Activated natural killer cells from patients with acute myeloid leukemia ...

Leukemia (2005) 19, 2215–2222 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu

Activated natural killer cells from patients with acute myeloid leukemia are cytotoxic against autologous leukemic blasts in NOD/SCID mice U Siegler1,3, CP Kalberer1,3, P Nowbakht1, S Sendelov1, S Meyer-Monard2 and A Wodnar-Filipowicz1 1 Department of Research, University Hospital Basel, Basel, Switzerland; and 2Division of Hematology, University Hospital Basel, Basel, Switzerland

Natural killer (NK) cells are implicated in the surveillance of hematological malignancies. They participate in the immune response against residual acute myeloid leukemia (AML) after hematopoietic stem cell transplantation with partial HLA class I disparity. However, the role of NK cells in autologous leukemiaspecific immunity remains poorly understood. We studied the function of NK cells in AML patients at diagnosis. Following isolation, CD56 þ CD3 cells exhibited a high proliferative potential in vitro in response to interleukin (IL)-2. The polyclonal population of activated AML-NK cells expressed normal levels of the activating receptor NKG2D and the major natural cytotoxicity receptor NKp46. AML-NK cells were highly effective with respect to interferon-c production, cytotoxicity against HLA class I-deficient K562 erythroleukemia cells in vitro and retardation of tumor growth in vivo in K562-bearing NOD/ SCID mice. Importantly, when AML blasts were injected into NOD/SCID mice, a single dose of adoptively transfered autologous AML-NK cells significantly reduced the AML load by 8–77%. Recognition of AML blasts may be related to the observed upregulation of ligands for NKG2D and natural cytotoxicity receptors in vivo. We conclude that AML patient-derived NK cells are fully functional, in support of exploring the benefit of AML immunotherapy with IL-2-stimulated autologous NK cells. Leukemia (2005) 19, 2215–2222. doi:10.1038/sj.leu.2403985; published online 13 October 2005 Keywords: AML; NK cells; NK activating receptors; NOD/SCID mice; antileukemia immunity; immunotherapy

Introduction Natural killer (NK) cells represent a CD56 þ CD3 peripheral blood (PB) lymphocyte subset that plays an important role in early innate immune defense against pathogens and cancer.1,2 They have the capacity to kill virus-infected or tumortransformed cells and to produce immunoregulatory cytokines without the need for prior sensitization of their targets. NK cell functions are regulated by signals delivered by an array of cell surface receptors. Activating receptors, represented by the natural cytotoxicity receptors (NCRs) and NKG2D, are responsible for NK cell activation and tumor cell killing.3 The cellular ligands recognized by NCRs on potential target cells remain unknown, whereas several NKG2D-specific ligands belonging to major histocompatibility complex (MIC) class I chain-related and UL-16-binding protein (ULBP) families have recently been described.4,5 Inhibitory receptors, represented by the killer immunoglobulin-like receptors (KIRs), can antagonize the activating pathways upon engagement of human leukocyte antigen (HLA) class I molecules.6 It is the balance between the opposing signals that defines the NK effector function in lysing Correspondence: Professor A Wodnar-Filipowicz, Experimental Hematology, Department of Research, Basel University Hospital, Hebelstrasse 20, CH-4031 Basel, Switzerland; Fax: þ 41 61 2652350; E-mail: [email protected] 3 These authors contributed equally to this work Received 30 March 2005; accepted 14 September 2005; published online 13 October 2005

the infected or transformed target cells, while sparing normal autologous cells.7,8 Recent progress in understanding the unique mechanism of receptor-dependent NK cell activity has induced interest in the potential value of these cells in immunotherapy of cancer, including hematological tumors.9,10 In patients with acute myeloid leukemia (AML), grafted with stem cells from haploidentical or unrelated donors, the mismatch between KIRs and HLA class I molecules facilitated NK cell-mediated killing of leukemic cells, reducing the risk of relapse.11–13 Consequently, adoptive transfer of haploidentical NK cells is undergoing clinical trials in stem cell recipients,14,15 and expansion of NK cells in vivo or ex vivo is being exploited to increase the dose and modulate the cytotoxicity of alloreactive effectors by the use of appropriate cytokines.16,17 These studies involve healthy donor-derived NK cells, while little is known regarding the role of patient-derived NK cells in the control and clearance of autologous leukemia. The NK-mediated recognition and killing of autologous leukemic cells is poor, and functional abnormalites at the level of activating receptor–ligand interactions may be responsible for the low susceptibility of leukemic blasts to NK-mediated lysis.18,19 The question of functional integrity of NK cells in leukemia is therefore important for understanding the mechanism of leukemia escape from NK cell immunity on the one hand, and for verifying their therapeutic potential on the other hand. The aim of the present study was to analyze the phenotype and function of NK cells from patients affected by AML. Our results demonstrate that purified AML-NK cells subjected to polyclonal expansion and activation with interleukin (IL)-2 are cytolytic against K562 erythroleukemia and against autologous AML blasts in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human leukemia. This work may help the development of immunotherapeutic strategies by adoptive transfer of autologous NK cells for the management of AML in patients not eligible for stem cell transplantation.

