Receptor Activator for NF-κB Ligand in Acute Myeloid Leukemia: Expression, Function, and Modulation of NK Cell Immunosurveillance This information is current as of September 27, 2021. Benjamin Joachim Schmiedel, Tina Nuebling, Julia Steinbacher, Alexandra Malinovska, Constantin Maximilian Wende, Miyuki Azuma, Pascal Schneider, Ludger Grosse-Hovest and Helmut Rainer Salih

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Receptor Activator for NF-kB Ligand in Acute Myeloid Leukemia: Expression, Function, and Modulation of NK Cell Immunosurveillance

Benjamin Joachim Schmiedel,* Tina Nuebling,* Julia Steinbacher,* Alexandra Malinovska,* Constantin Maximilian Wende,* Miyuki Azuma,† Pascal Schneider,‡ Ludger Grosse-Hovest,x and Helmut Rainer Salih*

The TNF family member receptor activator for NF-kB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK–RANKL interaction in NK cell–mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling Downloaded from via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti- leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity http://www.jimmunol.org/ with a more pronounced effect on IFN-g production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK–RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutral- ization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients. The Journal of Immu- nology, 2013, 190: 821–831.

atural killer cells are cytotoxic lymphocytes that play controlling leukemia in an autologous setting. This is also sup- an important role in anti-tumor immunity (1). Their in- ported by the observation that NK cell counts and activity are N volvement in immunosurveillance of hematopoietic ma- reduced in patients with leukemia and that activity levels of au- by guest on September 27, 2021 lignancies and in particular acute myeloid leukemia (AML) is tologous NK cells are associated with survival of leukemia patients highlighted by studies on haploidentical stem cell transplantation (7–9). Because NK cell reactivity is governed by a balance of mul- \(SCT), where the recipient’s leukemia cells fail to inhibit donor tiple inhibitory and activating receptors, interaction of NK cells NK cells via killer Ig-like receptors (KIRs), and KIR disparity is and leukemia cells is dependent on various immunoregulatory mol- associated with powerful graft versus leukemia effects and better ecules far beyond HLA class I–specific inhibitory KIR receptors clinical outcome (2–4). The observation that leukemia cells may (10, 11). Among others, many members of the TNF/TNFR family downregulate HLA class I molecules (5, 6), presumably to escape influence NK cell activation, and several TNF/TNFR family adaptive immunity, suggests that NK cells are also involved in members have been found to be expressed on AML cells and in- fluence anti-leukemia reactivity of NK cells that express their *Department of Hematology and Oncology, Eberhard Karls University, 72076 Tue- respective counterpart (12–14). bingen, Germany; †Department of Molecular Immunology, Tokyo Medical and Den- The TNFR family member receptor activator for NF-kB (RANK; tal University, Tokyo 133-8510, Japan; ‡Department of Biochemistry, University of x TNFRSF11A) and its ligand (receptor activator for NF-kB ligand; Lausanne, Epalinges, CH-1066 Switzerland; and Department of Immunology, Eber- hard Karls University, 72076 Tuebingen, Germany RANKL) are mainly known for their key role in regulating bone Received for publication June 28, 2012. Accepted for publication November 9, 2012. metabolism (15, 16) but were also found to influence the inter- This work was supported by grants from Deutsche Forschungsgemeinschaft action of dendritic cells (DCs) and T cells as well as the path- (SA1360/7-1, SFB685 TP A7), Wilhelm Sander-Stiftung (2007.115.3), and Deutsche ophysiology of hematopoietic malignancies and metastasis of solid Krebshilfe (109620). P.S. is supported by grants from the Swiss National Science tumors (17–25). Notably, available data indicate that RANK is Foundation. also expressed on NK cells (26), but to date nothing is known Address correspondence and reprint requests to Prof. Helmut Rainer Salih, Depart- ment of Hematology and Oncology, Eberhard Karls University, Otfried-Mueller regarding the functional relevance of RANK–RANKL interaction Strasse 10, 72076 Tuebingen, Germany. E-mail address: [email protected] for NK cell reactivity. We report in this study that AML cells tuebingen.de express RANKL in a high proportion of cases and that NK cells The online version of this article contains supplemental material. of AML patients display upregulated expression of its counterpart Abbreviations used in this article: AML, acute myeloid leukemia; CLL, chronic RANK. To account for the fact that several TNF/TNFR family lymphoid leukemia; DC, ; FAB, French–American–British classifica- tion; KIR, killer Ig-like receptor; mRANKL, membrane-bound RANKL; RANK, members may mediate different effects in mice and men (14, 27, receptor activator for NF-kB; RANKL, receptor activator for NF-kB ligand; SCT, 28), we set out to study the role of RANKL in AML pathophys- stem cell transplantation; SFI, specific fluorescence index; sRANKL, soluble iology by using primary malignant cells of leukemia patients and RANKL. examined the functional relevance of RANK–RANKL interaction Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 for NK cell immunosurveillance by using PBMCs of healthy www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201792 822 RANKL IMPAIRS NK CELL REACTIVITY donors as effector cells thereby mimicking the situation in patients instructions. Absorbance was measured at 450 nm with 650 nm as refer- that undergo allogeneic SCT. ence wavelength, and results are shown as means of triplicate measure- ments.

