Lenalidomide increases human dendritic cell maturation in multiple myeloma patients targeting monocyte differentiation and modulating mesenchymal stromal cell inhibitory properties

The use of Lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma-specific immunity is currently being explored. Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14+ cells obtained from patients with multiple myeloma or from a human monocytic cell line. LEN, at the concentration range reached in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3+ cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes. In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity.


INTRODUCTION
The Immunomodulatory drugs (IMiDs ® ) are a group of therapeutic agents, Thalidomide-derivatives, including Lenalidomide (LEN) and Pomalidomide (POM). The development of these drugs represented a paradigm shift in the treatment of multiple myeloma (MM) [1,2]. LENbased regimen is one of the standard of care for MM patients either in frontline or in relapsed setting [3][4][5]. Moreover, LEN has shown a significant impact on the progression free survival in maintenance after autologous stem cell transplantation [6].
Several mechanisms of action have been described, [7,8] including immunomodulatory effects as T helper 1 cell activation, T regulatory cell (T reg) suppression, and the induction of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells [9]. The molecular mechanisms involved in the anti-MM effect of IMiDs ® have been recently elucidated highlighting the role of Cereblon (CRBN) and its target factors [10,11]. LEN binds CRBN in MM cells and causes selective ubiquitination and degradation, by the CRBN-Cullin-RING E3 ubiquitin ligase (CRL) 4 complex, of two lymphoid transcription factors essential for MM survival, Ikaros and Aiolos [12]. Through the same mechanism, LEN enhances T cell proliferation and interleukin (IL)-2 production. The loss of Ikaros and Aiolos is necessary and sufficient for LEN therapeutic effect [13]. Recently, it has been also reported a distinct CRBN substrate, Casein Kinase 1 alpha (CK1-α) that is ubiquitinated and degraded after LEN treatment in myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) [14] and in MM, as reported by Manni S et al. [15].
Currently, few data are reported on the possible effects of LEN on dendritic cells (DCs) populations. [9,[16][17][18] Interestingly, it has been shown that DCs from peripheral blood (PB) were functionally defective in MM patients, since they had decreased expression of maturation markers and antigen presentation capacity [19,20]. Nevertheless, it is not known which is the role of DCs in the anti-MM effect of LEN. Different studies reported an increased incidence of acute Graft versus host disease (aGvHD), with a possible enhancement of the graft versus MM effect, in patients treated with LEN after allo-transplantation [21][22][23]. Since it is known that host and donor DCs are critical in the development of GvHD and also involved in the immunosuppressive properties of mesenchymal stromal cells (MSCs); [24][25][26] it is conceivable that LEN affects DCs.
Based on these evidences, in this study we investigated whether LEN may affect maturation, phenotype and functionality of DCs as antigen presenting cells (APCs), either directly or through the modulation of human mesenchymal stromal cell (hMSC) effect on DCs.

LEN enhanced in vitro DC differentiation from both bone marrow (BM) and PB monocytes of MM patients and increased their chemokine and cytokine production degrading Ikaros and Aiolos
We analyzed the expression of DC maturation markers on monocytes derived-DCs (mo-DCs) differentiated from BM aspirates and PB of MM patients. Despite a reduction of both number and % of mature DCs, LEN, at the concentration range reached in vivo in MM patients, [27] Figure 1B). Flow-cytometry histograms from one representative patient were reported in Supplementary Figure 1B. Any significant differences between BM and PB samples of the same patient were found on LEN effect on DC maturation markers (2way ANOVA) (data not shown).
Interestingly, the increased expression of DC maturation markers was abrogated when LEN was used in combination with Dexamethasone (Dex) at 10 - Figure 3).
Based on the literature data showing that LEN exerts the anti-MM activity through the selective ubiquitination and degradation of Cereblon targets, Ikaros and Aiolos [12], we assessed the protein levels of Cereblon, Ikaros and Aiolos on LEN-treated THP1-DCs. We showed that THP1-DCs expressed Cereblon ( Figure 4A) and that LEN treatment down-regulated Ikaros protein levels in a dose-dependent manner ( Figure 4B). Moreover, we found that the basal Aiolos protein level was very low in THP1-DCs and LEN further decreased its expression in these cells ( Figure 4C). We also tested the effect on two other Cereblon targets, Interferon regulatory factor (IRF) 4 and Sequestosome-1/SQSTM1 (p62), that were respectively down-regulated [28] and up-regulated [29] after LEN treatment in MM cells. We showed that THP1-DCs did not express IRF4 ( Figure 4D) and LEN did not affect p62 in THP-1 DCs ( Figure 4E).

