Increased effect of IMiDs by addition of cytokine-induced killer cells in multiple myeloma

Hematological OncologyHematol Oncol 2015Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/hon.2200

Original Research Article

Increased effect of IMiDs by addition of cytokine-inducedkiller cells in multiple myeloma

Katharina F. Bullok, Christoph Sippel and Ingo G.H. Schmidt-Wolf*Center for Integrated Oncology (CIO), Universitätsklinikum Bonn, Bonn, Germany

*Correspondence to: Prof. Dr. IngoG. H. Schmidt-Wolf, Center forIntegrated Oncology (CIO)University Hospital Bonn,Sigmund-Freud-Straße 25, 53105Bonn, Germany.E-mail: [email protected]

Received 31 January 2014Revised 10 December 2014Accepted 18 February 2015

Copyright © 2015 John Wiley & Son

AbstractImmunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide and pomalidomide,represent the basic principle of multiple myeloma treatment. However, the development ofresistance is a limiting factor. Over the last years, the efficient application of cytokine-induced killer (CIK) cells has been reported as an alternative strategy to treat hematolog-ical neoplasms. In this study, we tested for a potential synergistic effect by combining theIMiDs thalidomide, lenalidomide and pomalidomide with CIK cells in different myelomacell lines in vitro. Myeloma cells tested with CIK cells were significantly reduced. In thecombination, myeloma cells were significantly reduced compared with cells only testedwith IMiDs but not to the cells tested with CIK cells. Otherwise, the number of CIK cellswas significantly reduced when treated with IMiDs. Because IMiDs are active in patientswith myeloma, these results lead to the expectation that combination of IMiDs and CIKcells achieve better results in the treatment of multiple myeloma compared with the singleuse of IMiDs. Therefore, further examinations in an in vivo setting are necessary to have acloser look on the cellular interactions. Copyright © 2015 John Wiley & Sons, Ltd.

Keywords: multiple myeloma; cytokine-induced killer cells; immunomodulatory drugs;immunotherapy

Introduction

Multiple myeloma (MM) is a hematological malignancy,based on monoclonal expansion of plasma cells in the bonemarrow [1]. With the introduction of thalidomide (Thal)and other immunomodulatory drugs (IMiDs) such aslenalidomide (Lena) and pomalidomide (Pom), new op-tions for the treatment of MM were given [2–4]. Althoughthe molecular mechanism of those IMiDs remains unclear,several direct and indirect anti-myeloma effects have beendescribed such as anti-angiogenic, anti-proliferative, anti-inflammatory, pro-apoptotic and immunomodulatory ef-fects [5]. Nevertheless, only 30% of patients with MM re-spond to single used IMiDs [6], and the development ofresistances leads to further complications [7]. Thus, untiltoday, MM remains an incurable disease.The immunomodulatory effects of IMiDs, like stimula-

tion of T cells [8] and an enhanced lytic activity of naturalkiller (NK) and natural killer T (NKT) cells [9], lead to theexpectation that the combination of IMiDs and adoptiveimmunotherapy with cytokine-induced killer (CIK) cellsmight be a new option in the treatment of MM.CIK cells are a heterogeneous cell population consisting

predominantly of CD3+CD56+ cells and in minor amounts ofCD3+CD56� T cells as well as CD3�CD56+ NK cells [10].

s, Ltd.

They possess cytolytic capacities towards different typesof tumour cells in vitro and in vivo including MM cells[11–13]. This cytotoxicity is mediated via a non-majorhistocompatibility complex (MHC)-restricted mecha-nism [14].

In this study, we investigated the cytotoxic efficiency ofCIK cells in combination with IMiDs towards differentmyeloma cell lines in vitro; furthermore, we examinedthe effect of IMiDs on CIK cells.

Material and methods

Cell lines and culture conditions

The myeloma cell lines OPM-2, U-266 and KMS-11 [all ob-tained from Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Braunschweig, Germany] werecultured in RPMI-1640 medium (PAA, Cölbe, Germany)supplemented with 10% heat-inactivated fetal calf serum(FCS) (life technologies, Darmstadt, Germany), 100U/mlpenicillin (life technologies) and 100μg/ml streptomycin (lifetechnologies). The myeloma cell line KMS-12 PE (DSMZ)was cultured in RPMI-1640 medium with 20% heat-inactivated FCS.

K F Bullok et al.

The control cell line CCD-18Co (ATCC,Wesel, Germany)(human colon fibroblasts) was cultured in Eagle’s MinimumEssentialMedium (ATCC) consisting of 10% heat-inactivatedFCS, 100 U/ml penicillin and 100μg/ml streptomycin.Cells were incubated at 37°C in humidified 5% CO2

atmosphere.

