Aminobisphosphonates inhibit dendritic cell-mediated antigen-specific activation of CD1d-restricted iNKT cells

Clinical Immunology (2015) 158, 92–99

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

Aminobisphosphonates inhibit dendritic cell-mediated antigen-specific activation of CD1d-restricted iNKT cells☆,☆☆ Famke L. Schneiders a,⁎, Charlotte M. Huijts a , Aslihan Mantici a , Mica A.C. Menks a , Emmanuel Scotet b , Rob Veerhuis c,d , Henk M.W. Verheul a , Tanja D. de Gruijl a , Hans J. van der Vliet a,⁎ a

Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Institut National de la Santé et de la Recherche Médicale, UMR892, Centre de Recherche en Cancérologie Nantes-Angers, Nantes, France c Department of Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands d Department of Psychiatry, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands b

Received 15 September 2014; accepted with revision 10 March 2015 Available online 18 March 2015 KEYWORDS iNKT; Aminobisphosphonates; α-GalCer; Apolipoprotein E (apoE); dendritic cell (DC)

Abstract CD1d-restricted invariant natural killer T (iNKT) cells constitute an important immunoregulatory T cell subset that can be activated by the synthetic glycolipid αgalactosylceramide (α-GalCer) and initiate antitumor immune responses. As cancer patients are frequently treated with aminobisphosphonates (NBP), it is relevant to determine possible effects of NBP on CD1d-restricted glycolipid Ag-presentation to iNKT cells. We report a striking reduction of α-GalCer-induced iNKT cell activation by monocyte derived dendritic cells (moDC) upon their exposure to NBP during maturation. We found that production of apolipoprotein E (apoE), which is a known facilitator of trans-membrane transport of exogenously derived glycolipids, was significantly diminished in moDC exposed to NBP. As the inhibitory effect of NBP on iNKT cell activation was alleviated by exogenous apoE, our data indicate that reduced

Abbreviations: iNKT cells, invariant natural killer T cells; α-GalCer, alpha-galactosylceramide; Ag, antigen; APC, antigen presenting cell; NBP, aminobisphosphonates; moDC, monocyte derived dendritic cell; apoE, apolipoprotein E; IPP, isopentenyl pyrophosphate; SRA, scavenger receptor A; LDL-R, low density lipoprotein receptor; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; AP, allophycocyanin; SFM, serum-free medium; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline; LPS, lipopolysaccharide; sCAR-CD40L, soluble coxsackievirus-Ad receptor-CD40 ligand fusion protein; EBV, Epstein Barr virus; DF, dextran-FITC; MACS, magnetic activated cell sorting; CBA, Cytometric Bead Array kit; MFI, mean fluorescence intensity. ☆ None of the authors has any potential financial conflict of interest related to this manuscript. ☆☆ This work was supported by grant nr 90700309 from The Netherlands Organization for Health Research and Development (ZonMw) and grant VU 2010-4728 from the Dutch Cancer Society (KWF). ⁎ Corresponding authors. Fax: + 31 20 4444355. E-mail addresses: [email protected] (F.L. Schneiders), [email protected] (H.J. van der Vliet).

http://dx.doi.org/10.1016/j.clim.2015.03.007 1521-6616/© 2015 Elsevier Inc. All rights reserved.