Materials and Methods NK cell activation, degranulation, cytotoxicity, and cytokine Patients production PBMCs of AML patients obtained at the time of diagnosis before therapy Upregulation of CD69 and CD107a as markers for NK activation and de- and of healthy donors were isolated by density gradient centrifugation after granulation, respectively, was analyzed by FACS. NK cells within PBMCs obtaining informed consent in accordance with the Helsinki protocol. In were selected by staining for CD56+CD32. Cytotoxicity was analyzed by . functional analyses, PBMCs of patients with 80% blast count according 2 h BATDA europium release assays as previously described (13). IFN-g to differential blood count in blood smears were used without further production was analyzed using the ELISA mAb set from Thermo Scientific purification to avoid potential artifacts by Ab-based isolation techni- (Rockford, IL) according to the manufacturer’s instructions. Lysis rates and ques. The study was performed according to the guidelines of the local cytokine concentrations in supernatants are shown as means of triplicate ethics committee. measurements in each experiment. Transfectants and cell lines The RANKL-transfectants (L-RANKL) as well as parental controls (L Results cells) were previously described (29). The human AML cell line HL-60 Expression of RANKL in AML was obtained internally at Eberhard Karls University Tuebingen. Au- thenticity was determined by validating the respective immunophenotype Recent studies suggested an involvement of RANKL in disease described by the provider using FACS every 6 mo and specifically prior to pathophysiology of chronic lymphoid leukemia (CLL) and mul- use in experiments. tiple myeloma as well as metastatic spread of solid tumors (19– Downloaded from Abs and reagents 25, 32), but nothing was yet known on the role of the RANK– RANKL molecule system in AML. In this study, we analyzed The mAbs against RANKL (MIH24 and MIH23) and the RANK–Ig fusion protein were previously described (29, 30). The anti-RANK mAb (clone RANKL expression on primary AML cells by flow cytometry 80704) was from R&D Systems (Minneapolis, MN). The anti-mouse Ig–PE and selected malignant cells among PBMCs of leukemia patients and anti-human IgG–PE conjugates were from Jackson ImmunoResearch by staining for CD33 and CD34. Overall, substantial surface ex-

(West Grove, PA), and anti-human IgG1–PE and anti-mouse IgM–HRP pression (SFI $ 1.5) was detected in 53 of 78 (68%) investigated http://www.jimmunol.org/ were from Southern Biotech (Birmingham, AL). All other Abs were from AML patient samples. Individual SFI levels and the clinical BD Biosciences (San Jose, CA). Recombinant RANKL, IL-2, IL-10, and IFN-g were from ImmunoTools GmbH (Friesoythe, Germany). The IgG2 characteristics of each patient are given in Table I. In Fig. 1A, Abs Denosumab and Panitumumab as isotype control were obtained from RANKL surface expression on AML cells from selected patients Amgen (Thousand Oaks, CA). The IgG1 Ab Rituximab was obtained from is shown. Because RANKL expression patterns differed between Roche (Basel, Switzerland). All other reagents were from Sigma (St. Louis, individual patients, we studied whether RANKL was associated MO). with certain AML French–American–British classification (FAB) Flow cytometry types. The fractions of RANKL-positive (SFI $ 1.5) samples among that of the investigated patients with different FAB types

FACS was performed using specific mAb, RANK–Ig, and the respective by guest on September 27, 2021 isotype controls at 10 mg/ml followed by specific PE-conjugates (1:100). were as follows: M0, 2 of 5 (40%); M1, 10 of 11 (91%); M2, 16 of Analysis was performed using a FC500 (Beckman Coulter, Krefeld, Ger- 26 (62%); M3, 2 of 5 (40%); M4, 15 of 21 (71%); and M5, 8 of 10 many). Where indicated, specific fluorescence indices (SFI) were calcu- (80%). Statistical analysis revealed that RANKL expression was lated by dividing median fluorescences obtained with specific mAb by not significantly (p = 0.33, one-way ANOVA) associated with any median fluorescences obtained with isotype control. To exclude potential artifacts due to unspecific Ab binding, a threshold for defining surface posi- specific FAB type (Fig. 1B). Moreover, no significant association tivity was set at SFI $ 1.5. of RANKL expression with cytogenetic risk, a particular cytoge- netic abnormality, blast count, or white blood count was observed PCR analysis (data not shown). RANKL expression of leukemia cells was also RT-PCR was performed as described previously (31). The following primers confirmed at the mRNA level by RT-PCR. Amplicons of membrane- were used for nested PCR of RANKL splice variants: membrane-bound bound RANKL (mRANKL) were detected in all 30 investigated RANKL (accession number NM_003701; http://www.ncbi.nlm.nih.gov/ samples. Notably, this also comprised samples without detectable nuccore/), 59-CGTCGCCCTGTTCTTCTATT-39 and 59-TATGGGAACCA- GATGGGATG-39 (step 1; 353 bp) and 59-TCAGAAGATGGCACTCACTG- surface expression on leukemic cells, which could be due to con- 39 and 59-TGAGATGAGCAAAAGGCTGA-39 (step 2; 268 bp); soluble tamination with RANKL-expressing healthy cells like B and RANKL (accession number NM_033012; http://www.ncbi.nlm.nih.gov/ T cells (19, 29), but may also indicate that RANKL surface ex- nuccore/), 59-CTTAGAAGCCACCAAAGAATTG-39 and 59-TATGGGAA- pression is regulated posttranscriptionally. The mRNA splice 9 9 CCAGATGGGATG-3 (step 1; 347 bp) and 5 -TCAGAAGATGGCACT- variant specifically coding for sRANKL was detected in 5 (17%) CACTG-39 and 59-TGAGATGAGCAAAAGGCTGA-39 (step 2; 268 bp). Primers for 18S rRNA were 59-CGGCTACCACATCCAAGGAA-39 and of the samples (Fig. 1C and Table I). Comparative analysis using 59-GCTGGAATTACCGCGGCT-39 (186 bp). sera of leukemia patients and healthy donors (where mRNA for sRANKL was never detectable in PBMCs, data not shown) re- Determination of soluble RANKL, cytokines, and metabolic vealed that sRANKL protein levels are not significantly (p = 0.65, activity of AML cells Mann–Whitney U test) elevated in AML (Fig. 1D). Moreover, Levels of soluble RANKL (sRANKL) in sera of AML patients and healthy sRANKL was also not detectable in supernatants of patient AML donors were analyzed using the commercially available ELISA BI-20452 cells upon in vitro culture (data not shown). Notably, these ana- from Biomedica (Vienna, Austria) according to the manufacturer’s in- structions. Results shown are means of duplicates. lyses comprised those AML patient samples that displayed ampli- TNF, IL-6, IL-8, and IL-10 in culture supernatants, sometimes after cons of the alternatively spliced mRNA of sRANKL. This indicates dilution, were determined by ELISA using OptEIA sets from BD Bio- that presence of mRNA for RANKL does not necessarily result sciences (San Diego, CA) or by Matched Pairs for ELISA from Immu- in detectable protein expression, which lends further evidence that noTools GmbH according to the manufacturer’s instructions. Cytokine concentrations in supernatants are expressed as means of triplicates. RANKL expression may substantially be influenced by posttran- Metabolic activity of AML cells was measured using the Cell Prolif- scriptional and/or posttranslational mechanisms in individual AML eration Reagent WST-1 set from Roche according to the manufacturer’s patients. The Journal of Immunology 823