Generation of CIK cells

CIK cells were generated as previously described14. Inshort, non-adherent Ficoll-separated (Lymphoprep, PAA)human peripheral blood mononuclear cells from healthydonors were cultured in RPMI-1640 medium supple-mented with 10% heat-inactivated FCS, 25mmol/l Hepes(PAA), 100U/ml penicillin and 100μg/ml streptomycin.Cells were grown at a density of 5× 106 cell/ml. One thou-sand units per milliliter(U/ml) recombinant human inter-feron gamma (rh IFN-γ) (ImmunoTools, Friesoythe,Germany) was added at day 0. After 24 h, 300U/ml rhinterleukin-2 (rh IL-2), 100U/ml IL-1β (bothImmunoTools) and 50 ng/ml anti-CD3 (eBioscience,Frankfurt, Germany) were added. Every 3 days, further300U/ml rh IL-2 was added; after 14 days, the CIK cellswere matured and ready for use.

Figure 1. Effect of the IMiDs Thal, Lena and Pom on the viabilityPE. Cells (1 × 104) were cultured for 48 h with different concentr0,1μM, 1μM, 10μM, 100μM and 200μM; and Pom: 0,1μM, 1μM,Results represent data from three separate experiments. Data areANOVA)

Copyright © 2015 John Wiley & Sons, Ltd.

Cells were incubated at 37°C in humidified 5% CO2

atmosphere.

Drugs

The following drugs were used in this setting: Thal(Sigma-Aldrich, München, Germany), Lena (Selleck,München, Germany) and Pom (Sigma-Aldrich). All drugswere used at different concentrations for 24–72 h.

MTT assay

AnMTT assay was performed to measure the cytotoxicity ofthe different drugs and the CIK cells. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as-say is a colorimetric alternative to the 51Cr release assay,whereby the yellow MTT is reduced to purple formazan byviable cells [15].

In brief, 1–10×104 cells were plated in triplicates in flatbottomed 96-well plates and co-incubated with variousconcentrations of IMiDs and CIK cells over 24–72 h beforeMTT reagent was added. After 3 h of incubation, the plateswere centrifuged for 4min, 1200 rpm. The supernatant was

of the myeloma cell lines OPM-2, U-266, KMS-11 and KMS-12ations. Thal: 1μM, 10μM, 100μM, 200μM and 400μM; Lena:10μM and 100μM. Cell viability was measured via MTT assay.shown as mean ± standard deviation (SD). (p< 0.05, two-way

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Combination of IMiDs and CIK cells in multiple myeloma

abolished, and 80μl of MTT lysis buffer (isopropanol,HCl) was added. After shaking the plates for 10min at700 rpm, colorimetric analysis was performed on amultiwell scanning spectrophotometer (FluoStar Optima,BMG Labtech, Ortenberg, Germany) at 492 nm.

Figure 2. Cytotoxic effect of CIK cells on myeloma cell lines OPMCells were cultured for 24 h at different effector: target ratios of 1:represent four separate experiments for OPM-2, U-266, KMS-11 andare presented as mean ± SD. (p< 0.05, one-way ANOVA)


Enzyme-linked immunosorbent assay (ELISA)

For measurement of interferon gamma secretion, a sand-wich ELISA was performed (Thermo Fisher ScientificInc., Rockford, USA). Myeloma cells of 2 × 105 were

-2, U-266, KMS-11 and KMS-12 PE and control cells CCD-18Co.5, 1:2 and 1:1. Cell viability was measured via MTT assay. ResultsCCD-18Co and three separate experiments for KMS-12 PE. Data

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plated in triplicates in 96-well plates and co-incubated withThal, Lena or Pom. After 24 h, 1 × 105 CIK cells wereadded. After another 24 h, the plates were centrifuged for5min, 1100 rpm. Then 50μl supernatant was transferredto an antibody coated ELISA plate. Thereafter, the assaywas continued according to the manuals instructions. Ab-sorbance was measured with an ELISA reader at 450 nm(Glomax multidetection system, Promega, Mannheim,Germany).

Flowcytometric analysis

Phenotypes of treated and untreated CIK cells were deter-mined by fluorescence-activated cell sorting analysis(FACS Canto II, BD Bioscience, San Jose, USA). Cellswere stained with the monoclonal antibodies (mAb) anti-human CD45 PerCP-Cyanine5.5, anti-human CD3 FITC,anti-human CD56 APC, anti-human NKG2D (CD314)PE (eBioscience, Frankfurt, Germany), anti-human CD25

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Figure 3. Combined effect of IMiDs and CIK cells on viability of mwith different concentrations of IMiDs (Thal 100μM, Lena 100μM awas removed and cells were cultured for another 24 h at different efand 1:10 for D, E and F) of CIK cells. Cell viability was measured via Mfor U-266 and KMS-12 PE and two different buffy coats for KMS-(p< 0.001, two-way ANOVA)


PE and anti-human CD69 PE (BioLegend, Fell, Germany).FlowJo Data Analysis Software (Ashland, USA) was usedfor data analysis.