Aminobisphosphonates inhibit α-GalCer-mediated iNKT activation by moDC

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apoE production by antigen presenting cells (APC) through NBP limits glycolipid-induced iNKT cell activation. This should be taken into account in the design of iNKT cell-based anti-cancer therapies. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Invariant natural killer T (iNKT) cells constitute an important immunoregulatory T cell subset that is restricted by the CD1d antigen(Ag)-presenting molecule [1–3]. The synthetic glycolipid α-galactosylceramide (α-GalCer) can be presented to iNKT cells in the context of CD1d, resulting in their activation and subsequent production of large amounts of cytokines. Thereby, iNKT cells play an important role in initiating and orchestrating long-lasting Th1-biased pro-inflammatory antitumor immune responses [4]. In early preclinical models, i.v. administration of α-GalCer was found to induce antitumor activity against various cancer types (e.g. melanoma, sarcoma, colon carcinoma, and lymphoma) in multiple models, including spontaneous tumor models [5–8]. Interestingly, quantitative and qualitative defects in the iNKT cell pool have been observed in many cancer types, including colorectal cancer, lung cancer, breast cancer, melanoma, HNSCC, prostate cancer, myelodysplastic syndromes, and myeloma (reviewed in [8]). Numerical defects of the iNKT cell population observed in cancer patients correlated with poor clinical outcome in e.g. HNSCC, colorectal cancer, and neuroblastoma [9–11]. Several clinical trials demonstrated the in vivo occurrence of iNKT cell activation using α-GalCer loaded moDC and showed that an initial decrease in iNKT cell numbers was frequently followed by an expansion of the iNKT cell pool [12–14]. Clinical responses have also been observed after intranasal administration of α-GalCer loaded APC in patients with HNSCC, suggesting that the activation of iNKT cells in the proximity of the tumor could be effective [15]. Cancer patients are frequently treated with aminobisphosphonates (NBP) to prevent osteoporosis, to treat hypercalcemia, and to reduce the number of skeletal events in case of bone metastases. Since NBP are well-known activating compounds for a pro-inflammatory lymphocyte subpopulation termed Vγ9Vδ2-T cells, and we have previously found that activated iNKT cells are able to enhance activation of Vγ9Vδ2-T cells via the production of TNF-α [16], we reasoned that it was also important to evaluate possible effects of NBP on the iNKT cell-CD1d axis as this drug may affect the critical role of iNKT cells in the regulation of antitumor immune responses in these patients. Unexpectedly, we found a remarkable reduction of α-GalCer induced iNKT cell activation when monocyte derived dendritic cells (moDC) were exposed to NBP during maturation. Also, we found that production of the transport-protein apolipoprotein E (apoE) was significantly reduced in moDC exposed to NBP. As the inhibitory effect of NBP on iNKT cell activation was alleviated by exogenous apoE, our data suggest that reduced

moDC apoE production in patients treated with NBP may limit glycolipid Ag-induced iNKT cell activation in these patients. This should be taken into account in the design of novel iNKT-cell based anti-cancer therapies.

2. Methods 2.1. Antibodies and flow-cytometry Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridinin chlorophyll protein (PerCP)- or allophycocyanin (AP)-labeled mAb directed against CD1a, CD3, CD14, CD25, CD40, CD80, CD86 (BD Biosciences, New Jersey, USA), CD83, TCR-Vα24, -Vβ11, -Vδ2 (Beckman Coulter, Inc.), -Vγ9, CD1d (clone 51.1; eBiosciences, Inc.), hSRA (human SRA) and hLDL-R (human LDL-R; R&D systems; Minneapolis, USA), were used for flow-cytometry analysis. mAb staining was performed in PBS supplemented with 0.1% BSA and 0.02% sodium-azide (NaN3) for 30 min at 4 °C. All stained cells were analyzed on FACS Calibur (BD Biosciences) using CellQuest software.

2.2. Generation of moDC and moDC experiments Immature moDC were generated from monocytes isolated from PBMC using anti-CD14 magnetic microbeads, according to the manufacturer's instructions (Miltenyi Biotec). CD14+ cells were cultured for 5–7 days in the presence of recombinant human (rh)IL-4 (10 ng/ml, R&D systems, Minneapolis, USA) and rhGM-CSF (100 U/ml, Genzyme, Bayer HealthCare Pharmaceutics, Seattle, Washington, USA) in IMDM (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (Hyclone, Amsterdam, The Netherlands), 100 I.E./ml sodium penicillin (Astellas Pharma, Leiden, the Netherlands), 100 μg/ml streptomycin sulfate (RadiumfarmaFisiopharma, Naples, Italy), 2.0 mM β-glutamine (Life Technologies, Bleiswijk, the Netherlands) and 0.05 mM 2-mercaptoethanol (Merck, Darmstadt, Germany), hereafter referred to as complete medium. Immature moDC were suspended in complete RPMI medium or in serum-free medium (SFM), consisting of RPMI supplemented with 100 U/ml sodium penicillin, 100 μg/ml streptomycin sulfate, 2.0 mM β-glutamine, 0.05 mM 2-mercapoethanol and 0.4% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, USA), and matured with a cytokine mix containing 50 ng/mL TNF-α (R&D Systems), 100 ng/mL IL-6 (R&D Systems), 25 ng/mL IL-1β (R&D Systems) and 1 μg/mL PGE2 (SigmaAldrich, St. Louis, Missouri, USA) during 24 h at 37 °C in a humidified atmosphere under 5% CO2. Mature moDC were