Materials and methods

Patients and healthy donors In all, 19 patients with AML, including 16 newly diagnosed and three relapsed AML cases, and 11 healthy PB donors were enrolled in the study (Table 1 in Supplementary Information 1). The average blast content in PB was 74% (range 35–98%). Informed consent in compliance with the guidelines of the Ethical Committee of University Hospitals Basel was obtained for each donor.

NK cell isolation and expansion culture Mononuclear cells (MNCs) were isolated from PB by Ficoll– Histopaque (Sigma, St Louis, MO, USA) density-gradient

NK cells in AML U Siegler et al

2216 centrifugation and cryopreserved until use. NK cells from PB of healthy donors were purified by immunomagnetic negative selection (NK cell isolation kit; Supplementary Information 2; Miltenyi Biotec, Bergisch Gladbach, Germany) to a purity of 495%, as determined by staining with anti-CD3 and -CD56 monoclonal antibodies (mAbs) and analysis by FACS (see below). NK cells from PB of AML patients were isolated by negative selection followed by additional positive selection with CD16 or CD56 immunobeads (Miltenyi Biotec), resulting in a purity of 42–70%. A total of 2 104 to 2 105 of CD56 þ CD3 cells were seeded into 24-well plates in 2 ml of Iscove’s modified Dulbecco’s medium (IMDM) containing 5% human AB þ serum (Blutspendezentrum, Basel, Switzerland), 100 U/ml IL-2 (Novartis, Basel, Switzerland), phytohemagglutinin (PHA; 2 mg/ml; H16, Murex Biotech, Datford, England) and irradiated (30 Gy) allogeneic PB-MNCs as feeder cells. After 8–10 days, cells were transfered to six-well plates for further expansion. Cell numbers were determined weekly by Trypan blue dead cell exclusion and the content of CD56 þ CD3 NK cells was measured by FACS (see below). Between days 14 and 21, cultures of patient-derived NK cells were devoid of contaminating AML blasts and contained o5% of CD3 þ T cells. Controland AML-derived polyclonal populations of IL-2 and PHAactivated CD56 þ CD3 cells, hereafter termed control- and AML-NK cells, respectively, were used for phenotypic and functional analyses.

Flow cytometry (FACS) FACS analysis was performed with NK cells in 100 ml of fresh heparinized PB, with NK cells after purification and during culture, and with AML blasts in NOD/SCID PB and bone marrow (BM). NK cells were analyzed by gating on CD45brightCD56 þ CD3 cells, and AML blasts by gating on CD45 þ CD33 þ cells, as described.19 In the case of PB, red blood cell lysis (FACS Lysing solution; BD PharMingen, San Jose, CA, USA) followed the staining with appropriate mAbs. Cell staining was for 20 min in FACS buffer containing phosphate-buffered saline (PBS) and 2% fetal calf serum (FCS, Invitrogen, Carlsbad, CA) with fluorescein isothiocyanate (FITC), phycoerythrin (PE)- or allophycocyanin- and peridin chloropyll protein-conjugated mAbs against human CD3 (clone UCHT1), CD16 (clone 3G8), CD33 (clone WM53), CD45 (clone 2D1), CD56 (clone B159), CD69 (clone FN50), CD94 (clone HP-3D9) and CD161 (clone DX12) or isotype control (clone MOPC-21) mAbs. Staining with the unlabeled mAbs against CD158a (clone HP-3E4), CD158b (clone CH-L), NK-B1 (clone DX9; all from BD PharMingen), NKp46 (clone 9E2; provided by Marco Colonna, Washington University, St Louis, USA), NKG2D (clone M585), ULBP-1 (clone M295), ULBP-2 (clone M311) and ULBP-3 (clone M250; all IgG1 used at 10 mg/ml, provided by David Cosman, Amgen Inc., Seattle) was revealed with FITC- (Jackson ImmunoResearch, West Grove, PA, USA) or PE-conjugated (Southern Biotechnology Associates, Birmingham, AL, USA) goat antimouse (gtams) antibodies. Incubation with 10% mouse serum (Jackson ImmunoResearch) allowed the subsequent staining with directly labeled mAbs. NCR ligands were measured using dimeric complexes of recombinant soluble BirA1.4-tagged receptor molecules of NKp30 and NKp46 as staining reagents (5–10 mg/ml) and the binding was revealed with FITCconjugated gtams antibodies, as described.19 Propidium iodide (Sigma) was used to exclude dead cells from analysis. FACS data were analyzed using the FACSCalibur and CellQuest Pro software (Becton Dickinson). Cell surface expression was Leukemia