Statistics

GraphPad Prism (La Jolla, USA) was used for statisticalanalysis. One-way and two-way analysis of variance(ANOVA) with Bonferoni posttest was performed to ana-lyze statistical significance. The p-values<0.05 were con-sidered as significant.

Results

Effect of Thal, Lena and Pom on myeloma cell lines

The myeloma cell lines OPM-2, U-266, KMS-11 andKMS-12 PE were cultured with increasing concentrationsof Thal (1μM, 10μM, 100μM, 200μM and 400μM), Lena

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yeloma cells U-266, KMS-11 and KMS-12 PE. Cells were culturednd Pom 10μM) after 24 h (A, B and C)/72 h (D, E and F) mediumfector: target ratios (1:5, 1:2 and 1:1 for A, B and C and 1:5, 1:7,5TT assay. Results represent data from three different buffy coats

11 (in E also three buffy coats). Data are shown as mean ± SD.

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(0,1μM, 1μM, 10μM, 100μMand 200μM) or Pom (0,1μM,1μM, 10μM and 100μM) over 24–72 h (Figure 1). Never-theless, none of them had a significant effect on the viabilityof myeloma cells in vitro.

Effect of CIK cells on myeloma cell lines

CIK cells were co-cultured with myeloma cell linesOPM-2, U-266, KMS-11 and KMS-12 PE and the con-trol cell line CCD-18Co for 24 h (Figure 2). Therefore,CIK cells of four (three in KMS-12 PE) different buffycoats were used with different effector to target ratios(1:5, 1:2 and 1:1). CIK cells reduced significantly theviability of myeloma cells in vitro. The observed effectwas in correlation to the different effector to target ra-tios. However, the viability of CCD-18Co was alsosignificant reduced by CIK cells.

Figure 4. Significance between myeloma cells U-266, KMS-11and KMS-12 PE tested only with IMiDs (24 h) and cells tested withthe combination of IMiDs (24 h) and CIK cells at different effec-tor: target ratios (1:5, 1:2 and 1:1). Data represent results ofFigure 3. (p< 0.001, two- way ANOVA)

Combination of Thal, Lena and Pom with CIK cells

To investigate a synergistic effect of IMiDs and CIKcells, myeloma cell lines U-266, KMS-11 and KMS-12 PE were first cultured with Thal (100μM), Lena(100μM) or Pom (10μM) for 24 or 72 h. After 24 h,the whole medium was changed and the cells were cul-tured for another 24 h with CIK cells at different effec-tor to target ratios (1:5, 1:2 and 1:1) (Figure 3). Cellstested for 72 h with IMiDs were co-cultured with CIKcells for 24 h with different effector to target ratios(1:10, 7,5:1 and 5:1). The viability of myeloma cellstreated with IMiDs (24 h) and CIK cells was signifi-cantly lower compared with the viability of myelomacells co-cultured only with IMiDs (Figure 4). Myelomacells only cultured with CIK cells showed no signifi-cance towards the combined treated cells. The controlcells CCD-18Co showed the same results as the mye-loma cells (data not shown). In summary, no synergisticeffect of IMiDs and CIK cells was traceable in thissetting.In another setting, myeloma cells (U-266, KMS-11

and KMS-12 PE) were cultured for 72 h with previ-ously described IMiDs. Again, whole medium waschanged, and CIK cells with even lower effector to tar-get ratios (1:5, 1:7, 5 and 1:10) were added for 24 h(Figure 3).In all three cells lines, there was no significant differ-

ence in the samples treated with Thal. Cells treated withLena showed different results. In U-266, there were nosignificant differences. In KMS-11, cells treated withCIK cells (1:10) showed significantly lower viabilitycompared with cells only treated with Lena. KMS-12PE cells treated with Lena exposed significantly lowerviability compared with cells treated with CIK cells.


Samples treated both with Lena and CIK cells (1:5)showed lower viability compared with cells only treatedwith CIK cells (1:5).Cells treated with Pom showed the same results as cells

treated with Lena.