94 harvested by 5 mM ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (PBS, Braun Melsungen AG, Melsungen, Germany) and used for co-culture experiments, or irradiated (5000 Rad) and used for weekly stimulation of purified T-cells. For analysis of cell surface molecule expression immature moDC were cultured in RPMI medium and matured with lipopolysaccharide (LPS) or cytokine mix in the presence of 0 or 100 μM of the NBP Pamidronate (PCH, Pharmachemie BV, Haarlem, The Netherlands). MoDC were harvested after 24–48 hours of maturation and analyzed by flow cytometry for expression of CD14, CD1a, CD1d, CD40, CD80, CD83 and CD86. For evaluation of the production of cytokines by moDC, immature moDC were matured in SFM in the presence of 0, 50 or 100 μM NBP for 24 h. Supernatants of moDC were obtained after another 24 h incubation with 300 ng/ml of a soluble coxsackievirus-Ad receptor-CD40 ligand fusion protein (sCAR-CD40L, a kind gift of Dr. David Curiel, St Louis MO) and 1000 U/ml IFN-γ (BD Biosciences, New Jersey, USA). These supernatants were analyzed for inflammatory cytokines (e.g. IL-1β, IL-6, IL-8, IL-10, TNF and IL-12p70) by CBA using the Human Inflammatory Cytokines Kit, according to the manufacturer's guidelines (BD Biosciences, San Jose, USA). Sample data were obtained using flow-cytometry. In order to evaluate receptor mediated uptake of moDC matured in the presence or absence of NBP, mannose receptor–mediated endocytosis was measured by incubating mature moDC with 2 mg/ml FITC-dextran (DF; Sigma-Aldrich, St. Louis, USA) for 2 h at 37 °C before uptake was evaluated by flow-cytometry. In order to evaluate the effect of NBP on apoE production in moDC, immature moDC were matured with the cytokine maturation cocktail and supplemented with 0 or 100 μM NBP and Golgi Plug (BD Biosciences). For intracellular staining of apoE moDC were harvested after 18 h of maturation, fixed and permeabilized using the BD Fix-Perm kit (BD Biosciences) following manufacturer's guidelines. Intracellular staining was performed using an unlabelled mouse antibody recognizing human apoE (anti-human/monkey apoE mAb E981, Mabtech, Nacka Strand, Sweden) or an appropriate isotype control for 30 min at 4 °C in permeabilization buffer. After washing, cells were stained with the secondary AP-labeled goat Fab' anti-mouse IgG antibody (Beckman Coulter), for 30 min at 4 °C in permeabilization buffer. Cells were washed with permeabilization buffer and with FACS buffer before analysis. All stained cells were analyzed on FACS Calibur (BD Biosciences) using CellQuest software.

2.3. Generation of Vγ9Vδ2-T cell lines Vγ9Vδ2-T cell lines were generated from human PBMC by magnetic activated cell sorting (MACS) using either the murine anti-human Vδ2 TCR or anti-human Vγ9 TCR mAb, combined with a polyclonal goat–anti-mouse Ab or anti-PE Ab labeled with magnetic beads (Miltenyi Biotec). For culture/expansion of Vγ9Vδ2-T cells, 100 μM NBP was added to immature moDC during the last 2 h of maturation and co-cultured with Vγ9Vδ2-T cells with rhIL-2 (50 U/ml, BioVision, Mountain View, California, USA). Purified Vγ9Vδ2-T cells were re-

F.L. Schneiders et al. stimulated weekly with irradiated feeder-mix consisting of allogeneic PBMC and JY Epstein Barr virus (EBV)-transformed B cells in Yssel's medium supplemented with 50 ng/ml PHA (Murex Biotech, Dartford, U.K.) and 50 U/ml IL-2. Purity of Vγ9Vδ2-T cells used for experiments was N 90%.

2.4. Generation of iNKT cell lines For iNKT (defined as Vα24+Vβ11+) cell expansion, iNKT cells were isolated from PBMC through MACS-isolation using the 6B11 mAb (kind gift of Mark Exley, BIDMC, Harvard Medical School, Boston), or the murine anti-human TCR Vα24 mAb, combined with a polyclonal goat–anti-mouse Ab labeled with magnetic beads (Miltenyi Biotec). iNKT cells were expanded by co-culturing iNKT cells with moDC, pulsed with α-GalCer (100 ng/ml, Funakoshi Co, Tokyo, Japan) during maturation, and rhIL-2 (50 U/ml). Purified iNKT cells were re-stimulated weekly with irradiated feeder-mix consisting of allogeneic PBMC and JY EBVtransformed B cells in Yssel's medium supplemented with 50 ng/ml PHA and 50 U/ml IL-2. Purity of iNKT cells used for experiments was N 90%.