Table I. Patient characteristics and RANKL expression

mRNA Expression

RANKL sRANKL PBB WBC Hb Plt UPN FAB (SFI) mRANKL sRANKL (pM) Age (y) Sex (%) Karyotype (giga/l) (g/dl) (giga/l) 1 M0 1.6 ND ND ND 72 M 70 46, XY, del(29)(q11q13) 3.3 8.3 85 2 M0 1.3 ND ND ND 70 F 91 46, XX, del(11)(q13q23) 6.7 8.6 37 3 M0 1.2 + + 0 68 F 97 ND 22.6 8.1 66 4 M0 3.1 + 2 ND 68 M 93 46, XY 42.5 9.8 80 5 M0 1.1 + 2 ND 65 M 92 ND 58.7 7.7 30 6 M1 2.5 ND ND ND 67 M 78 Complex 11.1 8.9 48 7 M1 9.2 + 2 ND 61 M 63 47, XY, (+9) 59.4 13.0 69 8 M1 2.2 ND ND 0 71 F 80 46, XX / 47, XX, (+4) 25.8 7.8 50 9 M1 2.1 + + 0 50 M 98 46, XY 19.7 10.4 55 10 M1 1.6 + 2 ND 47 F 98 46, XX 56.3 9.4 60 11 M1 1.7 ND ND 0 50 F 89 46, XX, del(9)(q13,q22) 13.8 8.8 25 12 M1 1.8 ND ND 0 44 M 75 46, XY 10.7 7.9 234 13 M1 4.6 ND ND 0 47 F 91 47, XX, (+8) 27.6 10.0 26 14 M1 1.1 ND ND 0.176 52 M 98 46, XY 153.3 9.2 23 15 M1 2.6 ND ND 0.1675 41 F 94 46, XX, del(12p2) 68.7 8.1 55

