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Drug-induced senescent multiple myeloma cells elicit NK cell proliferation by direct or

exosome-mediated IL15 trans-presentation

Cristiana Borrelli1†, Biancamaria Ricci^, Elisabetta Vulpis

1, Cinzia Fionda

1, Maria Rosaria

Ricciardi2, Maria Teresa Petrucci

3, Laura Masuelli

4, Agnese Peri

1, Marco Cippitelli

1, Alessandra

Zingoni1, Angela Santoni

1#*, and Alessandra Soriani

1*

1Department of Molecular Medicine, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur

Italia – Fondazione Cenci Bolognetti, Rome, Italy; 2Hematology, Department of Clinical and Molecular

Medicine, Sapienza University of Rome, Italy; 3Division of Hematology, Department of Cellular

Biotechnologies and Hematology, Sapienza University of Rome, Italy; 4Department of Experimental

Medicine, Sapienza University of Rome, Rome, Italy; †Center for Life Nano Science@Sapienza, Istituto

Italiano di Tecnologia, Rome, Italy;^Department of Orthopaedics, Washington University in St. Louis-

School of Medicine; #Neuromed I.R.C.C.S.-Istituto Neurologico Mediterraneo, Pozzilli (IS), Italy

*these authors contributed equally to this work

Running title: Senescent MM promotes IL15-mediated NK cell proliferation

Keywords (5): NK cells, Multiple Myeloma, IL15 trans-presentation, proliferation, exosome

* Correspondence to: A. Soriani or A. Santoni, Department of Molecular Medicine, Sapienza

University of Rome, Viale Regina Elena 291, 00161, Rome, Italy. Telephone: +39-0649255152;

FAX: +39-0644340632. Email: [email protected] or [email protected]

Disclosure of potential conflicts of interest

The authors declare no potential conflicts of interest.

Funding

This work was supported by Italian Association for Cancer Research (AIRC 5x1000 cod.9962), the

Sapienza University of Rome (Progetto di ricerca 2016, cod.: RM116154C8F24748). E.V. is

supported by a fellowship from AIRC.

Abstract word count: 225

Body word count: 3311

Number of figures: 6

Number of tables: 1

Number of supplementary figures: 5

Number of supplementary tables: 2

Research. on November 15, 2020. © 2018 American Association for Cancercancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 24, 2018; DOI: 10.1158/2326-6066.CIR-17-0604

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Abstract

Treatment of multiple myeloma (MM) cells with sub-lethal doses of genotoxic drugs leads to

senescence and results in increased NK cell recognition and effector functions. Herein we

demonstrated that doxorubicin- and melphalan-treated senescent cells display increased expression

of IL15, a cytokine involved in NK cell activation, proliferation, and maturation. IL15 up-regulation

was evident at the mRNA and protein level, both in MM cell lines and malignant plasma cells (PCs)

from patients’ bone marrow (BM) aspirates. However, IL15 was detectable as a soluble cytokine

only in vivo, thus, indicating a functional role of IL15 in the BM tumor microenvironment. The

increased IL15 was accompanied by enhanced expression of the IL15/IL15RA complex on the

membrane of senescent myeloma cells, allowing the functional trans-presentation of this cytokine to

neighboring NK cells, which consequently underwent activation and proliferation. We

demonstrated that MM cell-derived exosomes, the release of which was augmented by melphalan

(MEL) treatment in senescent cells, also expressed IL15RA and IL15, and their interaction with NK

cells in the presence of exogenous IL15 resulted in increased proliferation. Altogether, our data

demonstrated that low doses of chemotherapeutic drugs, by inducing tumor cell senescence and a

senescence-associated secretory phenotype (SASP), promoted IL15 trans-presentation to NK cells

and, in turn, their activation and proliferation, thus, enhancing NK cell-tumor immune surveillance

and providing new insights for the exploitation of senescence-based cancer therapies.




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Introduction

Natural killer (NK) cells are innate lymphoid cells (ILCs) capable of killing malignant or

infected cells by an integrated interplay of activating and inhibitory receptor signaling and by

secreting a wide array of cytokines and chemokines (1-3). Ample evidence indicates NK cell

involvement in the protection against cancer in humans and experimental animals, and a number of

NK cell-based immunotherapeutic approaches have been exploited (4,5). NK cells, originally

identified as “naturally active,” are endowed with cytotoxic activity when incubated in vitro with

tumor target cells and can be increased by addition of stimulatory signals, such as cytokines (6).