Interferon γ secretion in the combination of IMiDsand CIK cells on myeloma cells

Myeloma cell lines U-266, KMS-11 and KMS-12 PE werefirst incubated with Thal (100μM), Lena (100μM) or Pom(10μM), after 24 h, the whole medium was changed andCIK cells were added at an effector to target ratio of 1:2.After another 24 h, interferon γ secretion was measuredby a sandwich ELISA (Figure 5).

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CIK + IMiDs

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igure 6. Effect of the IMiDs Thal, Lena and Pom on the viabilityf CIK cells. CIK cells (1 × 104) of four different buffy coats wereultured for 24 h with different concentrations of IMiDs. Thal00μM, Lena 100μM, and Pom 10μM. Cell viability was mea-ured by MTT assay. Results represent eight separate experi-ents for each buffy coat. Data are shown as mean ± SD.p< 0.05, one-way ANOVA)

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Figure 5. Interferon γ secretion in the combination of IMiDs andCIK cells on myeloma cell lines U-266, KMS-11 and KMS-12 PE.Myeloma cells were cultured with different concentrations ofIMiDs (Thal 100μM, Lena 100μM and Pom 10 μM), after 24 hwhole medium was removed and CIK cells (1:5, 1:7, 5, 1:10) wereadded for another 24 h. Interferon γ secretion was measured viaELISA. Results represent three different buffy coats for each cellline. Data are shown as mean ± SD. (p< 0.001, two-way ANOVA)

K F Bullok et al.

In all three myeloma cell lines, a stable IFN-γ secretionwas measured whether treated with IMiDs or not. CIK cellstreated with IMiDs acted in the same way, but IFN-γ secre-tion in total was higher.In all three cell lines, interferon γ secretion was signifi-

cantly higher in the combined samples compared with thesamples treated only with IMiDs. Samples treated withCIK cells showed a significantly higher IFN- γ secretion inthe cell line KMS-11 compared with the IMiD samples,but significance was not as high as in the combined samples.


In KMS-11, the combination with Pom also showedsignificantly higher IFN-γ secretion compared with un-treated CIK cells and compared with the combinationwith Thal.

In KMS-12 PE, the combination with Lena displayed asignificantly higher secretion of IFN-γ compared with un-treated CIK cells. Overall secretion of IFN-γ in KMS-12PE cells treated with CIK cells or the combination of CIKcells an IMiDs was much higher compared with the othercell lines. KMS-12 PE cells treated or untreated with IMiDsshowed the same INF-γ secretion as the other cell lines.

Effect of Thal, Lena and Pom on viability of CIKcells

CIK cells of four different buffy coats were tested withThal, Lena (both 100μM) or Pom (10μM) for 24 h (Figure6). All three IMiDs decreased the viability of CIK cells sig-nificantly. Pom had a significantly higher effect on CIKcells than Thal and Lena.

Effect of IMiDs on phenotypical determination ofCIK cells

CIK cells of five different buffy coats were treated withThal (100μM), Lena (100μM) or Pom (10μM). After24 h, treated and untreated cells were stained with mAbCD45, CD3, CD56, CD25, CD69 and NKG2D.

Foc1sm(

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CD3 and CD56 were used to characterize the differentCIK cell subsets (Figure 7). The flow cytometric analysisdid not show any difference between these cell subsets intreated and untreated cells. CD 3+CD56� cells representthe main group of cells followed by CD3+CD56+ andCD3�CD56+.CD 25, CD 69 and NKG2D were used as markers for the

activation of CIK cells. CIK cells of various buffy coatsshowed different results but same tendencies.CD 25 in general was expressed only by a small number

of cells. Expression of CD69 was higher especially inCD3�CD56+ cells. NKG2D showed high expression overall three cell subsets, most of all in CD3+CD56+ cells.In cells marked with CD25, treatment with Lena and

Pom caused an increased frequency of CD3+CD56+ andCD3+ CD56� cells. Cells treated with Pom even showeda slight enhancement of CD3�CD56+ cells. Thal had thesame effect but in an attenuated form.Cells marked with CD69 or NKG2D showed different

results. Treatment with Thal, Lena or Pom led to slightly

Figure 7. (A) Flowcytometric analysis was used to identify untreate24 h] CD3+CD56+ CIK cells. Data show representative dot plots. Fumarkers CD 25, CD69 and NKG2D. Data are representative for atreated and untreated CIK cells. Cells were treated for 24 h with TCIK cells of five different buffy coats. Data are shown as mean ± SDor NKG2D on CD3+CD56+ CIK cells. Cells were either untreate24 h. Data represent one generic buffy coat.


decreased frequencies of CD3+CD56+, CD3+CD56� andCD3�CD56+ cells compared with untreated controls(Figure 7).