2.5. iNKT cell activation experiments After harvesting with 5 mM EDTA, immature moDC were cultured in complete medium or in SFM, for indicated time periods in the presence of 0, 50 or 100 μM NBP. The moDC were loaded with 50 ng/ml α-GalCer or an equal volume of vehicle control and matured with the cytokine mix for 24 h at 37 °C. After maturation, the moDC were washed with PBS and co-cultured (ratio 1:1 or 1:2) with resting iNKT cells (defined as ≥ 6 days post re-stimulation) in RPMI medium or SFM for 24 h at 37 °C. iNKT cell activation was determined by assessing CD25 expression on iNKT cells by flow-cytometry. After 24 h supernatants were harvested and analyzed using the Th1/Th2/Th17 BD™ Cytometric Bead Array kit (CBA; BD Biosciences) for the simultaneous flow-cytometric detection of IL-2, IL-4, IL-6, IL-10, TNF, and IFN-γ, following the manufacturer's instructions and with the use of CBA analysis software (BD Biosciences). In order to evaluate the role of moDC expression of the co-stimulatory molecules CD80 and CD86 on iNKT cell activation we blocked them by means of a CD80/CD86 binding CTLA4-Ig fusion protein (a kind gift from Dr Lieping Chen, New Haven, CT). Here, mature NBP+ and NBP− moDC were pre-incubated with control-Ig or CTLA4-Ig fusion proteins (10 μg/ml) prior to the co-culture with iNKT cells. iNKT cell activation was determined by assessing CD25 expression using flow-cytometry. In experiments in which the role of exogenously added apoE was studied, immature moDC were cultured in SFM in the presence of 0 or 100 μM NBP. After 90 minutes incubation at 37 °C, moDC were pulsed with 50 ng/ml α-GalCer or an equal amount of vehicle control in the presence of 0, 1, 2.5 or 5 μg/ml human apoE (human plasma, very low density lipoprotein, Calbiochem) and matured with the cytokine cocktail for 24 h at 37 °C. After maturation, the moDC were washed with PBS and co-cultured with resting iNKT cells in SFM for 24 h at 37 °C. iNKT cell activation was determined by

Aminobisphosphonates inhibit α-GalCer-mediated iNKT activation by moDC assessing CD25 expression on iNKT cells; culture supernatant was harvested to evaluate cytokine production by iNKT cells.

2.6. Statistical analysis All data were analyzed using paired Student t-tests, 1-way ANOVA or Mann Whitney U tests, as appropriate. Findings were considered statistically significant when p-values were ≤ 0.05, as indicated with asterisks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Statistical analyses were performed using GraphPad Prism software (version 5.02, 2008).

3. Results 3.1. Aminobisphosphonates impair α-GalCer-induced iNKT cell activation mediated by moDC Vγ9Vδ2-T cells can be indirectly activated by NBP through inhibition of the mevalonate metabolism and the subsequent intracellular accumulation of isopentenyl pyrophosphate (IPP) in APC. We previously showed the ability of iNKT cells to contribute to this activation process through the release of TNF-α [16]. Here, we set out to assess the effects of NBP on moDC-induced and α-GalCer-mediated activation of iNKT cells through putative cross-talk with Vγ9Vδ2-T cells. To this end immature moDC were pulsed with α-GalCer during maturation and simultaneously exposed to NBP throughout maturation induction or for the final 2 h of cytokine mediated maturation induction (i.e. post-maturation), after which they were co-cultured with both Vγ9Vδ2-T and iNKT cells. As expected, NBP treatment during maturation of moDC resulted in Vγ9Vδ2-T cell activation in a concentration-dependent fashion (0 μM NBP vs 10 μM NBP and 200 μM NBP: relative increase in CD25 expression 3.7 ± 1.0 and 7.6 ± 2.6 fold, mean ± SEM,

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n = 2; data not shown). However, no increase in iNKT cell activation was observed after co-culture with Vγ9Vδ2-T cells and α-GalCer-pulsed and NBP-treated moDC. In fact, a striking inhibition of iNKT cell activation was observed when NBP was administered to moDC during maturation (0 μM NBP vs 200 μM NBP: 70% ± 10% reduction in CD25 expression, mean ± SEM, n = 2; data not shown), whereas iNKT cell activation remained unaffected when NBP was administered post-maturation (data not shown). Moreover, we found that the presence or the absence of Vγ9Vδ2-T cells did not affect the NBP-induced inhibitory effect on iNKT cell activation, indicating that the inhibitory effect of NBP administration on iNKT cell activation was not due to e.g. lysis of phospho-Ag expressing moDC by Vγ9Vδ2-T cells. Administration of NBP during maturation of moDC induced a dose-dependent decrease in glycolipidinduced iNKT cell activation (Fig. 1A). Furthermore, α-GalCerpulsed and NBP-treated moDC inhibited iNKT cells for the production of both Th1 or Th2 cytokines (mean relative cytokine production in the presence of 100 μM NBP compared to 0 μM NBP ± SEM: IL-2 0.4 ± 0.1 p = 0.01, IL-4 0.4 ± 0.1 p = 0.01; IL-10 0.3 ± 0.1 p = 0.01; and IFN-γ 0.3 ± 0.1 p = 0.01; (Fig. 1B; n = 4)). As this inhibitory effect of NBP might interfere with the efficacy of iNKT cell-based therapies, we next studied the possible underlying mechanisms.