Interleukin-15 (IL15) is central to the development, survival, and activation of NK cells (2,7-9) and

was first identified by its functional similarities to IL2 in promoting T and NK cell proliferation and

signaling through the same beta- and gamma-receptor subunits (10,11). These subunits are

important for signal transduction, whereas the alpha-receptor subunit is responsible for the

specificity and high affinity binding of IL15 to the receptor (12).

IL15 mRNA expression can be detected in a broad range of tissues, including adherent

mononuclear blood cells, activated macrophages, epithelial and fibroblast cells, placenta, and

skeletal muscle, with an expression pattern similar to IL15Rα (IL15R (12). However, IL15 is

rarely secreted but can be under pathologic conditions (11,13). In vivo, IL15 predominantly exists

associated to the plasma membrane, which explains the difficulty in its detection as soluble protein.

IL15 trans-presentation is a unique mechanism that stimulates IL15 signaling on neighboring cells

through cell-cell interactions, mediating different functions compared to conventional soluble

cytokine delivery (14). IL15 trans-presentation is critical to support NK cell development, survival,

and activation (15-17), suggesting that IL15 presented in trans may play a major role in augmenting

NK cell-mediated immunosurveillance (17).

NK cells have a well-established role in the clearance of a number of hematological

malignancies, including multiple myeloma (MM), a plasma cell tumor that mainly develops in the

bone marrow (BM). We previously demonstrated that treatment with sub-lethal doses of genotoxic




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drugs increases the expression of NKG2D and DNAM-1 activating ligands preferentially on

chemotherapy-treated MM senescent cells, which are preferentially killed by NK cells and trigger

IFN production (18-20). In a mouse model of MM, the genotoxic drug melphalan in vivo promotes

the establishment of a senescent tumor cell population that displays an increased expression of NK

cell activating ligands and becomes more susceptible to NK cell killing (21).

Senescence is a complex cellular program induced by genotoxic, replicative, or oncogenic

stress, in which, cell cycle-arrested cells remain metabolically active and secrete several soluble

factors, also known as the “senescence-associated secretory phenotype” (SASP), which mediates a

variety of cellular responses including modulation of cancer immune-surveillance. Studies have

shed a new light on the role of exosomes in mediating the cell-to-cell transmission of senescence

signals, suggesting that exosomes may act as new components of the SASP (22).

Herein, we observed that drug-induced senescent MM cells displayed higher IL15/IL15R

complex on the plasma membrane, thus, enhancing activation and proliferation of primary NK cells.

MM cell-derived exosomes also expressed IL15RA and had the capability to stimulate NK cell

proliferation in the presence of exogenous IL15. At present, the role of IL15 on myeloma cells

refers only to the existence of an autocrine IL15 loop promoting their survival (23). Overall, our

data demonstrated that beyond displaying activating ligands and increased susceptibility to NK

killing, drug-induced senescent MM cells also promoted NK cell activation and proliferation via

direct or exosome-mediated IL15 trans-presentation, thus, further potentiating NK cell antitumor

effector functions.

Materials and Methods

Cell lines and clinical samples

SKO-007(J3), ARK, RPMI8226 MM cell lines were provided by Prof. P. Trivedi (Sapienza

University of Rome, Italy). After thawing, cells were cultured for no longer than 4 weeks and tested

for mycoplasma monthly. SKO-007(J3) and RPMI8226 cell lines were authenticated by IRCCS




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Azienda Ospedaliera Universitaria San Martino-IST, S.S. Banca Biologica e Cell factory by STR

profile (Cell IDTM System, Promega). Detection of amplified fragments was obtained by ABI

PRISM 3100 Genetic Analyzer. Data analysis was performed by GeneMapper® software, version

4.0.

Bone marrow (BM) aspirates of 88 untreated MM patients were managed at the Department

of Cellular Biotechnologies and Hematology (Sapienza University of Rome). Informed and written

consent in accordance with the Declaration of Helsinki was obtained from all patients, and approval

was obtained from the Ethics Committee of the Sapienza University of Rome. No exclusion criteria

were used. Patient characteristics were described in Supplementary Table S1 and 2. The BM

aspirates were lysed to obtain bone marrow mononuclear cells (BMMCs), and CD138 MicroBeads

kit (cat. 130-097-616) (Miltenyi Biotec, Auburn, CA) were used to isolate malignant plasma cells

(PCs) as previously described (18) . Patients’ sera were obtained by centrifugation (2000 rpm) of

whole blood for 15 minutes at room temperature, collected and stored at -80°C before the assay.

MM Cell treatment

Sub-lethal doses of doxorubicin (DOX) and melphalan (MEL), determined by an MTT

assay as previously described (18), were used to treat SKO-007(J3), ARK, and RPMI8226 cell lines,

and patients’ PCs. Cell lines and patient-derived PCs were cultured for 48 hours at a density of

3x105 cells/mL and 5x10

5 cells/mL, respectively, and then washed and left for further 24 hours

without drugs.