quantified as the mean fluorescence intensity (MFI) ratio of values obtained with specific mAbs divided by values given by isotype controls or secondary antibodies.

IFN-g production and NK cell cytotoxicity NK cells from healthy donors and AML patients, prior to and following expansion cultures, were incubated (1 106 cells/ml) in 96-well plates for 36 h with 10 U/ml IL-12 and 100 ng/ml IL-18 (PeproTech, Rocky Hill, NY, USA) or with 100 U/ml IL-2. Brefeldin A (Sigma) was added at 5 mg/ml for the final 4 h of culture.20,21 Cells were fixed in 2% paraformaldehyde for 15 min, washed three times and permeabilized in FACS buffer containing 0.1% saponin. Anti-IFN-g FITC-conjugated and isotype control mAbs (BD PharMingen) were added for 30 min at room temperature. Cells were washed twice in permeabilization buffer and once in FACS buffer and were analyzed with FACSCalibur. Cytotoxicity of control- and AML-NK cells against the erythroleukemia cell line K562 and against primary AML blasts was determined in a 4-h 51Cr release assay.19,20 AML blasts from cryopreserved MNCs were maintained for 1–2 days in IMDM supplemented with 10% FCS prior to the assay. The MNCs were either used directly if the content of leukemic blasts was 485% or were enriched for blasts by the selection with anti-CD34- or CD33-specific immunobeads (Miltenyi Biotec) according to phenotype. NK effector:target (E:T) cell ratio ranged from 10:1 to 0.6:1 using 3 103 target cells in triplicate wells. In blocking experiments, AML blasts were preincubated with anti-HLA class I (clone W6-32, ATCC) or control anti-HLA class II (clone L423, ATCC; used with HLA-DR þ blasts) mAbs at 10 mg/ml for 15 min.

K562 and AML tumor formation in NOD/SCID mice and NK cell transfer NOD/LtSz-scid/scid (NOD/SCID) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were bred and maintained under pathogen-free conditions in the animal facility of the Department of Research, University Hospital Basel. K562 erythroleukemia cells, at 1 107 cells in 100 ml PBS, were injected subcutaneously into the dorsal lateral thorax of NOD/SCID mice.20 Control- or AML-NK cells, at 5 106 cells in 200 ml PBS, were injected intravenously (i.v.) 1 day after K562 inoculation. Tumor growth was monitored weekly by determining the tumor surface area.20 AML-MNCs, at 1 107 blasts in 200 ml PBS, were injected i.v. to 8- to 10-week-old NOD/SCID mice sublethally irradiated with 375 cGy (60Co source; 2 cGy/min). Engraftment of human leukemia was monitored on weeks 4, 8 and 12 by PB sampling and quantitation of human CD45 þ cells by FACS. In mice repopulated with AML blasts, as indicated by 40.5% of CD45 þ cells in the PB, BM samples were aspirated from one femur by puncture through the knee joint22 under intraperitoneal (i.p.) anaesthesia using Ketalar (75 mg/kg; Parke-Davies, Zu¨rich, Switzerland) and Xylasol (10 mg/kg; Gra¨ub AG, Bern, Switzerland) and analyzed by FACS. Autologous AML-NK cells from days 14 to 21 of culture (3–5 106 cells in 200 ml PBS) were injected i.v. 1 week after BM aspiration. Human IL-2 and IL-15 (10 mg each in 100 ml PBS; Amgen, Seattle) were administered i.p. in three doses: on the day of NK cell injection, 24 and 48 h later. Mice were killed on day 7 post NK cell transfer and the content of AML blasts in the BM was determined by FACS.