16 M1 1.5 ND ND 0 38 F 60 46, XX 26.0 9.6 153 Downloaded from 17 M2 3.0 ND ND ND 84 M 79 46, XY 115.2 7.8 107 18 M2 1.3 ND ND 0.028 38 M 28 46, XY 8.8 8.7 52 19 M2 1.0 ND ND ND 54 F 71 46, XX, t(8;21)(q22;q22) 9.0 8.9 40 20 M2 1.2 + 2 0 66 M 80 Complex 27.8 10.1 49 21 M2 1.0 ND ND 0.045 73 F 83 46, XX, inv(X)(p22q11) 5.0 6.3 95 22 M2 1.5 ND ND ND 73 M 83 46, XY 33.8 8.1 80 23 M2 1.7 ND ND 0 47 M 53 Complex 24.7 7.1 63 24 M2 1.6 ND ND 0 68 M 96 46, XY 85.5 9.5 146 http://www.jimmunol.org/ 25 M2 2.8 + 2 0.365 67 M 80 48, XYY, (+8) 21.1 6.4 29 26 M2 14.2 ND ND 0 41 M 43 46, XY 91.6 10.6 235 27 M2 1.1 + 2 0.5895 64 M 82 Complex 338.5 8.1 19 28 M2 2.7 ND ND 0.005 37 F 33 46, XX, del(1)(p32p34) 7.9 8.4 25 29 M2 2.3 ND ND ND 60 M 32 Complex 38.9 7.9 109 30 M2 1.2 + 2 0 48 M 87 46, XY, t(15;17), t(8;21) 36.9 7.1 66 31 M2 2.5 + 2 0.0745 68 F 57 46, XX, del(7)(q22q32) 125.3 13.9 71 32 M2 1.1 ND ND ND 76 F 59 46, XX 193.4 10.6 66 33 M2 1.1 ND ND 0 78 M 99 Complex 26.0 9.7 36 34 M2 1.3 + 2 ND 62 F 36 46, XX 83.2 7.8 51 by guest on September 27, 2021 35 M2 1.5 ND ND 0.0805 71 F 94 46, XX 105.4 10.4 83 36 M2 1.7 ND ND 0 73 F 44 Complex 22.2 12.3 66 37 M2 2.0 ND ND ND 73 M 59 ND 231.2 9.2 21 38 M2 1.0 ND ND 0.0125 74 F 78 46, XX 50.6 8.6 29 39 M2 2.7 + + 0.029 45 M 80 Complex 17.1 9.0 51 40 M2 3.3 + 2 0.0215 44 M 94 46, XY 138.5 10.3 57 41 M2 2.2 ND ND 0.014 68 M 93 47, XY, (+11) 110.9 8.0 27 42 M2 1.7 ND ND ND 70 F 20 47, XX, (+8), del(17)(p11-12) 4.3 12.6 205 43 M3 3.7 ND ND 0.0655 20 M 94 46, XY, t(15;17)(q22;q12) 67.6 8.8 29 44 M3 1.1 + 2 ND 46 M 51 46, XY, t(15,17), complex 23.1 13.3 25 45 M3 1.6 + 2 ND 67 F 94 46, XX, t(15;17) 99.0 9.9 128 46 M3 1.1 ND ND ND 29 M 61 46, XY, t(15;17) 30.3 9.4 39 47 M3 1.1 + 2 ND 69 M 82 46, XY, t(15;17) 12.8 9.1 73 48 M4 3.0 ND ND 0 66 M 72 Complex 61.9 5.8 18 49 M4 1.0 ND ND ND 49 M 82 ND 316.0 7.1 80 50 M4 4.9 + 2 0.212 69 M 66 Complex 49.5 8.7 42 51 M4 10.4 + + 0 74 F 71 Complex 37.0 12.4 121 52 M4 1.5 + 2 ND 71 M 93 47, XY, (+11) 79.7 9.9 40 53 M4 1.2 + 2 ND 55 M 72 46, XY 17.2 8.6 162 54 M4 2.2 + 2 0 74 F 67 Complex 30.4 11.0 72 55 M4 5.1 ND ND 0 66 M 47 45, XY, (27) 8.4 7.1 52 56 M4 1.4 ND ND ND 80 M 27 47, XY, (+4) / 48, XY, (+4), (+18) 4.6 9.1 115 57 M4 1.5 ND ND ND 41 F 73 Complex 112.7 8.5 30 58 M4 1.3 ND ND ND 70 M 64 Complex 11.4 9.5 11 59 M4 6.0 ND ND ND 37 M 28 Complex 72.5 11.2 42 60 M4 1.4 ND ND 0 53 M 77 ND 119.8 7.6 29 61 M4 2.1 ND ND ND 78 F 39 51, XX, (+6), (+9), (+9), (+11), (+13) 8.8 9.2 146 62 M4 1.6 ND ND 0.3415 33 M 94 Complex 65.7 12.0 88 63 M4 4.3 + 2 0.8495 72 M 78 46, XY 21.7 9.1 166 64 M4 1.6 ND ND ND 70 F 14 Complex 3.9 7.2 75 65 M4 1.1 + 2 ND 73 F 79 46, XX 40.3 9.6 63 66 M4 8.9 + + 0.2485 67 M 84 Complex 19.7 8.7 53 67 M4 12.1 ND ND ND 57 F 29 Complex 17.5 8.0 117 68 M4 2.1 + 2 0.5395 18 M 66 46, XY 45.1 8.0 75 69 M5 1.2 ND ND 0 32 M 33 Complex 105.2 6.7 71 70 M5 6.0 + 2 ND 64 F 91 46, XX 3.2 8.9 44 (Table continues) 824 RANKL IMPAIRS NK CELL REACTIVITY

Table I. (Continued)

mRNA Expression

RANKL sRANKL PBB WBC Hb Plt UPN FAB (SFI) mRANKL sRANKL (pM) Age (y) Sex (%) Karyotype (giga/l) (g/dl) (giga/l) 71 M5 1.5 ND ND 0 24 M 90 ND 96.3 9.4 168 72 M5 10.3 ND ND ND 54 M 89 46, XY, del(9)(q13q22) 109.4 8.2 78 73 M5 9.9 + 2 0.095 67 F 47 46, XX 18.2 11.4 12 74 M5 1.4 ND ND 0.2855 23 M 83 Complex 145.1 7.6 34 75 M5 2.6 + 2 ND 67 M 93 46, XY 263.5 14.7 63 76 M5 2.5 ND ND 0 85 M 92 46, XY 179.3 7.4 60 77 M5 2.1 + 2 ND 45 M 96 46, XY 70.3 8.1 139 78 M5 1.9 ND ND 0 43 M 97 46, XY 4.4 8.3 38 FAB, French–American–British classification; Hb, hemoglobin; PBB, peripheral blood blasts among nucleated cells; Plt, platelets; UPN, uniform patient number; WBC, white blood count.

RANKL transduces activating signals into AML cells RANK–Ig were specifically due to RANKL signaling (data not Many TNF family members including RANKL are able to mediate shown). Notably, we observed substantial interindividual differ- bidirectional signals (19, 33, 34). Therefore, we studied whether ences concerning the cytokine release of AML cells upon RANKL Downloaded from AML-expressed RANKL was able to transduce reverse signals signaling: Only with 8 of 17 investigated samples we observed that influence the cellular activity of the leukemia cells. To this release of all four cytokines by the AML cells. Release of TNF, end, primary AML cells of 10 different patients were cultured IL-6, IL-8, and IL-10 was observed with 16, 14, 15, and 10 of the alone, on isotype control or immobilized RANK–Ig, which ena- 17 samples, respectively (Fig. 2B). RANKL may thus (variably) bles RANKL multimerization. Subsequent analysis by ELISA contribute to the cytokine milieu associated with AML. Moreover, , revealed that RANKL signaling significantly (all p , 0.05, Mann– RANKL signaling potently and statistically significantly (p http://www.jimmunol.org/ Whitney U test) enhanced the release of TNF, IL-6, IL-8, and IL- 0.05, Mann–Whitney U test) stimulated cellular activity of AML 10, which are associated with AML pathophysiology (35, 36), into cells as revealed by WST-1 proliferation assays performed with the culture supernatants (Fig. 2A). No effects were observed with eight RANKL-positive patient samples, whereas no significant RANKL-negative AML cells, confirming that the effects of the (p = 0.7, Mann–Whitney U test) effects were observed in analyses by guest on September 27, 2021