LEN enhanced DC ability to stimulate autologous CD3 + cell proliferation
Next we evaluated whether the effect of LEN on DC maturation may affect DC functional properties. In order to evaluate LEN effect on DC ability to stimulate T cell proliferation, DCs differentiated from BM of 4 MM patients, were tested as stimulators in the autologous Mixed Lymphocyte Reaction (MLR) assay in the presence of LEN or vehicle.
Interestingly, CD3 + cell proliferation was significantly higher in co-culture with LEN-treated DCs,

LEN blunted hMSC inhibitory properties on DC differentiation inhibiting CK1-α levels
To investigate a possible MSC-mediated indirect effect of LEN on DCs, BM CD14 + cells of MM patients were We firstly demonstrated that hTERT-hMSC CM decreased DC maturation marker expression in this in vitro system ( Figure 6A) Figure 6B).
To investigate the molecular mechanism involved in the effect of LEN on MSCs, firstly we checked the expression profile of Cereblon and its target proteins in hTERT-hMSCs showing that they expressed Cereblon ( Figure 7A) but not Ikaros, Aiolos and IRF4 ( Figure 7B). We then focused our attention on another Cereblon substrate, CK1-α. [14] LEN, at the higher concentration, decreased CK1-α protein level in hTERT-hMSCs ( Figure 7C). We also found that LEN treatment did not affect p62 protein level, as observed in THP1-DCs ( Figure 7D).
To better clarify the correlation between the decreased CK1-α levels in hMSCs after LEN treatment and LEN effect on the immunosuppressive properties of MSCs on DCs, we down-regulated CK1α in hTERT-hMSCs through an IPTG inducible shRNA. We firstly checked CK1-α down-regulation by western blotting (Supplementary Figure 3A) and then we used the CM for in vitro DC differentiation from BM CD14 + cells of 2 MM patients. Interestingly, we found that the effect of hTERT-hMSCs on DC maturation markers were reverted by the down-regulation of CK1-α (mean MFI ± SEM: CNT vs wt hTERT-hMSCs vs hTERT-hMSCs 6044, HLA-DR: 108. 8

LEN treatment of MM patients increased ex vivo DC differentiation
Finally, to evaluate the effect of in vivo LEN treatment on DC maturation markers, we compared the expression profile of DCs differentiated from PB CD14 + cells of 9 MM relapsed patients, purified at the baseline (DAY 0) and after 7 days of LEN treatment, just before the start of the weekly treatment with Dex. All the patients were responsive to LEN treatment. Interestingly

DISCUSSION
DCs of MM patients are known to be functionally defective, with a decreased expression of maturation markers and antigen presentation ability [19]. The production by MM cells of several soluble factors with immunosuppressive properties on DCs, such as IL-6, IL-10, vascular endothelial growth factor and TGF-β, contributes to these immunological defects [19,30,31]. LEN exerts its immunomodulatory activity through several mechanisms of action on T and NK cells; [7,32] however, few literature data reported the effect of LEN on DCs in the contest of the anti-MM activity and in the increased incidence of GvHD observed in patients treated with this drug [33].
Studies performed on murine models, showed that both LEN and POM treatment increased the expression of DC maturation markers, enhanced DC endocytotic activity, increased the production of TNF-α and CCL2, and the DC-dependent T-cell expansion [16,17]. However, LEN effect on human DCs are still unclear. One study, recently published [18], reported that LEN (5 µg/ml ≈ 20 µM) enhanced the maturation of DCs generated from PB of MM patients and increased the ability to stimulate T cell proliferation in line with our results [18]. Particularly we found that LEN, at concentrations reached in vivo in MM patients treated with this drug 5-25 mg daily, [27,34] significantly increased the expression of DC maturation markers, HLA-DR, CD86 and CD209, that are involved in DC co-stimulatory function and trafficking, [35][36][37] both in BM and PB of MM patients. This effect was abrogated in the presence of Dex at 10 -8 M, consistent with several studies that reported Dex inhibitory effects on DC maturation and functions [38,39]. However, contradictory data were reported on the antagonistic effects of Dex on LEN immunomodulatory properties. A study from Paiva et al. [40] on high-risk Smoldering MM (SMM) patients reported no significant differences in the immune profile of patients treated with LEN/Dex vs LEN alone [40]. Conversely, different studies showed a profound inhibition   [41,42].
Consistent with the effect of LEN on DC maturation markers, we found that LEN treatment increased DC production of IL-8, CCL2, CCL5, and TNF-α, in line with data observed on murine models [16,17]. These factors regulate the antigen uptake and the activation of the innate immune system, and are chemo-attractants for various immune cells, [43] suggesting that LEN treatment enhanced the role of DC in linking innate and adaptive anti-tumor-antigen immune responses. In line with this hypothesis, we showed that LEN effect on in vitro DC differentiation was associated with an increased DC functional activity to stimulate T cell proliferation that was abrogated by the combination with Dex.
Subsequently, we investigated the molecular mechanism beyond LEN effects on DCs. It is widely demonstrated that LEN exerts its anti-MM activity through the modulation of Ikaros and Aiolos [12,13]. Moreover, Ikaros deficiency in host APCs failed to enhance GvLeukemia despite increased GvHD severity, in a murine model of allo-transplantation [44]. Similarly, we found that both Ikaros and Aiolos were degraded in DCs after LEN treatment, as previously showed for MM cells and T cells [12,13] [21]. Along with a direct effect of LEN on DC maturation, our data suggested a potential indirect effect, through the modulation of MSC immunomodulatory properties such as the production of cytokines and chemokines. [24][25][26]45] When DCs were cultured with LEN-treated MSC CM, we observed an increased expression of DC maturation markers suggesting the ability of LEN to blunt the inhibitory effect of MSCs on DC differentiation. Moreover, we found that the treatment of MSCs with LEN significantly decreased the expression levels of PTGS2, known to inhibit the transitional processes of differentiation of monocytes into DCs [46,47]. The molecular mechanisms involved in the effect of LEN on MSC was also investigated. Surprisingly, we lacked to find the expression of the main Cereblon targets, Ikaros and Aiolos, by MSCs but we showed that LEN caused the degradation of a novel described Cereblon substrate, CK1-α in MSCs. CK1-α is a component of the beta (β)catenin destruction complex and a negative regulator of p53, and several studies [15,48] recently reported that reduced CK1-α levels decrease MM cell survival and inhibit cell cycle progression [15,48]. Interestingly, this factor seems to be involved in the resistance of plasma cells to LEN after long-term exposure [49].
Lastly, our in vitro evidences were expanded and confirmed by ex vivo DC cultures in relapsed MM patients treated with LEN 25 mg/day, as mono-therapy for one week, just before the start of the weekly treatment with Dex. After 7 days of treatment we found an increased PB DC differentiation. Of note, all analyzed patients were responsive to LEN treatment. This early effect was in agreement with recent data reporting the in vivo increase of T and NK cells, with a rapid decline of Ikaros, after 7 days of POM treatment without Dex in MM patients [50].
In conclusion, our data indicate that LEN increases the expression of mature DC markers both in vitro and in ex vivo cultures, enhancing DC ability to stimulate T cell proliferation and to release chemokines involved in the immune response. LEN treatment also reduces the immunosuppressive properties of hMSCs, suggesting new possible effects of IMiDs ® on the allo-reactivity against MM cells.