Statistical analysis Unpaired Student’s t-test was used to analyze the NK receptor expression in control- vs AML-NK cells and NK cell cytotoxicity in groups of K562-transplanted mice. Paired Student’s t-test was used to compare AML content in the NOD/SCID BM prior and after treatment with NK cells.

Results

Expansion of AML-NK cells in vitro We first examined the proliferative potential of NK cells from patients with newly diagnosed or relapsed AML. The number of CD56 þ CD3 NK cells in patients’ PB was in the normal range (0.2470.09/l), but owing to the high blast content of 7474.2%, NK cells constituted only 0.870.25% of MNC, as compared to 7.071.5% measured in healthy donor PB. Following purification, AML-NK cells were subjected to expansion in vitro in the presence of IL-2 and PHA. No difference in the expansion kinetics of AML- and control-NK cells was observed (Figure 1). In both culture types, the strongest expansion phase was between 10 and 14 days, and the proliferation reached a plateau on days 21–28. On day 14, CD56 þ CD3 AML-NK cell number was increased by 8167272-fold. It was also possible to repeat the in vitro expansion process by a consecutive PHA/IL-2 stimulation of cultured NK cells, although their proliferative capacity decreased to 30710.8-fold (data not shown). Altogether, these results demonstrate that despite a more complex isolation process than that with control-NK cells, stimulation with IL-2 and PHA in the presence of irradiated feeders results in a very efficient expansion of the polyclonal population of activated CD56 þ CD3 AML-NK cells.

Expression of major inhibitory and activating receptors by AML-NK cells The cell surface phenotype of AML-NK cells was examined by multicolor FACS analysis to define the expression of NK cell

2217 maturation and activation markers (Figure 2). At days 14–21 of culture, expression levels of CD16, CD69, CD161 and CD94 and the inhibitory receptors CD158a, CD158b and NK-B1 did not significantly differ from the phenotype of control-NK cells (Figure 2a). Also at day 0 prior to expansion cultures, the AML patient-derived CD56 þ NK cells contained a normal proportion of CD16 þ cells (average 70%; not shown), as well as CD158a, CD158b and NKB1 receptor-expressing cells (average 10%; not shown). Analysis of the major activating NK cell receptors, NKp46 and NKG2D, was performed at 14–21 days of expansion culture and in fresh PB prior to culture (day 0; Figure 2b). NKp46, representing the major NCR, was expressed in AML PB at an average MFI ratio of 8.371.4, as compared to 6.870.7 in controls, with NKp46 levels below all normal values in 3/13 analyzed patients. The average expression level of NKG2D in AML PB was 6.470.9 compared to 10.070.7 in controls (Po0.05), with 7/13 patients below normal. Following the in vitro expansion, there was a significant three- to five-fold upregulation of NKG2D, but not of NKp46, and there were no apparent differences in the NKp46 and NKG2D receptor levels between control- and AML-NK cells. After the 2- to 3-week-long culture period, all control- and AML-NK cells acquired a CD56brightCD16bright phenotype (data not shown). Taken together, these data demonstrate that the IL-2-activated polyclonal population of AML-NK cells does not differ from controlNK cells in terms of surface expression of the analyzed markers, including the activating receptors NKG2D and NKp46.

IFN-g production by AML-NK cells We assessed IFN-g production by NK cells after in vitro expansion cultures (days 14 and 21) subjected to a 36 h stimulation with IL-12 and IL-18. The percentage of IFN-gproducing cells was measured by intracellular FACS analysis (Figure 3). IFN-g production was observed in a similar proportion of AML-NK cells (26.1713.2%) and control-NK cells (22.778.6%) and was significantly higher than the baseline response of o1% seen without cytokine stimulation or with IL-2 alone. Analogously, fresh NK cells from PB of AML patients and healthy controls prior to culture (day 0) responded equally to the stimulation with IL-12 and IL-18 (Figure 3). Hence, AML-NK cells are capable of producing IFN-g in response to appriopriate cytokine stimulation.

The cytolytic activity of AML-NK cells against K562 targets in vitro and in NOD/SCID mice

Figure 1 Expansion of purified NK cells in vitro. AML patientderived NK cells (AML-NK; n ¼ 9) or healthy donor-derived NK cells (control-NK; n ¼ 5) were purified and stimulated with IL-2 and PHA, as described in Materials and methods. The number of CD56 þ CD3 NK cells was determined at the indicated time points and expressed as mean7standard error of the mean (s.e.m.).