FIGURE 1. RANKL expression in AML. (A and B) RANKL surface expression on patient AML cells was investigated by FACS using the RANKL mAb MIH24 with mouse IgG2b serving as isotype control. Malignant cells were gated based on CD33 and CD34 staining. (A) Representative results from exemplary AML patients with different FAB types; numbers in histograms represent uniform patient number (UPN) as shown in Table I. Shaded peaks, anti-RANKL; open peaks, isotype control. (B) SFI levels obtained by analysis of 78 AML patients grouped according to their FAB subtypes and medians of results obtained with all patients analyzed (bar). The dotted line represents SFI 1.5 as defined threshold for surface positivity. No significant association with specific FAB types was observed (p = 0.33, one-way ANOVA). (C) PBMCs from AML patients (.80% blast count) were investigated for RANKL mRNA expression by RT-PCR with 18S rRNA serving as control. Exemplary results obtained with RANKL surface-positive and RANKL surface-negative cells of patients with different FAB types (identified by their respective UPN) are shown. mRANKL, splice variant for membrane-bound RANKL; sRANKL, splice variant for soluble RANKL; +, positive control. (D) Levels of sRANKL in sera of healthy donors and AML patients (n = 20 and 44, respectively) were analyzed by ELISA. Results obtained with single patients are depicted; the bar indicates the median of the results. Statistical analysis was performed using the Mann–Whitney U test. The Journal of Immunology 825

RANKL positivity (Fig. 2C and data not shown). Together, these data indicate that signaling via RANKL may contribute to AML pathophysiology. AML-derived factors released upon RANKL signaling inhibit NK cell reactivity Next, we studied how the factors released by AML cells upon RANKL signaling influence NK cell anti-tumor immunity. PBMCs of healthy donors were cultured with the RANKL-negative (data not shown) leukemia cell line HL-60 in the presence of super- natants from patient AML cells that had been generated upon RANKL-signaling as described earlier. Target cell lysis was markedly decreased by the factors released upon culture on RANK– Ig compared with experiments with supernatants derived from AML cells cultured alone or on human IgG1 as isotype control. This effect was clearly dependent on RANKL expression by AML cells, as no effects were observed with supernatants from RANKL- negative leukemia cells (Fig. 3A). Reduced anti-leukemia reac-

tivity upon exposure to RANKL-induced factors was also observed Downloaded from when CD107a expression as surrogate marker for granule mobi- lization on CD56+CD32 cells among treated PBMCs was ana- lyzed, which also confirmed that specifically the reactivity of NK cells was affected (Fig. 3B). Statistical analysis of the effects on cytotoxicity and degranulation in independent experiments with

RANKL-positive and RANKL-negative patient cells (cytotoxicity: http://www.jimmunol.org/ n = 46 and n = 8, respectively; degranulation: n = 34 and n =7, respectively) revealed that only supernatants obtained upon reverse signaling into RANKL-positive patient cells caused a statistically significant (both p , 0.05, Mann–Whitney U test) reduction of NK cell reactivity (Fig. 3C). Thus, RANKL enables leukemia cells to release factors that impair NK cell immunity. Denosumab blocks immunomodulatory effects of RANKL

signaling by guest on September 27, 2021 On the basis of the central role of RANKL in bone metabolism, the mAb Denosumab, capable of blocking RANKL, was developed and recently proved to be effective for treatment of nonmalignant and malignant osteolysis (37, 38). We found that Denosumab specifi- cally bound to patient AML cells, thereby also confirming our results on RANKL expression by primary AML cells (Fig. 4A). We further confirmed that Denosumab is able to prevent binding of RANK to its ligand in our experimental system (Supplemental Fig. 1). Next, we studied whether Denosumab was able to block RANK-induced signaling via RANKL into primary AML cells. FIGURE 2. RANKL signaling induces cytokine release and metabolic Denosumab treatment statistically significantly (all p , 0.05, activity of AML cells. PBMCs of AML patients (all .80% blast count) Mann–Whitney U test) diminished the RANKL signaling–induced were cultured alone, on immobilized RANK–Ig or human IgG1 as control. release of TNF, IL-6, IL-8, and IL-10 by the AML cells, whereas A B ( and ) The levels of TNF (after 6 h), IL-6, IL-8, and IL-10 (all after 24 h) isotype control had no effect (Fig. 4B). Importantly, also the in- in culture supernatants were determined by ELISA. (A) Results obtained hibitory effects of the factor(s) released by AML cells upon with RANKL-positive AML cells of 10 different patients and medians of results (bar) for each cytokine are shown. (B) Analysis of results obtained RANKL signaling on NK reactivity as determined by analyses of , with 17 RANKL-positive patient samples with regard to release of specific degranulation were significantly (p 0.05, Mann–Whitney U cytokine combinations. Positive response (+) was defined as $3-fold in- test) reduced when Denosumab was present (and thus able to block crease of each individual cytokine upon RANKL signaling. The percentage RANK–RANKL interaction) during generation of the culture of samples responding with the indicated cytokine pattern is depicted. (C) supernatants that were subsequently used in assays with RANKL- Metabolic activity of AML cells was determined by WST proliferation negative HL-60 cells and PBMCs of three independent donors assays after 48 h. Results of one representative experiment each with leu- (Fig. 4C). Thus, Denosumab may serve to decrease the release of kemia cells of RANKL-positive and RANKL-negative patients and com- RANKL-induced factors from AML cells that contribute to dis- bined results obtained in independent experiments with AML cells of ease pathophysiology and immune evasion from NK cell reac- different RANKL-positive (n =8,upper panels) and RANKL-negative (n = tivity. 3, lower panels) patients are shown. *p , 0.05 (Mann–Whitney U test). AML-derived factors induce RANK expression on NK cells with leukemia cells of three RANKL-negative patients. Notably, Numerous TNF/TNFR family members are expressed by NK cells basal proliferation rates observed with samples of different and are upregulated in malignant disease, where they influence NK patients varied substantially without a significant association with reactivity upon interaction with their target cell–expressed coun- 826 RANKL IMPAIRS NK CELL REACTIVITY