3.2. Aminobisphosphonate-mediated inhibition of iNKT cell antigen-dependent activation is not caused by differences in co-stimulation, CD1d expression, soluble- and receptor-mediated endocytosis, or cytokine production by moDC In order to determine how NBP negatively affect iNKT cell antigen-dependent activation, we compared the phenotype and functions of moDC matured in the presence or absence

Figure 1 Aminobisphosphonates interfere with the activation and cytokine production of glycolipid-reactive iNKT cells. A) Immature moDC were matured in SFM with 0 or 50 ng/ml α-GalCer and with different concentrations of NBP before co-culturing with resting iNKT cells for 24 h. After co-culture MFI of CD25 expression on iNKT cells was assessed by flow-cytometry. Shown are means of CD25 expression (MFI) ± SEM (vehicle vs 0 μM NBP 64.0 ± 11.4 vs 204.6 ± 36.1 p b 0.001; 0 μM NBP vs 100 μM NBP 204.6 ± 36.1 vs 118.4 ±30.0 p b 0.01; 0 μM NBP vs 200 μM NBP 204.6 ± 36.1 vs 75.9 ± 17.9 p b 0.001; n = 7). B) Immature moDC were matured in SFM in the presence of 50 ng/ml α-GalCer and 0 or 100 μM NBP before co-culturing with resting iNKT cells. After a 24 h co-culture supernatants were harvested and analyzed using a Th1/Th2/Th17 CBA kit. Shown is the relative cytokine production of IL-2, IL-4, IL-10 and IFN-γ. White bars represent iNKT cells co-cultured with α-GalCer pulsed mature moDC. Black bars represent iNKT cells co-cultured with α-GalCer pulsed moDC matured in the presence of 100 μM NBP. The mean values of the cytokine production are listed (in pg/ml) for the co-culture of iNKT cells with α-GalCer pulsed mature moDC. Mean relative cytokine production in the presence of 100 μM NBP compared to 0 μM NBP ± SEM is shown (IL-2 0.4 ± 0.1 p = 0.01, IL-4 0.4 ± 0.1 p = 0.01; IL-10 0.3 ± 0.1 p = 0.01; and IFN-γ 0.3 ± 0.1 p = 0.01; n = 4). Four separate experiments were performed using iNKT cell lines from three different donors.

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Figure 2 Effect of NBP on the expression of CD1d, the maturation marker CD83 and co-stimulatory markers on moDC. A) MoDC were matured in SFM in the presence of 0 (white bars) or 100 μM (black bars) NBP for 24–48 h before marker expression was assessed by flow-cytometry. Shown are means of marker expression (MFI) ± SEM (CD80 0 μM NBP vs 100 μM NBP 60.2 ± 9.6 vs 41.8 ± 7.7, p = 0.027; n = 10). B) Immature moDC were matured in SFM in the presence of 0, 50 or 100 μM NBP and 0 or 50 ng/ml α-GalCer for 24 h and subsequently incubated with control-Ig (black bars) or CTLA4-Ig fusion protein (10 μg/ml; white bars) prior to the onset of iNKT cell co-culture. After 24 h co-culture CD25 expression on iNKT cells was assessed by flow-cytometry. Means of CD25 expression ± SEM of relative data are shown. Also the mean values of MFI of CD25 expression on iNKT cells are listed (50 μM NBP vs 100 μM NBP 417.0 ± 82.3 vs 141.3 ± 22.9, p = 0.05; 100 μM NBP/CTLA4-Ig vs 100 μM NBP/control Ig 141.3 ± 22.9 vs 314.1 ± 24.1, p b 0.05, n = 3).