The cytolytic capacity of AML-NK cells was investigated first against the erythroleukemia cell line K562, which represents a highly susceptible NK cell target due to HLA class I deficiency. In vitro cytotoxicity of AML-NK cells was as high as of controlNK cells, and even exceeded the control values at low E:T ratios (Figure 4a). The cytotoxicity of AML-NK cells was further evaluated in vivo in a K562 tumor model. We showed previously that IL-2-stimulated NK cells from healthy donors significantly reduced the growth of K562 tumors in immunodeficient NOD/SCID mice.20 This tumor model was now used by injecting AML-NK cells 1 day after K562 inoculation. The effect of NK cells was followed at 1–4 weeks by determining the surface area of subcutaneous K562 tumors (Figure 4b). In mice that did not receive any NK cells, tumors reached an average size of 322748 mm2, whereas they were markedly smaller in response to control-NK cells (138741 mm2) and AML-NK cells Leukemia


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Figure 2 AML-NK cells and control-NK cells have a similar expression pattern of surface markers and NK receptors. (a) Phenotype of NK cells on days 14–21 of culture is illustrated in representative histograms. Shaded areas correspond to staining with specific mAbs, and thin lines to staining with control mAbs. The MFI ratios (7s.e.m.) of positive cell populations are indicated. CD158a and CD158b were expressed by 23–30% of control- and AML-NK cells. (b) Cell surface expression of NKp46 and NKG2D receptors by control-NK (n ¼ 7–11) and AML-NK (n ¼ 12–13) cells determined in fresh PB (day 0) and following in vitro expansion (days 14–21); average MFI ratios are indicated. Significant difference in NKG2D expression by control- and AML-NK cells on day 0 is indicated (*Po0.05); expression of NKp46 on days 0 and 14–21, as well as of NKG2D on days 14–21, was not different in control- and AML-NK cells.

The cytolytic activity of AML-NK cells against autologous leukemic blasts in vitro and in NOD/SCID mice

Figure 3 IFN-g production by AML-NK cells. The proportion of IFN-g-positive NK cells (7s.e.m.) from AML patients and from healthy controls was measured by intracellular FACS analysis prior to (day 0; n ¼ 3) and on day 14 of culture (n ¼ 7). NK cells were either stimulated with IL-12 and IL-18 for 36 h or cultured with IL-2 or without the addition of any cytokine. nd: not done.

(169753 mm2; P ¼ 0.05). These results indicate that AML-NK cells derived from expansion cultures are fully functional with respect to cytotoxicity against HLA class I-deficient K562 cells in vitro and retardation of tumor growth in vivo upon adoptive transfer to K562 tumor-bearing NOD/SCID mice. Leukemia

Our main goal was to define whether AML-NK cells derived from expansion cultures are cytolytic against autologous leukemic blasts. With NK cells and the corresponding blasts from five AML patients tested in cytotoxicity assays in vitro over the range of E:T ratios of 10:1–0.6:1, only low lysis of 11.373.8 to 1.170.7% was observed (Figure 5a). Cytolysis was significantly increased to 53.973.4 and 21.372.3%, respectively, when blasts were preincubated with anti-HLA class I mAb, indicating that HLA class I molecules displayed by leukemic blasts exert a dominant inhibitory effect through interactions with KIRs on NK effector cells. This conclusion was further confirmed in control experiments in which an addition of antiHLA class II mAb did not result in any significant induction of cytolysis, demonstrating that the observed effect of anti-HLA class I mAb was not due to mAb-dependent cellular cytotoxicity (ADCC) via CD16. To investigate whether AML blasts are potential targets for autologous AML-NK cells in vivo, NOD/SCID mice were transplanted with 1 107 patient-derived AML cells. The content of human blasts, which engrafted in the NOD/SCID BM at 8–12 weeks, varied from 0.5 to 70% (mean: 2977.5%, n ¼ 22). Mice were divided into three groups with comparable ranges of tumor engraftment and were treated as follows: group