Immunofluorescence and flow cytometry

The expression of IL15 and IL15R on MM cells was evaluated with unconjugated anti-IL15

(MAB2471) and anti-IL15R, respectively (R&D systems, Minneapolis, MN).

Allophycocyanin (APC)-conjugated Goat affinity purified F(ab’)2 fragment to Mouse IgG (GAM)

was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). APC-conjugated

anti-CD69 (FN50) was from Biolegend (San Diego, CA). To detect IL15 and ILRA expression on

the MM cell surface, 1 x 106 of untreated and MEL/DOX-treated cells were incubated for 20




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minutes at 4°C with unconjugated IL15 and IL15RA monoclonal antibodies, then washed and

incubated for further 20 minutes with secondary GAM-APC. For the intracellular staining of IL15,

1x106

of untreated and MEL/DOX-treated cells were fixed with Flow Cytometry Fixation Buffer

(cat. FC004) (R&D systems), permeabilized with Flow Cytometry Permeabilization Buffer (cat.

FC005; R&D systems), and incubated with unconjugated IL15 (MAB2471) for 30 minutes. Cells

were then washed and incubated for 20 minutes with secondary GAM-APC for final flow

cytometric analysis. In some experiments, cells were stained with propidium iodide (PI; 50 g/mL)

to assess cell viability. PE-conjugated anti-CD38 (HIT2) and FITC-conjugated anti-CD138 (MI15)

antibodies were purchased from BD Biosciences (San Jose, CA). NK cells were purified using

human NK cell isolation kit (Miltenyi Biotec, San Diego, CA) from healthy donor PBMCs, isolated

through Lymphoprep (Stemcell technologies, Vancouver, Canada). Cell population was routinely

more than 90% CD56+ CD3

–, as assessed by immunofluorescence and flow cytometry analysis. In

some experiments, NK cells were fixed and permeabilized with 30% methanol plus 0.4%

paraformaldehyde (PFA) in PBS for 30 minutes, washed, and then incubated in 0.05% Tween plus

1% PFA in PBS and stained with anti-Ki67-FITC (clone MIB-1; Dako, Santa Clara, CA). Cells

were analyzed with a FACS Canto II (BD Biosciences). Flow cytometric analysis was performed

using the FlowJo software version 8.8.7 (TreeStar, Ashland, OR).

Real-time PCR

Total RNA was isolated from MM cells (SKO-007(J3), ARK, RPMI8226) using TRIzol

reagent (Invitrogen) according to the manufacturer’s protocol, and 1 g was used for cDNA first-

strand synthesis in a 25 μL reaction volume (M-MLV reverse transcriptase, cat. M170A; Promega).

1 μL of resulting cDNA was used in a 25 μL PCR reaction. IL15 (Hs00174106_m1) and IL15R

(Hs00542604_m1) mRNA expression was analyzed by real-time PCR using specific TaqMan Gene

Expression Assays (Applied Biosystems, Foster City, CA). Relative expression of each gene versus

-actin was calculated according to the ΔΔCt method (24).

SDS-PAGE and Western blot




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Drug-treated MM cells were lysed for 20 minutes at 4°C in 1X RIPA lysis buffer (1% NP-

40, 0.1% SDS ,50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM EDTA)

plus complete protease inhibitor mixture used at 1X (cat. P2714) and phosphatase inhibitors sodium

orthovanadate and sodium fluoride (Sigma-Aldrich, St. Louis, MO). The Bio-Rad Protein Assay

(Bio-Rad Laboratories; Hercules, CA) was used to measure protein concentration. 50 g of total

lysates was resolved by SDS-PAGE and transferred with transfer buffer (25 mM Tris/HCl, 20 mM

glycine, and 10% (v/v) methanol) (25) to nitrocellulose membranes (Whatman GmbH; Dassel,

Germany). After blocking with BSA, membranes were probed with the following specific

antibodies (1 g/mL): -actin (AC15; Sigma-Aldrich), IL15 (MAB2471),

IL15RHSP70 (SC24; Santa Cruz Biotechnology, Dallas, Texas), and calreticulin

(PA3-900; Thermo Fisher Scientific, Waltham, MA)A horseradish peroxidase (HRP)-conjugated

secondary antibody (cat. NA931V and NA934V; GE Healthcare Life Sciences, Buckinghamshire,

UK) and an enhanced chemiluminescence kit (cat. RPN2106; GE Healthcare) were used to reveal

immunoreactivity.