of NBP. As shown in Fig. 2A, NBP treatment of moDC during maturation did not significantly alter cell surface expression of CD1d, the maturation marker CD83 or co-stimulatory markers, with the exception of the expression of CD80, which was moderately but significantly less expressed on the surface of moDC treated with 100 μM NBP (mean MFI CD80 ± SEM: 60.2 ± 9.6 vs 41.8 ± 7.7, in the absence or presence of 100 μM NBP respectively, p = 0.027; n = 10). In order to determine whether the reduced CD80 expression on moDC might be responsible for the observed inhibition of iNKT cell activation, CD80 and CD86 were blocked by means of CTLA-4-Ig fusion protein prior to the co-culture of α-GalCer pulsed moDC with iNKT cells. Blocking of the CD80/CD86 co-stimulatory pathway did not significantly reduce the antigen dependent activation of iNKT cells in the absence of NBP, implying that the reduced expression of CD80 on moDC was not responsible for the striking decrease in iNKT cell activation observed in the presence of NBP (Fig. 2B). Interestingly however, blocking of CD80/CD86 did further impair the already diminished iNKT cell activation caused by 100 μM NBP treatment of moDC, revealing that in this setting CD80/CD86 co-stimulation does contribute some relevant stimulatory signals to iNKT cells, likely in a bid to compensate for the detrimental effects on iNKT cell activation of NBP. Since moDC have the ability to manipulate their microenvironment by producing a wide array of cytokines and iNKT cells have been shown to be highly responsive to the cytokines produced upon their interaction with moDC [17], we compared CD40L-induced moDC cytokine production (i.e. IL-1β, IL-6, IL-8, IL-10, TNF, IL-12) when moDC were matured in the presence or absence of NBP. As shown in Fig. 3, following a 24 h moDC maturation in SFM in the presence of 0 or 100 μM NBP, no differences were observed in the production of cytokines by maturing moDC. Next, we evaluated whether NBP treatment of moDC would affect their ability for receptor-mediated uptake. For

this purpose, mannose receptor-mediated uptake of dextran FITC by moDC and cell-surface expression of scavenger receptor A (SRA) and LDL-receptor (LDL-R) were assessed on moDC, both of which have been implicated in the uptake of glycolipids by APC [18,19]. As indicated in Fig. 4A, maturation of moDC, when performed either in the presence or the absence of NBP, resulted in comparable dextran-FITC uptake. Likewise, the expression of either SRA or LDL-R was not affected by NBP treatment (Fig. 4B).

Figure 3 No significant differences in the production of cytokines by moDC upon treatment with NBP. Immature moDC were matured in SFM in the presence of 0 or 100 μM NBP for 24 h before moDC were cultured another 24 h in the presence of sCAR-CD40 fusion protein and IFN-γ in order to stimulate cytokine release. Subsequently supernatants were harvested and analyzed for the production of inflammatory cytokines (i.e. IL-1β, IL-6, IL-8, IL-10, TNF and IL-12p70) by CBA using the Human Inflammatory Cytokines Kit. Bars represent moDC matured with 0 μM NBP (white bars) or 100 μM NBP (black bars). Shown are means of cytokine production (pg/ml) ± SEM (n = 4).

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Figure 4 No significant differences in receptor-mediated endocytosis and the expression of SRA and LDL-R by moDC upon treatment with NBP. A) MoDC were matured in SFM in the presence of 0 or 100 μM NBP for 24 h. Dextran FITC was administrated during the last two hours of maturation after which dextran FITC positivity of moDC was assessed by flow-cytometry. Shown are means of dextran FITC positive moDC (MFI) ± SEM (0 μM vs 100 μM NBP 553.2 ± 105.5 vs 474.4 ± 62.3, p = 0.15, n = 7). B) MoDC were matured in SFM in the presence of 0 (white bars) or 100 μM (black bars) NBP for 24 h before marker expression of SRA and LDL-R was assessed by flow-cytometry. Shown are means of marker expression (%) ± SEM (LDL-R 0 μM vs 100 μM NBP 90.5 ± 2.9 vs 89.6 ± 4.1, p = 0.82; SRA 0 μM vs 100 μM NBP 18.7 ± 9.0 vs 23.1 ± 11.6, p = 0.24, n = 3).

Taken together, our results do not support the involvement of an NBP-induced decrease of co-stimulatory marker expression, cytokine production, receptor-mediated uptake or SRA and LDL-R expression by moDC that could account for the reduced antigen-mediated activation of iNKT cells.