FIGURE 3. Factors released by AML cells upon RANKL signaling impair NK cell reactivity. Primary RANKL-positive and RANKL-negative AML cells of patients with .80% blast count were cultured alone, on immobilized RANK–Ig or human IgG1 as control for 24 h before harvest of culture supernatants (sn). Then, PBMCs of healthy donors as effectors were cultured with HL-60 cells in culture supernatants di- luted 1:3 with fresh medium. Exemplary results with leukemia cells of the indicated (UPN) patients in analyses of (A) cytotoxicity by 2 h BATDA europium release assays and (B) CD107a upregulation on NK cells (CD56+CD32) after 3 h by FACS (percentages of CD107a-positive NK cells are given) are shown. (C) Downloaded from Combined analysis of the effects of supernatants from RANKL-positive and RANKL-negative patient AML cells. Results obtained with untreated PBMCs in each individual data set were set to 1. Left, Cytotoxicity (n = 46 and n = 8, respectively). Right, CD107a expression (n = 34 and n = 7, respectively). *p , 0.05 (Mann–

Whitney U test). http://www.jimmunol.org/

terparts (13, 14, 39–41). As available data indicated that RANK leased upon RANKL signaling also modulate NK cell RANK can be expressed by NK cells (26), we comparatively analyzed expression (Fig. 5D). Taken together, RANK expression on NK by guest on September 27, 2021 RANK expression on NK cells of healthy donors and our AML cells can be induced by factors released by AML cells, which turn patients. Only low levels of RANK were detected on NK cells of may facilitate RANK–RANKL interaction that subsequently could healthy donors, whereas significantly (p , 0.05, Mann–Whitney directly influence NK cell reactivity via RANK. U test) increased expression was observed on NK cells of the leukemia patients (Fig. 5A). To unravel the mechanism(s) re- RANK directly impairs NK reactivity upon interaction with sponsible for the differential RANK expression on NK cells of RANKL-expressing target cells patients, freshly isolated PBMCs of healthy donors were incubated To determine whether and how forward signaling via RANK di- with supernatants of primary AML cells that had been cultured rectly influenced NK cell reactivity, we used murine tumor cells alone, on immobilized RANK–Ig or isotype control. Afterwards, transfected with human RANKL (L-RANKL) or RANKL-negative RANK expression on NK cells was determined by FACS analyses. control cells (L cells) as targets for NK cells within PBMCs that Notably, NK cells cultured in fresh medium acquired low levels of were previously cultured for 72 h to induce RANK expression. RANK, likely due to unspecific effects of the isolation procedure Cytotoxicity assays revealed that lysis was profoundly reduced by and/or in vitro culture. Control supernatants derived from AML target cell–expressed RANKL. Similarly, release of IFN-g, the cells only slightly modulated RANK expression, whereas that second major mechanism by which NK cells contribute to anti- obtained upon RANKL signaling into the leukemia cells signifi- tumor immunity, was significantly reduced in the presence of cantly (p , 0.05, Mann–Whitney U test) induced RANK upreg- target-expressed RANKL (Fig. 6A). Next, we used primary AML ulation on NK cells (Fig. 5B). Next, we treated PBMCs from cells and determined whether Denosumab was able to improve healthy donors with the cytokines that we had found to be released the reactivity of RANK-expressing NK cells within allogeneic upon RANKL signaling by AML cells. Again, only low levels of PBMCs. This was facilitated by the fact that Denosumab does not RANK were detected on resting NK cells and expression in- affect NK cell reactivity via its Fc part allowing for attribution of creased over time upon in vitro culture in medium alone. Whereas its effects in our functional analyses specifically to blocking TNF, IL-6, and IL-8 had no effect, RANK expression on NK cells RANK–RANKL interaction (Supplemental Fig. 1). In cytotoxicity was strongly upregulated in the presence of IL-10. Induction of assays, we observed increased NK lysis of primary RANKL- RANK expression was not associated with NK cell activation, as expressing AML cells upon blocking RANK–RANKL interaction no substantial upregulation of the activation marker CD69 was with Denosumab (Fig. 6B). In addition, IFN-g production was observed upon IL-10 treatment (Fig. 5C and data not shown). substantially increased upon blocking RANK–RANKL interaction Notably, RANK was also upregulated by supernatants of AML (Fig. 6C). Statistical analysis of the effects on cytotoxicity and cells that did not respond with secretion of IL-10 to RANKL cytokine production in 36 and 13 independent experiments, re- signaling. This indicates that IL-10 is sufficient but not necessary spectively, revealed that both NK effector functions were signif- to induce RANK, and other yet unidentified factors that are re- icantly (both p , 0.05, Mann–Whitney U test) enhanced by The Journal of Immunology 827 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Denosumab blocks RANKL signaling into malignant cells and its NK-inhibitory effects. (A) Binding of Denosumab to the indicated RANKL-positive or RANKL-negative primary AML cells was analyzed by FACS. Shaded peaks, Denosumab; open peaks, isotype control. (B) PBMCs of AML patients (all .80% blast count) were cultured alone, on immobilized RANK–Ig to induce RANKL signaling (black bars) or human IgG1 as control (white bars). Where indicated, AML cells were treated for 1 h with Denosumab or isotype control (10 mg/ml each) followed by washing prior to induction of RANKL signaling. Release of TNF (after 6 h), IL-6, IL-8, and IL-10 (all after 24 h) was determined by ELISA of culture supernatants. Data of one representative experiment each are shown in the upper panels, and lower panels depict results of experiments with leukemic cells of at least four different patients. (C) PBMCs of healthy donors were cultured with HL-60 cells in the presence or absence of culture supernatants (sn) generated as described in (B) diluted 1:3 with fresh medium. CD107a expression levels on NK cells (CD56+CD32) were analyzed after 3 h by FACS. To account for donor variation and to enable statistical analysis, combined data of three independent experiments with PBMCs of different donors after normalization by setting results obtained with PBMCs cultured in control supernatants to 100% are shown. *p , 0.05 (Mann–Whitney U test).