Proliferation assay

Freshly isolated peripheral blood NK cells were purified as previously described, adjusted to

1x106 cells/mL and cocultured overnight (o.n.) with untreated or MEL-treated SKO-007(J3) cells at

E:T ratio of 1:1. The day after, MM cells were removed from the culture by CD138+

magnetic

beads positive selection (Miltenyi Biotec), and NK cells were incubated for a further 5 days at 37°C

and 5% CO2. For blocking experiments, MM cells were pre-treated 1 hour with 1 g/mL of IL15

neutralizing monoclonal antibody (mAb) (MAB2471; R&D systems), then washed, and left o.n.

with primary NK cells (E:T=1:1). Five days later NK cell proliferation was evaluated by using the

BrdU flow kits (cat. 552598) according to the instruction manual (BD Biosciences, San Jose, CA).

In some experiments, 1x106 NK cells were incubated with exosomes (5-10 g/mL) derived from

untreated or drug-treated MM cells (as described before) in the presence or absence of human




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recombinant IL15 (5-50 ng/mL; cat. 200-15) or IL2 (200 U/mL; cat. 200-02; PeproTech, London,

UK), and cell proliferation was measured after five days by CFSE (cat. 150347-59-4; Sigma-

Aldrich) assay (26) or evaluating the Ki67 (MIB-1; Dako, Santa Clara, CA) proliferation marker.

Exosome isolation and purification

Exosome-free medium was obtained as follows: fetal bovine serum (FBS; ThermoFisher

Scientific) was centrifuged at 100,000 x g for 3 hours in a Beckman ultracentrifuge (Beckman

Coulter, Brea, CA) to remove microvesicle-like exosomes. RPMI 1640 was supplemented with

10% FCS-exosome free medium and antibiotics (penicillin, streptomycin, glutamine used at 1X;

Thermo Fisher Scientific). ARK and SKO-007(J3) MM cell lines were cultured at 0.8-1 x 106

cells/mL in exosome-free medium for 48 hours. Exosome purification consists of different

sequential centrifugations as previously reported (27). Briefly, cells were harvested by

centrifugation at 300 x g for 10 minutes and supernatants were collected. Cell-free supernatants

were then centrifuged at 2,000 x g for 20 minutes, followed by centrifugation at 10,000 x g for 30

minutes to remove cells and debris. Supernatants were filtered using a 0.22 μm filter and

centrifuged at 100,000 x g for 70 minutes at 4°C in a Beckman ultracentrifuge to pellet exosomes.

The resulting pellet was washed in a large volume of cold PBS and again centrifuged at 100,000 x g

for 70 minutes at 4°C. Finally, exosomes were resuspended in PBS for further analyses and

functional studies.

Transmission Electron microscopy

Transmission electron microscopy was performed as previously described (28). Briefly,

exosomes were fixed in 2% PFA and adsorbed on formvar-carbon-coated copper grids. The grids

were then incubated in 1% glutaraldehyde for 5 minutes, washed with deionized water eight times,

and then negatively stained with 2% uranyl oxalate (pH 7) for 5 minutes and methyl

cellulose/uranyl for 10 minutes at 4°C. Excess methyl cellulose/uranyl was blotted off, and the grids

were air-dried and observed with a TEM (Philips Morgagni268D) at an accelerating voltage of 80

kV. Digital images were taken with Mega View imaging software.




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ELISA/Luminex

Detection of IL15 in sera or plasma of MM patients was performed using specific ELISA

kits from R&D Systems. Plates were developed using a peroxidase substrate system (DuoSet

ELISA Development Kit, DY247-05; R&D systems), and then read with the Victor3 multilabel

plate reader (Model # 1420-033; Perkin Elmer, Santa Clara, CA) capable of measuring absorbance

in 96-well plates using dual wavelengths of 450-540 nm. Results were expressed as picograms per

milliliter (pg/mL) and referred to a standard curve obtained by plotting the mean absorbance for

each standard on the y-axis against the concentration on the x-axis and drawing the best fit curve

through the points on the graph. Detection of IL15 in the MM conditioned supernatants was

performed with a Milliplex MAP

Human Cytokine/Chemokine Magnetic Bead Panel - Immunology

Multiplex Assay, according to the manufacturer's instructions (Millipore, Burlington, MA). Plates

were read with Bio-Plex MAGPIX Multiplex Reader (Bio-Rad) and analyzed by Bio-Plex Manager

MP software.

Statistics

Error bars represent standard deviation (SD) or standard error of the mean (SEM). Data were

evaluated by paired Student t tests, with the exception of ELISA data, which were analyzed by

Mann-Whitney test. For statistical analysis, GraphPad Prism software was used. Statistical

significance is indicated with the p values<0.05.