Discussion

IMiDs are already widely used in the treatment of MM. Itsanti-myeloma effects in vivo have been recently shown. Inclinical studies, the combination of Thal/dexamethasoneand Lena/dexamethasone achieved partial response ratesof 61.2 and 80.3 % respectively, with tolerable side effects[16]. However, we observed a slightly lower anti-myelomaeffect of IMiDs in vitro. This might be explained by thecomplex mechanisms of action of IMiDs. The direct anti-tumour effect just represents a small part of the whole po-tentiality of IMiDs. Effects on the microenvironment ofMM or immunomodulatory effects cannot be revealed byin vitro testing. Nevertheless, chemotherapy with IMiDsremains one of the backbones in the treatment of MM.

d and treated [thal (100μM), lena (100μM) and pom (10μM) forrthermore, flow cytometry was used to determine the activationll activation markers. (B) Distribution of different phenotypes inhal (100μM), Lena (100μM) or Pom (10μM). Results represent(p< 0.001, one-way ANOVA). (C) Expression of CD25, CD69d or treated with Thal100μM, Lena 100μM or Pom 10 μM for

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K F Bullok et al.

A different approach is the use of CIK cells in patientswith MM [17]. We investigated the anti-myeloma effectof CIK cells from healthy donors on different myeloma celllines in vitro. It was recently shown that CIK cells have thecapability to lyse myeloma cells when previously co-cultured with dendritic cells (DCs) [11]. In this study, wedemonstrate the anti-myeloma effect of untreated CIK cellson MM cells at very low effector to target ratios. CIK cellswere able to lyse MM cells to a significant extent evenwhen tested with an effector to target ratio of 1:2. The lyticeffect of CIK cells on tumour cells is mediated via non-MHC-restricted, NK-like recognition. The expression ofNK cell receptors, like NKG2D, seems to play an importantrole in the recognition of myeloma cells [18]. Nevertheless,other studies showed that CIK cells keep their TCR-mediated specific cytotoxicity during maturation [19]. Insummary, these complex mechanisms, how CIK cells elim-inate tumour cells, still remain unclear.Furthermore, we investigated the anti-myeloma effect of

IMiDs and CIK cells when applied in combination. Theanti-myeloma effect in the combination is significantlyhigher compared with the effect of only IMiDs. In contrastto this, other data show significant reduction of CIK cellswhen tested with IMiDs. These results lead to the possiblemechanisms how IMiDs interact with CIK cells. The immu-nomodulatory effect of IMiDs occurs on different levels, oneis the costimulation of T cells. Costimulation is mediated viaT-cell receptor but also requires further signals of antigenpresenting cells. Activation of T cells leads to increased pro-liferation and production of IL-2 and INF-γ [8,20]. Increasedproduction of IL-2 and INF-γ induces proliferation of NKcells and improves their lytic capability towards myelomacells [21,22]. Another effect is the enhancement of NKTcells. In the presence of Lena, DC-mediated NKT cell expan-sion as well as INF-γ production is increased and causes fur-ther activation and proliferation of NK cells [9]. Therefore,the immunomodulatory effect of IMiDs affects at least mostcell types in the heterogeneous population of CIK cells. Nev-ertheless, it has to be considered that the outcome of thestudy is strongly influenced by other variables like cell–cellinteraction or endogenous mediators.An additional feature of IMiDs is their anti-

inflammatory potential. They are able to block the tran-scription factor NF-κB and thereby the expression of in-flammatory genes that leads to a reduced secretion ofinflammatory cytokines [23]. In our setting, this may causea restricted expansion of the CIK cells full activity.It has been already proposed that IMiDs have an effect

on regulatory T cells (Tregs). Different studies have shownconflicting outcomes. On the one hand, IMiDs inhibitedproliferation of Tregs [24]. Other studies showed an in-creased number of Tregs in patients treated with IMiDs[25]. The impact of IMiDs on the Treg population in CIKcells is another interesting point that needs to be further in-vestigated. The anti-inflammatory effect of IMiDs, as well


as the interaction with Tregs, may cause the decreasednumber of CIK cells we observed in this study.

In summary, the combination of IMiDs and adoptive im-munotherapy seems to be a promising alternative for treat-ment of MM. Because of the complex mechanisms ofinteraction, it is essential to detect the right timing for theapplication of CIK cells in an experimental setting, wherethe synergistic effects prevail over negative interactions.This opens new questions that have to be addressed in anin vivo model.

Conf lict of interest

All authors declare no conflict of interest.

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Hematol Oncol 2015

DOI: 10.1002/hon

Increased effect of IMiDs by addition of cytokine-induced killer cells in multiple myeloma

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