did not result in any significant increase of lysis (Figure 4). This is in agreement with the low levels of HLA class I molecules on the surface of MSCs. In addition, mAb-mediated blocking of the HLA-E-specific inhibitory receptor CD94/NKG2A did not result in increased cytotoxic activity. Given that the levels of surface HLA-E usually parallel the HLA class I surface density, our results suggest that MSCs are characterized by low or absent expression of HLA-E molecules.
Decreased susceptibility of MSCs to NK-cell-mediated lysis after exposure to IFN- : role of HLA class I molecules
During infection or, more generally, during inflammatory responses, cells are exposed to proinflammatory cytokines, such as IFN-
Cytokine production by NK cells on interaction with MSCs
To analyze whether NK cells could produce IFN-
Our data provide information on the result of the interaction between NK cells and MSCs. We show that MSCs are susceptible to NK-mediated lysis and that different NK receptor-ligand interactions contribute to NK-cell activation and MSC lysis. In addition, proliferative responses of resting NK cells to IL-2 or IL-15 were blocked even by low numbers of MSCs. In the case of activated NK cells, only a partial decrease in proliferative capacity could be detected. The mechanism(s) by which MSCs would inhibit NK-cell proliferation has not yet been identified. It is conceivable that inhibition may reflect the production of soluble factors, as has been shown for MSC-T-cell interaction. The role of TGF-  , IL-10, PGE2, and IDO are being investigated in our laboratory. A previous study reported that MSCs were resistant to allospecific CTLs capable of lysing PHA-blasts from the same MSC donor.43 This finding is compatible with the low surface expression of HLA class I molecules. On the other hand, the surface expression of low levels of HLA class I molecules favors the NK-mediated lysis of MSCs, as described in the present study. Moreover, MSCs expressed different ligands recognized by activating NK receptors, including PVR and Nectin-2 (DNAM-1 ligands) and ULBPs (NKG2D ligands). Accordingly, mAb-mediated blocking of NKG2D and DNAM-1 resulted in the partial inhibition of lysis. In addition, receptor-blocking experiments using specific mAbs revealed that lysis of MSCs was also dependent on NKp30 and NKp46, though the cellular ligands of these receptors have not been identified so far. Remarkably, NK cells could lyse not only allogeneic but also autologous MSCs. This finding is reminiscent of previous data regarding the susceptibility of immature DC (iDC) to lysis by autologous NK cells.46 NK-mediated lysis of iDC is thought to represent a mechanism of quality control, allowing positive selection of those DCs undergoing full maturation and thus capable of promoting optimal T-cell priming.47,48 In the case of iDC, however, NK-cell activation involved primarily the NKp30 receptor, whereas the lysis of MSCs is mediated by different receptor-ligand interactions. The fact that NK cells can lyse autologous MSCs may prompt one to ask why MSCs are not killed by NK cells in vivo. This may reflect differences between MSCs cultured in vitro and those existing in vivo. The latter represent an infrequent cell type that could perhaps be localized in niches, thus escaping the NK-mediated attack, or could express higher levels of HLA class I molecules, or lack ligands for activating receptors. This would render MSCs resistant to NK-mediated killing. Moreover, in vivo NK cells might not reach, under normal conditions, an activation state sufficient for killing MSCs. In this context, resting, nonactivated NK cells failed to kill MSCs in vitro. Whatever the explanation, cytolytic interactions between NK cells and MSCs might occur with BM transplantation in which cytokines capable of inducing NK-cell activation can be released. Indeed, our present data clearly show that even short-term exposure to IL-2 can induce strong NK cytotoxicity against MSCs. Thus, our data suggest that, in an inflammatory environment, NK cells may become potentially capable of lysing MSCs. Notably, MSCs are the precursors of stromal BM cells and are thought to favor hematopoietic-cell engraftment while preventing GVHD.49 Accordingly, MSCs, obtained under culture conditions similar to that of the present study, have been used in experimental protocols together with CD34+ hematopoietic-cell precursors.39,40 However, under these conditions, NK cells generated from donor CD34+ cells beyond 2 to 3 weeks after transplantation should not cause any relevant damage to MSCs because, during the same time interval, the latter might have undergone differentiation to BM stromal cells and other tissues. The adoptive transfer of NK cells has been proposed in the therapy of acute myeloid leukemias in allogeneic bone marrow transplantation to eradicate leukemic cells.50-52 The adoptive infusion of activated NK cells could potentially kill MSCs if they are infused shortly before or simultaneously with NK cells. Note, however, that this effect could be, at least in part, counteracted by the effect of IFN-. Indeed this cytokine, which is released by NK cells and other cell types, induced the up-regulation of HLA class I molecules in MSCs, thus rendering these cells resistant to NK-mediated lysis. It would be of interest to analyze the final outcome of the in vivo interactions between NK cells and MSCs in suitable murine models, in experimental settings reproducing conditions that occur in BM transplantation-associated adoptive immunotherapy involving donor NK cells and MSCs. In conclusion, our results provide relevant information on the possible effect of NK cells on MSCs. Whether these interactions are relevant in physiologic conditions remains to be determined; however, our data offer an interesting clue regarding possible interferences occurring when the two cell types are used in combination in approaches of adoptive immunotherapy.
Submitted July 13, 2005; accepted October 5, 2005.
Prepublished online as Blood First Edition Paper, October 20, 2005; DOI 10.1182/blood-2005-07-2775.
Supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Istituto Superiore di Sanità (ISS), Ministero della Salute, Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Fondazione Compagnia di San Paolo, Turin, Italy; European Union FP6, LSHB-CT-2004-503319-Allostem (the European Commission is not liable for any use that may be made of the information contained); and Fondo per gli Investimenti della Ricerca di Base (FIRB)-MIUR progetto cod.RBNE017B4C.
G.M.S. designed and performed the research and wrote the paper; A.C. performed research; S.B. provided selected samples; M.C.M. provided financial support and analyzed the data; and L.M. designed the research, analyzed the data, and wrote the paper.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Lorenzo Moretta, Istituto Giannina Gaslini, Largo Gerolamo Gaslini 5, 16147 Genova, Italy; e-mail: lorenzomoretta{at}ospedale-gaslini.ge.it.
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Abstract
Introduction
P9, IgG2b) were purchased from Becton Dickinson (Mountain View, CA). Anti-CD56-PC5 (N901, IgG1), anti-CD48 (J4-57, IgG1), and anti-CD54-PE (84H10, IgG1) were provided by Immunotech-Coulter (Marseille, France). To evaluate the surface expression of HLA class I and HLA class II molecules, W632 and D1-12 (kind gift from R. Accolla) mAbs were used. The anti-CD56-PE (B159, IgG1) and, for intracellular cytokine staining, the anti-huIFN-
) or after 20-hour (
) or 7-day (
) culture in 100 U/mL IL-2 for cytolytic activity against 3 different allogeneic MSC populations. (D-F) Results of 3 independent experiments in which different MSC populations were used as targets while either autologous (squares) or allogeneic (triangles) IL-2-activated NK cells were used as effectors. Data are expressed as percentage of lysis.
) or in the presence of anti-CD94/NKG2A (
) or anti-HLA class I (