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Figure 4 Cytolytic activity of AML-NK cells against the erythroleukemia cell line K562. (a) In vitro cytotoxicity of AML-NK cells (n ¼ 10) and control-NK cells (n ¼ 7) on days 14–21 of culture was determined against K562 cells in a 51Cr-release assay (7s.e.m.) at the indicated E:T ratios. (b) In vivo NK cell activity in NOD/SCID mice inoculated subcutaneously with 1 107 K562 cells. Tumor formation was determined as the mean surface area7s.e.m. at weeks 1–4 in mice without NK cell treatment (no NK; n ¼ 16) and in mice injected i.v. with 5 106 control-NK cells (n ¼ 3) or AML-NK cells (from three different AML patients tested in eight mice) on day 1 post K562 inoculation. *Significant reduction (P ¼ 0.05) of tumor sizes after AMLNK cell infusions was obtained when comparing untreated mice with all treated mice (n ¼ 8) and also with a subgroup of mice (n ¼ 4) treated with AML-NK cells of patient 13 (see Supplementary Information 1).

A was left untreated, group B received three i.p. doses of IL-2 and IL-15 and group C was injected i.v. with a single dose of 3–5 106 autologous NK cells along with three i.p. doses of IL-2 and IL-15, administered to support the viability and maintain the activation of the transfered NK cells (Figure 5b). The percentage of AML blasts in the BM in two control groups remained stable during the 2-week-long observation period (group A: 26711.3 vs 19.578.3%; group B: 25.577.0 vs 22.477.8%). In contrast, upon infusion of a single dose of AML-NK cells, the tumor load was significantly decreased from 29.576.7 to 9.472.2% (Po0.05). Remarkably, tumor regression was seen in all mice injected with NK cells, independent of the initial tumor load (8–77% reduction; see inset in Figure 5b). Dilution effect of human cells with infused NK cells could be excluded, because all human cells were CD33 þ blasts (Supplementary Information 2). We conclude that therapy with IL-2-stimulated autologous AML-NK cells was effective even with a single NK cell administration.

Figure 5 Cytolytic activity of AML-NK cells against autologous AML blasts. (a) In vitro cytotoxicity of AML-NK cells measured in 51Crrelease assays without addition of any mAbs (n ¼ 5), in the presence of anti-HLA class I mAb (n ¼ 5) or control anti-HLA class II mAb (n ¼ 3). Patient blasts that displayed a loading efficiency of 15007375 cpm and o10% of spontaneous 51Cr release were included in the analysis. (b) In vivo cytotoxicity of AML-NK cells in AML-transplanted NOD/ SCID mice. Engraftment with human AML blasts was determined by FACS analysis of CD45 þ CD33 þ cells in BM aspirates at weeks 8–12 post transplantation (open bars). AML-engrafted mice were either left untreated (group A; n ¼ 7; 26 þ 11.3% blasts), received three doses of IL-2 and IL-15 i.p. (group B; n ¼ 8; 25.577.0% blasts) or were injected with 3–5 106 autologous AML-NK cells from expansion cultures along with IL-2 and IL-15 administration (group C; n ¼ 7; 29.576.7% blasts). At 2 weeks after BM aspiration and 1 week after NK cell injections, mice were killed and BM was analyzed for the content of human AML blasts by FACS (filled bars). Significant difference between mice in group C prior to and after NK cell infusion is indicated (*Po 0.05); there were no significant differences in groups A and B. The inserted graph illustrates the blast content in the BM of individual mice in group C before (open symbols) and after the transfer of NK cells (filled symbols).

Increased surface expression of NKG2D and NCR ligands by transplanted AML blasts recovered from NOD/SCID mice The antileukemic effect of AML-NK cells against autologous blasts in NOD/SCID mice was more pronounced than the cytolytic activity measured in vitro, which was mostly dependent on blocking the interaction of HLA class I with inhibitory KIRs. To explain the increased sensitivity of AML blasts to autologous NK cells in vivo, we hypothesized that in the mouse BM microenvironment, the KIR-derived signals might be effectively counteracted by signals delivered by activating receptors. To address this issue, AML blasts of two patients, which engrafted in mice and subsequently responded to therapy Leukemia


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Figure 6 Upregulation of ligands for activating NK cell receptors on AML blasts following engraftment in NOD/SCID mice. Using mAbs against ULBP1, -2, -3, and dimers of soluble (s) NKp30 and NKp46, the ligand expression on AML blasts was determined by FACS analysis in PB of two patients at diagnosis (open bars) and in BM of NOD/SCID mice (filled bars) at 8–12 weeks after transplantation with the same blasts. The average MFI ratios (7s.e.m.) obtained with AML blasts of patient 1 (n ¼ 10 mice) and patient 2 (n ¼ 3 mice) are indicated.