Denosumab (Fig. 6B, 6C). Notably, the stimulatory effect of Discussion RANKL blocking (defined as .20% increase) on cytokine pro- Activity of both tumor cells and immune effector cells including duction occurred in a statistically significantly (p , 0.05, Mann– NK cells is substantially influenced by various members of the Whitney U test) higher number of experiments compared with TNF/TNFR family (12). RANKL and its receptors RANK and cytotoxicity [10 of 13 (77%) versus 12 of 36 (33%) independent osteoprotegerin play a central role in regulating bone turnover experiments, respectively] (Fig. 6D). When RANKL-negative (16), but available data indicate that RANKL also contributes to AML cells were used as targets, NK cytotoxicity and cytokine the pathophysiology of malignant diseases. It has been shown that production were not affected by the presence of Denosumab, the RANK–RANKL system substantially influences regulation of thereby excluding that the above-described effects were due to modulation of effector cells by the Ab (Supplemental Fig. 1). cancer cell migration and metastasis of solid tumors (21–25, 32). Thus, RANK inhibits both cytotoxicity and cytokine production With regard to hematopoietic malignancies, RANKL was reported of NK cells upon interaction with its leukemia-expressed ligand to be expressed in membrane-bound and soluble form in multiple with the effect of RANKL on the two major NK effector functions myeloma and may contribute to disease pathology, for example, by being more pronounced with regard to cytokine production. induction of osteolysis (20, 42, 43). In CLL, RANKL expression 828 RANKL IMPAIRS NK CELL REACTIVITY

FIGURE 5. Factors released by AML cells upon RANKL signaling enhance RANK expression on NK cells. Expression of RANK on CD56+CD32 NK cells was analyzed by FACS. (A) Comparative analysis of PBMCs from AML patients and healthy donors (n = 60 each). The percentage of RANK-positive NK cells is indicated. Results obtained with single patients are depicted; bar indicates medians of measurements. (B) Results obtained with PBMCs of healthy donors after 72 h of culture in the presence of 1:3 diluted super- natants (sn) generated by incubation of AML cells of different patients Downloaded from alone, on immobilized RANK–Ig or human IgG1 as control (n = 12). Results were normalized in each in- dividual data set by setting expres- sion in PBMCs cultured in medium to 1. (C) Results obtained upon in-

cubation of healthy PBMCs in the http://www.jimmunol.org/ presence or absence of recombinant IL-10 (10 ng/ml) for the indicated times. The activation marker CD69 was determined in parallel. The per- centages of RANK-positive and CD69- positive NK cells are indicated. (D) Comparative analysis of RANK in- duction on NK cells within PBMCs as determined in (B) upon exposure to supernatants of AML cells that did by guest on September 27, 2021 (IL-10 +, n = 9) and did not (IL-10 2, n = 3) respond to RANKL signaling with release of IL-10. *p , 0.05 (Mann–Whitney U test).

has been reported to influence the release of IL-8, which acts as posttranscriptional and/or posttranslational mechanisms in the autocrine and paracrine growth and survival factor for the ma- individual patients. This again is in line with findings on the ex- lignant cells (19). pression and release of other TNF family members in leukemic In this study, we report for the first time to our knowledge that cells (13, 14, 40, 41). RANKL is expressed on primary leukemia cells in a high pro- When we set out to determine whether the leukemia-expressed portion of AML cases. No significant association with specific RANKL was functional, we found that RANKL signaling sig- FAB types or parameters associated with disease severity was nificantly induced the release of TNF, IL-6, IL-8, and IL-10 as well observed. The latter is in line with results regarding the expression as metabolic activity of primary AML cells. This is in line with of other TNF family members in AML (e.g., Refs. 13, 14). findings that many ligands of the TNF family mediate bidirectional RANKL can also be released as soluble form due to alternative signals (33) and with reports that RANKL alters cytokine pro- splicing or by shedding from the cell surface due to the activity of duction of T cells and CLL cells (19, 34). The cytokines that we metalloproteinases. We detected expression of the mRNA for the found to be induced by RANKL signaling into AML cells are alternatively spliced form of RANKL in 17% of the investigated known to act as autocrine/paracrine growth and survival factors in cases. However, neither release of sRANKL protein by AML cells AML and are, at least in part, associated with development and in vitro nor elevated RANKL levels in sera of AML patients were progression of the disease (35, 36). Notably, RANKL signaling observed in our study. The mRNA for mRANKL was found in all did not always induce release of the same cytokines with all investigated AML samples including cases where surface ex- RANKL-positive patient samples. Rather, we found distinct pat- pression was not detectable. Although the latter may be due to terns of cytokine release upon RANKL signaling, and whereas contamination with RANKL-expressing healthy cells, the lack of TNF, IL-6, and IL-8 were released in most of the investigated correlation between mRNA expression and prevalence of the re- cases, IL-10 was only released by 59% of the patient samples. spective soluble and membrane-bound protein points to a potential Only 47% of the patient AML cells released all four cytokines regulatory or mutational blockade of RANKL expression by investigated in our study, but all investigated RANKL-positive The Journal of Immunology 829