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Results

Upregulation of IL15 and IL15Rexpression on drug-induced senescent MM cells

We previously demonstrated that sub-lethal doses of genotoxic drugs, such as doxorubicin

(DOX) and melphalan (MEL), induce a senescence-associated secretory phenotype (SASP) in

primary malignant PCs and SKO-007(J3) MM cells (18,29). We then asked whether drug-induced

MM cells could also exhibit a SASP phenotype and release soluble mediators capable of affecting

NK cell functions and focused our attention on IL15, a cytokine involved in NK cell activation and

proliferation/maturation. We evaluated IL15 mRNA expression by real-time PCR analysis on SKO-

007(J3), ARK, and RPMI8226 MM cell lines treated with DOX and MEL for 24, 48, and 72 hours

(48 hours of treatment plus 24 hours without drug). Our findings showed that upregulation of IL15

mRNA with both drug treatments was evident by 24 hours, with a peak at 48 hours (Fig. 1A). All

these cell lines underwent senescence in response to genotoxic drugs (Supplementary Fig. S1,

panel A)(19) . Intracellular staining on DOX- or MEL-treated MM cells after overnight incubation

with brefeldin A revealed an increase in IL15 intracellular protein expression (Fig. 1B) that was

confirmed by Western blot analysis (Fig. 1C). However, augmented IL15 mRNA and intracellular

protein expression was not accompanied by a parallel increase in cytokine release, as determined by

Luminex technology (value <4 pg/mL, lower detection limit).

The absence of detectable IL15 in the culture supernatants of MM cell lines suggested that

IL15 could be trapped by the surface-expressed IL15Rand could signal in trans. Thus, IL15

surface expression was determined by immunofluorescence and flow cytometry, and we found that

all the MM cells analyzed exposed this cytokine on the plasma membrane upon drug treatment (Fig.

1D). We then investigated the ability of genotoxic drugs to regulate the expression of

IL15RUpon 72 hours of drug treatment of SKO-007(J3), ARK, and RPMI8226 MM cells, we

found increased IL15R protein levels (Fig. 2A) and a significant augmentation of its cell-surface

expression, as revealed by FACS analysis (Fig. 2B and C). Similar results were obtained on drug-




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treated, primary malignant PCs derived from the BM of patients at different states of disease, which

displayed increased SA-Gal activity by FACS analysis (Supplementary Fig. S1B) and a clear

perinuclear blue staining by microscopy (Supplementary Fig. S1C), both associated to a senescent

phenotype. The upregulation of IL15 mRNA (Fig. 3A) was accompanied by a concomitant increase

of both IL15 and IL15R cell surface expression on MEL-treated primary PCs (Fig. 3B and C).

We also investigated whether IL15/IL15R expression could be associated with variation in patient

clinical characteristics (Supplementary Table S1). Our data demonstrated that a more pronounced

basal and drug-induced IL15/IL15R expression is independent of the clinical stage, age, and/or

the percentage of malignant PCs. Bortezomib and lenalidomide, two commonly used therapeutics

that did not induce a senescence phenotype in our model (Supplementary Fig. S1D), did not

upregulate IL15/IL15R expression in MM patients (Supplementary Fig. S2A and B).

Collectively, these data indicated that drug-induced senescent myeloma cells express increased

IL15/IL15R complex on the surface membrane.

Drug-treated MM cells elicit NK cell activation and proliferation by trans-presenting IL15

To identify the functional role of IL15/IL15R complex increased expression on drug-

treated MM cells, we investigated whether IL15 trans-presentation could induce NK cell activation

and proliferation. MEL-treated MM cells were incubated overnight to allow for trans-presentation

of IL15 to freshly isolated peripheral blood NK cells, and then CD138+ cells were removed from

the culture, and the expression of the CD69 activation marker on NK cells was evaluated by

immunofluorescence and FACS analysis. As observed with soluble recombinant IL15 alone, used

as control, primary NK cells in contact with MM cells showed an increased expression of CD69,

which was higher for MEL-treated cells (Fig. 4A).