with autologous NK cells, were recovered from NOD/SCID BM and the expression of ligands for major activating receptors was examined. These included ULBP ligands of NKG2D as well as yet unknown NCR-specific ligands detected by the use of dimers of soluble recombinant NCRs.19 In comparison with ligand expression prior to transplantation, blasts recovered from mice showed significantly higher levels of several of the tested ligands, in particular those of ULBP1 and NKp46-specific ligand, the levels of which increased by 2.5- to 5.2-fold (Figure 6). It is therefore possible that the increase in ligand levels taking place in the murine BM microenvironment is rendering the transplanted AML blasts more susceptible to NK cell lysis.

Discussion The NK cell vs leukemia response provided in the context of haploidentical donor stem cell transplantation has received substantial recognition, stimulating studies on the molecular mechanism and the clinical impact of NK cell alloreactivity.23 In contrast, autologous leukemia-specific NK cell immunity remains poorly understood and the concept of autologous immunotherapy with NK cells has not been well explored.24 With this study, we addressed the antileukemic cytotoxicity of NK cells from patients with AML. The results demonstrate that purified AML-NK cells subjected to polyclonal expansion and activation with IL-2 are cytotoxic against K562 erythroleukemia and against autologous AML blasts in NOD/SCID mice engrafted with human leukemia. The CD56 þ CD3 NK cells in AML are a minor population of PB cells.18 At diagnosis, NK cell accounted for less than 1% of PB MNCs, which was 10-fold below normal levels. However, in absolute numbers, NK cells in AML PB were within the normal range, arguing that the development of NK cells is not affected by the disease. In accord, the AML-derived NK cells do not carry the underlying cytogenetic changes of the respective AML blasts (see Supplementary Information 2). AML-NK cells retained a high proliferative potential in vitro, which was indistinguishable from control-NK cells, as previously observed with NK cells from patients in remission.25 In response to IL-2 and PHA, AMLNK cell numbers increased about 1000-fold. Hence, unlike in Leukemia

chronic myelogenous leukemia, in which the proliferative potential of NK cells decreases significantly with the disease progression,26,27 AML-NK cells can be expanded in vitro with high efficacy. The capacity to produce proinflammatory cytokines and to kill NK-sensitive targets are important characteristics of activated NK cells. Previous studies documented the ability of AMLNK cells in complete remission to produce IFN-g and TNF-a after polyclonal activation and to efficiently kill HLA class I-deficient K562 erythroleukemia cells.25,28 We assessed the IFN-g production by AML-NK cells isolated at diagnosis and observed a normal responsiveness to stimulation with IL-12 and IL-18. AML-NK cells were also indistinguishable from controlNK cells with respect to their cytotoxic potential against K562 cells in vitro. Their cytolytic properties were further confirmed in vivo in a NOD/SCID mouse tumor model,20 in which K562 tumor growth was efficiently retarded upon a single injection of AML-NK cells. The killing of K562 tumors provides a strong argument that the NK cell population in AML is functionally mature. This is reinforced by recent findings of reduced cytotoxicity against K562 cells related to the immaturity of NK cells developing during the initial months after allogeneic stem cell transplantation.29 The capacity of NK cells to recognize and kill autologous malignant leukemic cells, including AML blasts, is generally poor as observed in this and previous studies.18,28,30 Our analysis of the phenotype of AML-NK cells derived from patients at diagnosis revealed no significant differences in the expression levels of several receptors important for NK cell function, as compared to NK cells from healthy individuals. These receptors included the NK cell maturation and activation markers, CD16, CD161, CD69 and CD94, as well as KIR receptors, CD158a (KIR2DL1), CD158b (KIR2DL2/3) and NKB1. This cell surface screening of the polyclonal NK cell population with specific antibodies cannot exclude differences in KIR expression at the clonal level, as implicated by skewed KIR genotypes in human leukemias.31 Concerning the impact of KIR ligands on susceptibility of blasts to NK cell lysis, selective downregulation or loss of HLA class I alleles on leukemic blasts has been reported, but is infrequent.32,33 Also, AML blasts in our study expressed HLA class I at MFI ratios of 115726, which were below 232720 found on healthy PB MNCs (data not shown), but nevertheless