Patients
BM and/or PB were obtained from 30 consecutive patients with active MM (50% female, 50% male; median age: 71 years, range 43-94), including both newly diagnosed and relapsed MM, admitted to our hematological Unit. Patient samples were obtained after informed consent, according to the Declaration of Helsinki. The study was approved by the Institutional Ethical Review Board of our Hospital.
Moreover, PB were obtained from 9 patients with relapsed MM (4 female, 5 male; median age: 73 years, range: 56-82; International Staging System (ISS) I: 4, International Staging System (ISS) II: 3, International Staging System (ISS) III: 2), at the baseline and after 7 days of treatment with LEN 25 mg/day (days 1-21), just before the start of the weekly treatment with Dex.
Mononuclear cells (MNCs) were isolated from BM and PB samples after Ficoll gradient separation and used for further in vitro studies.

Cell lines
The human myeloma cell line (HMCL) JJN3, purchased by DSMZ (Braunschweig, Germany) and the human monocytic cell line THP-1, obtained from the American Type Culture Collection (Rockville, MD), were maintained in culture in RPMI 1640 medium with 10% FBS; hTERT-hMSCs were kindly gifted from Dr Giuseppe Gaipa (Monza, Italy) and maintained in culture with RPMI 10% FBS with hydrocortisone (10 -6 M). All cell lines were authenticated and tested for mycoplasma contamination.

Cell purification
BM and PB CD14 + cells were purified from total MNCs by an immuno-magnetic method using anti-CD14 mAb coated microbeads (MACS, Miltenyi Biotec; Bergisch-Gladbach, Germany). CD3 + cells were isolated following the same protocol, using anti-CD3 mAb from PB of MM patients. The presence of potential contaminating cells in each fraction was evaluated by flow cytometry analysis, using the fluorescence-activated flow cytometer BD FACS Canto II with Diva software (Becton, Dickinson and Company (BD); Franklin Lakes, NJ). Purity of cell samples was > 92%.