To examine the direct role of IL15 in NK cell activation induced by drug-treated MM cells,

SKO-007(J3) cells were pre-treated with an IL15 blocking monoclonal antibody (mAb) or with a

non-reactive isotype control before coculture with the NK cells (Fig. 4B). We found that sub-lethal




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doses of MEL promoted IL15 trans-presentation by MM cells to NK cells, thus, stimulating their

activation. We also observed augmented proliferation of NK cells cocultured with drug-treated MM

cells compared to untreated MM cells (Fig. 4C and D), which was associated with an increased

proportion of cells in the S phase of the cell cycle (Supplementary Fig. S3). Blocking experiments

with an anti-IL15 demonstrated a direct role of trans-presented IL15 in the enhanced NK cell

proliferation (Fig. 4E). Similar results were obtained with the ARK MM cell line, where an

increased CD69 expression (Supplementary Fig. S4A and B) and proliferation (Supplementary

Fig. S4C and D) on primary NK cells was observed upon coculture with MEL-treated myeloma

cells. Altogether these data indicated that drug-treated MM cells, by displaying increased IL15R

acquire the capability to induce NK cell activation and stimulate NK cell proliferation with a

mechanism dependent on IL15 trans-presentation.

MM cell exosomes express IL15RA and increase IL15-induced NK cell proliferation

IL15R has been shown to be associated to exosomes derived from DCs (30) or different

IL15R transfectants (31). Thus, we further investigated the expression of IL15/IL15RA complex

on MM cell-derived exosomes. To this purpose, exosomes were isolated from the conditioned

media of SKO-007(J3) cells as previously reported (28) and characterized by transmission electron

microscopy and Western blot. Ultrastructural analysis showed that exosomal preparations contained

typical nano-sized cup-shaped vesicles (Fig. 5A). Heat-shock protein 70 (HSP70), a canonical

exosomal marker, was detected on exosomes, and the absence of calreticulin, which is exclusively

associated to the endoplasmic reticulum (ER), confirmed the purity of the nanovesicle preparations

(Fig. 5B). Both IL15R and IL15 were associated with MM-derived exosomes, although at lower

expression compared to the cell lysates (Fig. 5B). No differences were found on IL15R

expression on exosomes derived from drug-treated SKO-007(J3) and ARK MM cells

(Supplementary Fig. S5).

As next step, we endeavored to address whether MM-derived exosomes have the capability

to stimulate NK cell proliferation. Our findings revealed a slight increase of cell proliferation in the




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presence of exosomes alone that was further stimulated by IL15(Fig. 5C). To further investigate,

freshly isolated human NK cells were labelled with CFSE and cultured with exosomes in the

presence of exogenous IL15, and we observed a significant increase of IL15-induced NK cell

proliferation (Fig. 5D and E).

Because IL2 and trans-presentation of IL15 by IL15RA are required for NK cell

proliferation (32), we asked whether exosome-mediated NK cell proliferation was dependent only

on the presence of exogenous IL15 or if it could also be mediated by IL2. The augmentation of NK

cell proliferation, measured by the expression of the Ki67 marker, was observed only with IL15 and

not with IL2, strongly suggesting that this effect could be mediated by IL15 trans-presentation (Fig.

5F). We also asked whether IL15 could be found in the sera from MM patients with different

clinical characteristics (Supplementary Table S2). We observed that about 21% of patients with

active MM exhibited variable serum IL15 (Fig. 6A), and IL15 was also found in the plasma from

BM aspirates isolated from different MM patients (Fig. 6B), thus, indicating the presence of this

cytokine in the BM tumor microenvironment. Serum IL15 was detectable in MM patients with a

less favorable disease progression (Table 1). Altogether, these data highlight that IL15RA harbored

by MM-cell derived exosomes is functional, leading to NK cell proliferation when associated with

IL15.

Discussion

Senescent cells show numerous changes in gene expression, leading to an increased

expression of many proteins that can promote or repress tumor progression (33,34). This secretory

program is a hallmark of senescence and is referred to as the senescence-associated secretory

phenotype (33). Herein, we provide evidence that drug-induced multiple myeloma senescent cells

overexpress IL15, a cytokine involved in NK cell activation and proliferation/maturation.

Upregulation of IL15 expression was accompanied by an increased expression of the IL15/IL15RA

complex on the membrane of senescent myeloma cells, which also released exosomes carrying both




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IL15 and IL15RA, leading to increased activation and proliferation of NK cells. Unsurprisingly, no

increased release of the cytokine was observed. Accordingly with the current theory of trans-

presentation proposing that IL15 and IL15RA are pre-associated within presenting cells prior to be

shuttled to the cell surface (14), our data showed increase of IL15 both at mRNA and protein levels

upon MEL treatment, whereas IL15R upregulation was only attributable to an increase of the cell

surface protein.

The IL15 trans-presentation by drug-treated MM cells leads to NK cell activation and

proliferation, as demonstrated by the enhanced expression of the CD69 activation marker and the

increased incorporation of BrdU by freshly isolated NK cells upon coculture with MEL-treated MM

cells. Our findings also demonstrated that both IL15Rand IL were present on the exosomes

released by SKO-007(J3) MM cells at steady state conditions, as well as upon MEL treatment.