2221 high enough to provide a dominant signal. Indeed, antibodymediated masking of HLA class I molecules promoted the autologous killing of AML blasts, indicating that KIR signaling prevents the cytotoxic effect of NK cells. According to a recent study, IL-2-activated NK cells from most of the analyzed AML patients expressed low levels of the NCRs.18 In our analysis of the polyclonal population of AML-NK cells following their expansion in vitro, we were unable to confirm the prevalence of NKp46dull cells. We therefore extended the analysis to fresh patients’ PB, and found that NKp46 were present at normal levels, but there was a tendency to lower expression of NKG2D receptor. In analogy to downregulation of NKG2D in T cells by MIC ligands released from solid tumors,34 levels of this receptor in NK cells may be influenced by ligands shed from blasts in AML. Following NK cell expansion in culture, expression of NKG2D was strongly upregulated while that of NKp46 remained stable, similarly to NK cells from healthy donors.35,36 Further investigations involving a large patient cohort are warranted to assess the contribution of activating receptors to inadequate NK interaction with leukemic targets. Our recent study demonstrated that AML blasts from about 80% of patients expressed very low levels of ULBPs and NCR ligands.19 We conclude at present that low density of NKG2D and NCR ligands, due to incomplete process of myeloid lineage maturation19 or ligand shedding,37 together with the abundance of HLA class I molecules, is compromising the recognition of blasts by NK cells. The immunodeficient NOD/SCID mice have previously been used to demonstrate the cytolytic potential of human haploidentical NK cells against CML blasts,38 but adoptive transfer of NK cells to mice engrafted with AML39 has not been reported. We employed NOD/SCID mice to assess the cytotoxicity of AML-NK cells and found consistent reduction of AML load upon adoptive transfer of autologous NK cells into tumor-transplanted mice. Up to five-fold upregulation of ligands for activating NKG2D and NCR receptors, which has taken place in the murine microenvironment, may provide an explanation for a partial clearance of leukemic cells in vivo. Exposure of AML blasts to myeloid-specific growth factors upregulates expression of activating ligands and increases the susceptibility to NK-mediated killing.19 Therefore, it might be hypothesized that growth factors produced in the murine BM have an influence on ligand expression by transplanted AML blasts, which is relevant for overcoming the inhibitory HLA class I signals. The importance of triggering ligands for the susceptibility to NK-mediated lysis has been substantiated by potent rejection of tumor cells after ectopic expression of murine NKG2D ligands, Rae1 and H60.40 Similarly, the cytolysis of human leukemic B cells by autologous gd T cells was increased upon trans-retinoic acid-induced upregulation of ULBP3 and MICA ligands in vitro.41 The possibility of substantial ex vivo expansion of highly cytotoxic AML-NK cells implicates their usefulness as cellular therapeutics for clearance of autologous leukemia. AML is characterized by a poor prognosis, especially in the elderly who cannot undergo high-dose chemotherapy and are not eligible for stem cell transplantation.42,43 New immunotherapeutic approaches are needed to improve the cure rates, possibly by raising protective immune responses. Infusions of donor-derived NK cells, including NK cells activated by 2-week-long treatment with IL-2 in vitro, have been reported as safe and also effective in increasing the donor chimerism in the transplanted patients.15,44 The results presented in our study may be important for the design of immunotherapeutic treatment strategies based on infusions of autologous NK cells. Given the

importance of activating ligand–receptor interactions for the tumor recognition process, clinical use of low dose of IL-245 and IL-1546 to upregulate the receptors and support the maintenance of adoptively transfered NK cells along with administration of myeloid growth factors to upregulate the respective ligands19 may be beneficial in enhancing the effectiveness of leukemia therapy with ex vivo IL-2-activated autologous NK cells.

Acknowledgements We thank Alois Gratwohl, Jakob R Passweg and Michael Gregor for AML samples, Gennaro De Libero and Lucia Mori for soluble NCRs, Marco Colonna and David Cosman for mAbs, Martine Jotterand for cytogenetic analysis of AML-NK cells and Andre´ Tichelli and Linda Kenins for critical reading of the manuscript. This work was supported by grants from the Swiss National Science Foundation (4046-058689 and 3100-067072.01), Krebsliga beider Basel (7/2003), Swiss Cancer League (OCS01282-08-2002) and Stiftung fu¨r Krebsbeka¨mpfung (Nr 195).

Supplementary Information Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).

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