DC differentiation and cell treatment
DCs were differentiated from purified CD14 + cells, cultured in vitro at 1 × 10 6 cells/ml in RPMI 10% FBS, with recombinant human (rh) granulocyte macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and IL-4 (50 ng/ml) (all purchased by Peprotech, Rocky Hill, NJ), for 8 days (replacing half media with fresh cytokines every 2-3 days), in the presence of LEN (purchased by Celgene, Italy Corporation, Milan, Italy) or vehicle (DMSO), at concentration 0.1 and 1 µM. TNF-α at 10 ng/ml (OriGene; Rockville, MD) was added to the culture medium for the last 24 h, in order to induce DC terminal maturation. At the end of culture period, both cells and CM were collected for further analysis. In some experiment, the combination of LEN and Dex (obtained by Sigma Aldrich, Milan, Italy) was tested on DC differentiation. Briefly, DCs were differentiated from BM CD14 + cells of MM patients, in the presence of LEN (0.1 and 1 µM) or vehicle, as reported above. At the end of culture period, cells were collected and reseeded (5 × 10 4 /ml) in fresh medium with Dex (10 -8 M) or vehicle (EtOH) for 48 h. After Dex treatment, cells were collected and analyzed for DC maturation markers. For the ex vivo studies, PB CD14 + cells were isolated from MM patients at day 0 and after one week (day 7) of LEN (25 mg/day) treatment, just before the start of the weekly treatment with Dex. Cells were then differentiated into DCs, following the above protocol, without LEN in vitro treatment.
In some experiments DCs were differentiated from BM CD14 + cells of MM patients, in the presence or absence of the CM (ratio 1:2 with RPMI 10% FBS, with IL-4 and GM-CSF) of hTERT-hMSCs treated with LEN or DMSO. Briefly, 1 × 10 4 hTERT-hMSCs were seeded in T75 flasks and cultured in RPMI 10% FBS, in presence of LEN (0.1 and 1 µM) or DMSO, for 5 days. At the end of culture period, the medium was replaced with RPMI 10% FBS in order to discard LEN, and after 48 h, the CM was collected and used during DC differentiation, as previously reported. In some experiments, after 5 days of LEN treatment, hTERT-hMSC pellets were collected for immunoblotting and RT-PCR analysis.

Autologous mixed lymphocyte reaction and cell proliferation assay
DCs were differentiated, as previously reported, in presence of LEN or vehicle (alone or in combination with Dex), from BM of 6 MM patients, for 8 days. Then, treated cells were collected, analyzed by flow-cytometry and partly re-seeded (3 × 10 3 cells/w) in round-bottomed 96well-plates, in RPMI 15% AB human serum. DCs were co-cultured with autologous PB CD3 + cells (1 × 10 4 ) for 6 days. At the end of culture period, an MTT assay (Cell Counting Kit-8; Alexis, Vinci-Biochem s.r.l., Italy) was performed in order to measure T cell proliferation.

Western blotting
Nuclear and cytosolic extracts were obtained using a commercial kit (Active Motif, Carlsbad, CA) following the manufacturer's protocol from THP1-DCs and hTERT-hMSCs, treated with LEN or DMSO. Immunoblotting was performed as previously reported [51] using the following antibodies: rabbit monoclonal anti-Aiolos (1:714) (code n.

mRNA silencing
RNAi was performed through the generation of inducible shRNA stable cell lines. hTERT-hMSC were transduced with the IPTG inducible lentiviral particles carrying CSNK1A1-specific shRNA (pLKO_ IPTG_3XLacO, Sigma-Aldrich, Milan, Italy). Two independent shRNAs (TRCN0000006044, and TRCN0000006042) sequences were chosen. 3 × 10 4 cells were infected with a multiplicity of infection (MOI) of 4, in the presence of 8 µg/ml polybrene (Sigma-Aldrich, Milan, Italy). 24 h later, the infected medium was replaced with fresh growing medium. Puromycin selection (0.5 µg/ml) was initiated 2 days after transduction. Once a cellular clone was established, to induce CK1α silencing, cells were incubated with 500 µM IPTG (Sigma-Aldrich, Milan, Italy) every 2-3 days for a total of one week. Then, fresh medium without IPTG and puromycin was added for further 48 h. At the end of culture period, cell pellets were collected and analyzed by western blotting to check CK1-α down-regulation and select the more efficient clone. CM was also collected and used for in vitro DC differentiation.

RT-PCR
Total RNA was extracted from hTERT-hMSCs, after all different experimental conditions, using the RNeasy total RNA isolation kit (Qiagen; Hilden, Germany). To normalize the differences in RNA quality and reverse transcription efficiency, we applied the comparative Ct method using the endogenous reference gene GAPDH.

Statistical analysis
Data were expressed as mean ± SEM or median values. Paired Student's t-test was used to analyze flow cytometry data of in vitro DC differentiation from BM and PB of MM patients and for the ex vivo studies. Nonparametric Friedman test, Wilcoxon test and Mann-Whitney test were used for the other experiments with a lower number of samples. Results were considered significant at p < 0.05. GraphPad Prism 6.1 ™ (GraphPad Software Inc., La Jolla, CA, USA) was used for all the statistical analyses.