MM-derived exosomes had the capability to promote NK cell proliferation induced by exogenous

IL15 but not by IL2, strongly suggesting a key functional role of IL15RA.

In accordance with the expression of IL15RA, no differences were observed between

exosomes derived from untreated and MEL-treated cells in terms of stimulatory effects on NK cell

proliferation. However, based on our previous findings demonstrating that MEL-treated MM cells

can release a higher number of exosomes with respect to untreated cells (28), we propose that low-

dose chemotherapy promotes stronger IL15-dependent NK cell responses as a consequence of the

enhanced exosome release by drug-treated senescent tumor cells. Our results also provided

evidence of soluble IL15 presence in about 21% of patients affected by active myeloma, and we

cannot rule out that it is complexed to IL15RA on the exosome surface, as previously reported (31).

IL15 infusion in cancer patients has been shown to affect mainly the expansion of the CD56high

NK

cell subset and stimulate cytotoxic activity (35).

Complexed IL15, as compared with IL15 alone, has been demonstrated to more efficiently

reduce tumor burden (36). It has been also shown that in vivo delivery of IL15/IL15RA complexes

triggers rapid and significant regression of established solid tumors in two murine models (37). The




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physiologic trans-presentation of IL15 has been mimicked by generating a construct of IL15 linked

to the extended sushi domain of IL15RA, and it has been established a more effective stimulation of

NK and T cells, in terms of proliferation and cytotoxicity, than the targeted presentation of IL15

alone (38). In this respect, a clinical trial of ALT-803, an IL15 super-agonist, in patients with

relapsed or refractory MM is currently ongoing (ClinicalTrials.gov NCT02099539). However,

according to previous data (39), we found a correlation between IL15 release in the sera of MM

patients and disease progression. To this regard, it is important to underline that the induction of

senescence by genotoxic agents on the one hand inhibits tumor cell growth, and on the other,

enhances the IL15-driven effect on NK cell antitumor activity. The findings that bortezomib and

lenalidomide, two drugs with different mechanisms and unable to induce senescence in our model,

did not upregulate IL15/IL15R expression in MM patients, further strengthening the hypothesis

that increased IL15/IL15RA complex is driven by senescence.

Induction of cellular senescence by chemotherapeutic agents has emerged as an appealing

option to arrest cancer cell proliferation. Immunosurveillance of senescent cells in cancer and other

pathological processes by the innate immune system has been highlighted. Our group contributed to

report the importance of NK cells in senescent cell recognition and elimination (19,21). Using a

model of oncogene-induced senescence (OIS), Iannello et al. demonstrated that p53-restored

senescent tumor cells recruit NK cells by secreting CCL2 (40). Our findings indicated that in

addition to these immune modulatory activities, it can be envisaged that the success of senescence-

based anticancer therapies might be also rely on the ability of senescent cells to trigger an IL15-

dependent antitumor innate immune response.

Acknowledgements

The expert technical assistance of Bernardina Milana is gratefully acknowledged. We also thank

Lucilla Simonelli for technical assistance in electron microscopy procedures.




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Figure legends

Fig 1. Doxorubicin- and melphalan-treated MM cells upregulate IL15 expression. (A).

SKO-007(J3), ARK, and RPMI8226 MM cells were left untreated (NT) or treated with doxorubicin

(DOX; 0.05 M) and/or melphalan (MEL; 20M) for the indicated times. IL15, both at the (A)

mRNA and (B-D) protein level was evaluated. (A) Real-time PCR (24 hours (h), 48h, and 48+24h);

(B) Representative FACS histograms of intracellular (intra) IL15 (1 g/1x106 cells) (48+24h); (C)

Western blot (48+24h); and (D) Representative FACS histograms of extracellular (extra) IL15 (1

g/1x106 cells) (48+24h). (A) The average (±SEM) of three independent experiments. Isotype

control antibodies in (B) and (D) have been used at same concentration than specific antibodies.

Statistic was calculated by paired Student t test (*p0.05; **p0.01). Results in panel B, C, and D

are representative of 1 out of at least 3 independent experiments.

Fig 2. IL15R expression is increased on drug-treated MM cells. IL15R surface expression on

SKO-007(J3), ARK, and RPMI8226 was analyzed upon 48 h treatment with DOX (0.05 M)

and/or MEL (20M) agents plus 24h without drug by (A) Western blot and (B-C) FACS analysis.