signaling pathways or cytokine production in the leukemia cells that may be associated with development and progression of dis- ease. Thus, RANKL expression and/or signaling may play a par- ticular role in individual patients. The factors released by AML cells upon RANKL signaling were further found to suppress NK cell reactivity, which points to a functional relevance of RANKL for NK cell immunity in AML. This is of importance as NK cells play an important role in im- munosurveillance of AML, both in an autologous setting and after therapeutic intervention, for example, with allogeneic SCT (2–4). Notably, certain TNF family ligands may transduce signals even in the absence of their cognate counterpart, as exemplified by the role of CD137 ligand in promoting cytokine production by macrophages (44). In addition, RANK has been shown to be expressed by various cell types in peripheral blood and bone marrow and may thus readily be available to interact with AML- expressed RANKL (17, 26). By inducing the release of immu- nomodulatory factors, RANKL may thus substantially affect the

reciprocal interaction of leukemia cells with the immune system, Downloaded from and preventing RANKL signaling might serve to influence the clinical course of AML. Our data indicate that this can be achieved with the mAb Denosumab capable of blocking RANKL, which was recently approved for treatment of osteolysis (37, 38). Denosumab reduced the release of immunomodulatory factors by AML

cells and this reinforced NK cell anti-leukemia reactivity in our http://www.jimmunol.org/ analyses. Further rationale for neutralization of RANKL as an approach to enhance NK cell reactivity against AML cells is provided by our analyses on the expression and function of RANK on NK cells. RANK was previously shown to be expressed on NK cells, but nothing was yet known regarding its functional role (15, 17, 26). We report in this study that RANK is upregulated on NK cells of AML patients compared with healthy controls. This may, at least in part, be due to factors released from AML cells, as RANK was by guest on September 27, 2021 found to be induced on healthy NK cells by factors released from AML cells upon RANKL signaling. In line, IL-10, a cytokine FIGURE 6. RANK mediates inhibition of NK cells, and disruption of known for its distinct immunomodulatory/immunosuppressive RANK–RANKL interaction by Denosumab reinforces NK cell anti-leu- effects that we found to be released by AML cells upon RANKL kemia reactivity. PBMCs were cultured for 72 h to induce RANK ex- signaling, caused upregulation of RANK on NK cells. However, A pression on NK cells. Then ( ) cocultures with RANKL-transfectants supernatants of AML cells that did not release IL-10 in response to (L-RANKL) or RANKL-negative control cells (L cells) were performed. RANKL signaling also caused upregulation, indicating that other Cytotoxicity was determined by 2 h BATDA europium release assays (left); IFN-g levels in supernatants were analyzed by ELISA after 24 h factors produced by AML cells upon RANK–RANKL interaction (right). (B–D) Cocultures with RANKL-positive primary AML cells and that yet remain to be elucidated also modulate RANK ex- (.80% blast count) in the presence or absence of Denosumab or isotype pression in NK cells. It is tempting to speculate that interaction of control (10 mg/ml each): (B) Cytotoxicity was determined by 2 h europium NK cell–expressed RANK with its AML-expressed ligand may release assays. Results of one exemplary experiment with pronounced cause a “vicious RANK–RANKL cycle” of NK immunosub- effects of RANKL blocking (left) and combined results of 36 independent version, disruption of which by blocking RANKL may restore experiments (right) are shown (E:T ratio 80:1). (C) IFN-g levels in NK cell reactivity. This is even more as we found that RANKL supernatants as determined by ELISA after 24 h. Left, Results of one also directly diminishes the reactivity of NK cells by mediating representative experiment. Right, Combined results of 13 independent inhibitory forward signals via RANK. The inhibitory effects of experiments (E:T ratio 6:1). (D) Comparative analysis of the effects of RANK in NK cells were revealed by analysis of cytotoxicity and Denosumab on cytotoxicity and cytokine production. Lysis rates and cy- tokine levels obtained with untreated PBMCs in each individual data set IFN-g production in experiments with RANKL-transfectants as were set to 1 to account for effects due to the allogeneic setting in the targets. Moreover, blocking RANK–RANKL interaction in cul- different experiments. More than 20% increase by blockade of RANK– tures of allogeneic NK cells and primary RANKL-positive AML RANKL interaction in each individual assay (dotted line) was defined as cells by Denosumab increased target cell lysis and IFN-g pro- positive response. *p , 0.05 (Mann–Whitney U test). duction. Notably, we confirmed that Denosumab did not induce Fc-mediated effects in NK cells (45), and no modulation of NK reactivity by Denosumab was observed when RANKL-negative AML patient samples responded to RANKL signaling by release cells were used as targets. This clearly attributes the effects of of at least one of the cytokines. Together with our finding that Denosumab in our functional analyses to disruption of RANK– RANKL stimulates leukemia cell metabolism, these data strongly RANKL interaction. Notably, the effects of blocking AML-ex- point to an involvement of RANKL in AML pathophysiology. The pressed RANKL by Denosumab on NK function were significantly differing cytokine response to RANKL signaling could be due to less pronounced with regard to cytotoxicity compared with cyto- regulatory or mutational blockades of RANKL or the associated kine production. 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