3.3. Aminobisphosphonate treatment impairs apolipoprotein E production by moDC and the subsequent α-GalCer-mediated iNKT cell activation We next set out to determine whether inhibition of glycolipidmediated iNKT cell activation by NBP could be caused by differences in the ability of moDC to scavenge glycolipids for processing. It has been reported that apoE secretion by moDC is involved in facilitating the transport and subsequent SRAand LDL-R-mediated uptake of α-GalCer by moDC [18]. Of note, mevalonate pathway inhibition can reduce cellular apoE secretion by inhibiting protein prenylation, as was shown in brain glia cells [20]. In addition, transcriptional regulation of apoE in humans is, among other factors, mediated by the concentration of intracellular free cholesterol [21], and it is therefore very well conceivable that NBP, through inhibition of the cholesterol generating mevalonate pathway, can negatively affect moDC apoE production resulting in a subsequent impairment in α-GalCer uptake by moDC. By means of ELISA, we could not detect statistically significant differences in apoE levels in the culture supernatants of moDC matured in the presence or absence of NBP (data not shown), possibly due to swift and dynamic binding of secreted apoE to both serum constituents and receptors present on moDC in our culture system. For this reason, we evaluated the impact of NBP treatment on apoE production by moDC using intracellular flow-cytometry. As shown in Fig. 5A, NBP-treated moDC produce significantly less apoE when compared to untreated moDC (mean percentages ± SD 35.1 ± 3.4 vs 15.3 ± 2.6, relative percentages ± SD 1 ± 0 vs 0.5 ± 0.2;

in the absence or presence of 100 μM NBP respectively, p b 0.05, n = 3; representative histogram is shown). Based on this observation, we evaluated whether reduced iNKT cell activation caused by NBP treatment of maturing moDC could be reversed by addition of exogenous apoE. As depicted in Fig. 5B, iNKT cell activation was completely restored (in a dose dependent manner) when apoE was administered during moDC maturation in the presence of NBP. Furthermore, inhibition of cytokine production by iNKT cells was also alleviated when NBP-treated moDC were matured in the presence of exogenous apoE (mean relative cytokine production in the presence of 100 μM NBP and in the presence of 100 μM NBP and 5 μg/ml apoE respectively compared to 0 μM NBP (set at 1) ± SEM: IL-2 0.82 ± 0.24 and 0.77 ± 0.03, IL-4 0.45 ± 0.01 and 1.22 ± 0.30; IL-6 0.46 ± 0.04 and 0.82 ± 0.13; IL-10 0.46 ± 0.00 and 1.05 ± 0.06; TNF 0.43 ± 0.04 and 0.66 ± 0.07; IFN-γ 0.50 ± 0.13 and 1.02 ± 0.08 (Fig. 5C; n = 2)). In order to correct for differences in the cytokine producing capacity between iNKT cell lines from different donors and between iNKT cell lines from the same donor used at different time points, the observed differences in iNKT cell cytokine production are presented relative to the control condition in each experiment where iNKT cells were activated by α-GalCer loaded moDC matured in the absence of NBP. The absolute values of iNKT cell cytokine production in the different conditions are shown in the Supplementary Table S1.

4. Discussion The results presented in this study demonstrate for the first time a dose-dependent inhibitory effect of NBP on the capacity of moDC to activate iNKT cells in response to α-GalCer exposure. This inhibition of iNKT cell activation was evidenced by a reduction of CD25 cell surface expression levels, as well as a significant inhibition of the production of both Th1- and Th2-type cytokines.

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Figure 5 Exogenous ApoE administration restores NBP-induced defective iNKT cell activation by α-GalCer pulsed moDC. A) Addition of NBP (100 μM) during maturation of moDC in SFM results in a mean decrease (mean decrease in percentages ± SD 54.4 ± 19.9) in intracellular apoE (mean percentages ± SD 35.1 ± 5.9 vs 15.3 ± 4.4, relative percentages ± SD 1 ± 0 vs 0.5 ± 0.2, in the absence or presence of 100 μM NBP respectively, p b 0.05, n = 3). The gray histogram represents staining with isotype control, the thin line represents apoE staining in mature moDC cultured without NBP and the bold line represents apoE staining in mature moDC cultured with 100 μM NBP. A representative histogram of one out of three independent experiments is shown. B) Administration of apoE during maturation of moDC in the presence of NBP leads to a concentration dependent alleviation of inhibition of glycolipid-induced iNKT cell activation. CD25 expression by iNKT cells (mean % ± SEM) was reduced in the presence of NBP (0 μM NBP vs 100 μM NBP 46.3 ± 10.3 vs 26.7 ± 5.4; p b 0.01 n = 5). Alleviation of the reduced iNKT cell activation was seen when apoE was added (100 μM NBP vs 100 μM NBP + 5 μg/ml apoE 26.7 ± 5.4 vs 44.3 ± 8.5; p b 0.05, n = 5). C) Administration of apoE during maturation of NBP+ moDC leads to an alleviation of impaired glycolipid-induced iNKT cell cytokine production. Shown is the relative cytokine production of IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ. iNKT cells were co-cultured with α-GalCer pulsed moDC, matured in the absence (white bars) or presence of 100 μM NBP (black bars), or in the presence of 100 μM NBP and 5 μg/ml apoE (gray bars). The mean values of the cytokine production are listed (in pg/ml) for the co-culture of iNKT cells with α-GalCer pulsed mature moDC. Mean relative cytokine production in the presence of 100 μM NBP and in the presence of 100 μM NBP and 5 μg/ml apoE respectively as compared to 0 μM NBP (set at 1) ± SEM is shown (IL-2 0.82 ± 0.24 and 0.77 ± 0.03, IL-4 0.45 ± 0.01 and 1.22 ± 0.30; IL-6 0.46 ± 0.04 and 0.82 ± 0.13; IL-10 0.46 ± 0.00 and 1.05 ± 0.06; TNF 0.43 ± 0.04 and 0.66 ± 0.07; IFN-γ 0.50 ± 0.13 and 1.02 ± 0.08; n = 2).