(B) Representative histograms of IL15RA surface expression on SKO-007(J3), ARK, and

RPMI8226 MM cells. (C) The MFI (±SEM) was calculated based on at least three independent

experiments and evaluated by paired Student t test (*p0.05).

Fig 3. MM patients’ malignant PCs express augmented levels of the IL15/IL15R complex.

(A) IL15 mRNA expression upon MEL (20 M) treatment (72h) was evaluated in isolated PCs. The

average (±SD) of different patients is shown as fold increase (f.i.) (Pt.; n=6). Paired sample t-test

was used (*p0.05). Representative histograms of (B) IL15 and (C) IL15R plasma membrane

surface expression was detected on untreated (NT) and MEL-treated (20 M) patient PCs (n=4) by

immunofluorescence and flow cytometry, gating on CD138+CD38

+ cells.

Fig 4. MEL-treated MM cells elicit NK cell activation and proliferation by IL15 trans-

presentation. (A) Freshly isolated primary NK cells were cocultured with untreated- or MEL (20




17

M)-treated SKO-007(J3) MM cells. CD69 expression was evaluated after 5 days by

immunofluorescence and FACS analysis. NK cells were also supplemented with human

recombinant IL15 (10 ng/mL) as control. Results from a representative donor is shown. Numbers

represent the percentage of CD69+cells. (B) SKO-007(J3) cells were incubated with anti-IL15

blocking antibody or isotype control before the coculture with primary NK cells as in (A). The

average (±SEM) of at least three independent experiments is shown. (C) Cell proliferation was

analyzed by in vitro labeling of NK cells with BrdU and then coculturing with SKO-007(J3) cells as

in (A). Results from a representative donor is shown. Numbers represent the percentage of BrdU+

cells. (D) The average (±SEM) of at least three independent experiments is shown for NK cell

proliferation. (E) SKO-007(J3) cells were incubated with anti-IL15 blocking antibody or isotype

control before the coculture with primary NK cells and BrdU incorporation was analyzed on NK

cells. The average (±SEM) of at least three independent experiments is shown. Paired sample t-test

was used in panel (B), (D), and (E) (*p0.05).

Fig 5. IL15RA- and IL15-expressing exosomes released by MM cells increase NK cell

proliferation. (A) Electron microscopy of exosome morphology and size. A representative picture

of SKO-007(J3)-derived exosomes is shown. Scale bar: 50nm. (B) IL15R IL15, HSP70, and

calreticulin expression was evaluated in cell or exosome lysates by Western blot analysis. (C) Cell

proliferation of NK cells exposed to exosomes derived from MEL (20M)-treated MM cells alone

or in presence of IL15 (10ng/mL) was evaluated by BrdU assay on freshly isolated primary NK

cells. (D) Representative histograms of cell proliferation analyzed by CFSE detection on NK cells

cultured for five days with exosomes in the presence of IL15 (10ng/mL). (E) The average (±SEM)

of at least three independent experiments is shown for NK cell proliferation (*p0.05). (F)

Representative histograms of NK cell proliferation measured after five days with the Ki67 marker

on cells exposed to exosomes alone or in the presence of IL15 (10ng/mL) and IL2 (200U/mL).




18

Fig 6. MM patients release variable levels of IL15. (A) The presence of the IL15 was evaluated

in the sera of MM patients (Pt.; n=78). at different states of the disease or (B) in the bone marrow

(BM) or peripheral blood (PB) plasma by ELISA. Mann-Whitney statistical test was used (*p0.05).

State of

disease

IL15

(pg/mL)

Response to

therapy

Clinical

outcome

(at 1 year)

Smoldering 116,9 NE Lost FU

Relapse 3,6 NE Death

MGUS 4,9 NE Onset


Smoldering 1,86 NE Onset

Relapse 12,4 PR +Relapse


Onset 14,2 VGPR +Relapse

Relapse 446,2 PD +++Relapse

Relapse 12,4 PR +Relapse

Onset 8,9 NE Death

Table 1. Correlation of IL15 expression in the serum with patient clinical outcomes.

Serum IL15 levels were compared with clinical outcomes at 1 year in patients with multiple

myeloma. IL15 was measured by enzyme-linked immunosorbent assay (ELISA).

NE=Not Evaluable; Lost FU=Lost Folllow-up; PR=Partial Response; VGPR= Very Good Partial

Response; PD= Progressive Disease. +=1 relapse; +++=>1 relapse.




19

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Published OnlineFirst April 24, 2018.Cancer Immunol Res Cristiana Borrelli, Biancamaria Ricci, Elisabetta Vulpis, et al. trans-presentationproliferation by direct or exosome-mediated IL-15 Drug-induced senescent Multiple Myeloma cells elicit NK cell

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