As the inhibition of iNKT cell activation induced by NBP could result from an impairment of several moDC functions, these were further assessed. We show that NBP treatment of moDC does not significantly affect the surface expression of CD1d, CD83 and CD40, but does influence CD80 expression, which was moderately, but significantly, reduced. However, functional blockade of CD80 on untreated moDC could not mimic the NBP-mediated inhibition of α-GalCer-induced iNKT cell activation. Interestingly, additional blocking of CD80 by a CTLA-Ig fusion protein on NBP-treated moDC, led to a significant increased reduction of iNKT cell activation levels, thus suggesting the importance of co-stimulatory signals when activation by the primary activating ligand is repressed. Moreover, other processes involved in Ag presentation and iNKT cell activation were not affected, e.g. cytokine production and the expression of SRA, LDL-R and mannose receptor. In line with these data, a previous study reported comparable viability, phenotype, and function (in terms of cytokine production and endocytic potential) of moDC that matured in the presence or absence of the NBP zoledronic acid [22].

As previously reported, apoE is an important molecule involved in promoting the capture and the presentation of glycolipids to iNKT-cells [18,23]. We analyzed the effect of NBP on the production of apoE by moDC and we showed a significant inhibition of apoE expression in moDC treated with NBP during maturation. These data are in line with previous reports showing reduced apoE mRNA expression in human PBMC [21], and a decreased secretion of apoE by brain glia cells [20], both under the influence of statins, another upstream mevalonate pathway inhibitor. In our model exogenous replenishing of apoE restores iNKT cell activation and cytokine production, which thus confirms that inhibition of apoE production induced by NBP treatment is critically involved in the observed inhibition of glycolipid-induced iNKT cell activation. Replenishing mevalonate pathway intermediates like geranylgeranyl-pyrophosphate, farnesyl-pyrophosphate or squalene did not result in consistent recovery of iNKT cell activation (data not shown), as was the case in a report describing the inhibition of glycolipid-induced iNKT cell activation by statins in mice [24], suggesting that other inhibitory mechanisms might be involved when different

Aminobisphosphonates inhibit α-GalCer-mediated iNKT activation by moDC parts of the mevalonate pathway are repressed and/or other cell types are involved. Of note, we found that exposure of moDC to both statins and NBP resulted in a further reduction of iNKT cell activation (data not shown). Cancer patients are frequently treated with aminobisphosphonates (NBP) to prevent osteoporosis, to treat hypercalcemia, and to reduce the number of skeletal events in case of bone metastases. iNKT cells have been demonstrated to play a critical role in the regulation of antitumor immune responses, and various strategies are being developed to exploit iNKT cell based immunotherapies. Preclinical data showed that i.v. administration of α-GalCer-pulsed moDC resulted in greater antitumor responses compared to i.v. α-GalCer alone. Therefore, this approach, with variations in the APC platform, was subsequently evaluated in clinical trials, and was found to be safe and capable of inducing clinical anti-tumor responses in several patients [13–15,25]. Our data suggest that treatment of patients with NBP could hamper the efficacy of in vivo or ex vivo moDC treatment with α-GalCer by impairing apoE production. Therefore, the effects of NBP on iNKT cells are relevant to consider in the design of future iNKT-cell based anticancer therapies. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clim.2015.03.007.

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Acknowledgments [16]

We thank Sinéad Lougheed, MSc and Dafni Chondronasiou, MSc for technical assistance. [17]

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