IL-6 Produced by Type I IFN DC Controls IFN-γ Production by Regulating the Suppressive Effect of CD4+ CD25+ Regulatory T Cells

IL-6 Produced by Type I IFN DC Controls IFN-␥ Production by Regulating the Suppressive Effect of CD4ⴙ CD25ⴙ Regulatory T Cells Olivier Detournay, Naima Mazouz, Michel Goldman, and Michel Toungouz ABSTRACT: The dendritic cell family is composed of different subsets differentially governing the immune response. Type I interferon (IFN) dendritic cells (DC) are endowed with the ability to trigger both Th1 and Th2 type responses. In view of the pivotal role of regulatory T cells in limiting the effectiveness of effector cells, we analyzed the interactions between these cells and type I IFN DC. DC were generated from monocytes in the presence of IFN-␤ and interleukin (IL)-3 (DCI3) or granulocyte macrophage– colony-stimulating factor and IL-4 (DCG4) and activated by poly(I: C). Despite the release of lower amounts of IL-12 after maturation, DCI3 were able to induce a higher IFN-␥ production by T lymphocytes during the mixed leucocyte reaction (MLR) as compared with DCG4. mRNA analysis disclosed that DCI3 overtranscribed the IL-6 ABBREVIATIONS DC dendritic cells DCG4 dendritic cells generated in the presence of GM-CSF and IL-4 DCI3 dendritic cells generated in the presence of IL-3 and IFN-␤ MLR mixed leucocyte reaction

INTRODUCTION Dendritic cells (DC) are professional antigen-presenting cells (APC), endowed with the unique capacity to trigger From the Department of Immunology-Hematology-Transfusion, Erasme Hospital, Brussels, Belgium (O.D., N.M., M.G., M.T.). O.D. is a fellow of the “Fonds National de la Recherche Scientifique-FNRS” (Grant Télévie). Address reprint requests to: Michel Toungouz MD, PhD, Department of Immunology-Hematology-Transfusion, Erasme Hospital, 808 Route de Lennik, B-1070 Brussels, Belgium; Tel: ⫹32 2 555 38 62; Fax: ⫹32 2 555 44 99; E-mail: [email protected]. Olivier Detournay and Naima Mazouz contributed equally to this paper. Supported by the HPVAC program of the “Région Wallonne” and BruCells SA/NV. Received October 26, 2004; revised December 24, 2004; accepted January 7, 2005. Human Immunology 66, 460 – 468 (2005) © American Society for Histocompatibility and Immunogenetics, 2005 Published by Elsevier Inc.

gene and secreted high amounts of the protein. Neutralization of IL-6 revealed that this cytokine specifically contributed to the IFN-␥ release induced by DCI3. Finally, depletion of CD25⫹ T cells before the MLR identified these cells as a target for IL-6. We conclude that DCI3 are endowed with the property of regulating the suppressive effect of regulatory T cells through high IL-6 production. This novel mechanism of T cell control is relevant for the use of DCI3 in vaccination strategies. Human Immunology 66, 460 – 468 (2005). © American Society for Histocompatibility and Immunogenetics, 2005. Published by Elsevier Inc. KEYWORDS: Dendritic cells; IFN-␥; IL-6; regulatory T cells; type I IFN

PDC Th1 Th2 TLR Treg

plasmacytoid dendritic cells T helper 1 T helper 2 Toll-like receptors regulatory T cells

naive T cells. This property has lead to their use in many clinical trials mainly targeting cancer (reviewed by O’Neill et al. [1]). The effectiveness of such immunointervention remains limited. Crucial parameters to consider to optimize the immunogenicity of DC are the nature of the DC subset and its interaction with other immune cells. DC are diverse belonging to different subsets that are phenotypically and functionally different. DC precursors are present at low levels in human peripheral blood. They comprise myeloid and lymphoid derived cells. On appropriate stimulation, immature myeloid DC mature 0198-8859/05/$–see front matter doi:10.1016/j.humimm.2005.01.012

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into fully competent DC, produce large amounts of interleukin (IL)-12, and preferentially induce T-helper 1 cell (Th1) differentiation. Myeloid DC can be differentiated in vitro from monocytes by a 7-day culture in the presence of granulocyte macrophage– colony-stimulating factor (GM-CSF) and IL-4 (DCG4) [2]. Lymphoid DC precursors differentiate into plasmacytoid DC (PDC) that reveal a plasma cell-like morphology and lack myeloid markers, but highly express CD123 (IL-3 receptor). Immature PDC activated by IL-3 and CD40-ligand preferentially promote Th2 differentiation [3], and, in some instances, Th1 responses [4]. After maturation on recognizing viral components, via Toll-like receptors (TLR), they release high amounts of IFN-␣ [5]. More recently, an additional DC subset resulting from monocytes differentiation in the presence of type I IFN has been described. These “type I IFN DC” include DC differentiated from monocytes in the presence of type I IFN combined either with IL-3 [6] or GM-CSF [7]. Belardelli et al. showed that GM-CSF/IFN-␤ DC (DCG␤) are endowed with potent functional activities, superior with respect to the IL-4/GM-CSF treatment, notably through IL-15 production strongly promoting Th1 responses [7]. Buelens et al. showed that these cells have a mature phenotype. In vitro, these IL-3/IFN-␤ (DCI3) DC induce strong allogeneic responses characterized by both Th1 (IFN-␥) and Th2 (IL-5) type cytokine production. The amounts of IFN-␥ released during the mixed lymphocyte reaction (MLR) is very high despite a limited capacity of DCI3 to release IL-12 [6]. Cytokine released by DC are crucial for the interaction with other immune cells (i.e., regulatory T [Treg] cells). These cells have been recently shown to be partially controlled by IL-6 derived from DCG4 [8]. In the present work, we investigated the response of type I IFN DC to maturation in terms of cytokines production both at the mRNA and protein level. We next analyzed the mechanism of T-cell activation during MLR involving this DC subset. Finally, we studied the consequences of type I IFN DC activation on the interaction with Treg cells. Taking into account the recent data showing that DCI3 and DCG␤ show similar phenotypic and functional properties (Mazouz et al., in press), all experiments were performed using DCI3. MATERIALS AND METHODS DC Generation and Characterization Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood of healthy donors by centrifugation over Lymphoprep density gradient (Nycomed, Oslo, Norway). Monocytes were obtained by a 2-hour adhesion in 75-cm2 flasks and cultured during 5 days in RPMI 1640 (Cambrex, Verviers, Belgium) supple-

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mented with 2 mM L-glutamine (Gibco, Paisley, Scotland), 50 ␮M 2-mercaptoethanol (Gibco), 1% nonessential amino acids (Gibco), and 10% fetal bovine serum (BioWhittaker Europe) in presence of either GM-CSF (800 UI/ml) (Leukomax, Novartis, Switzerland) and IL-4 (200 UI/ml) (Cell Genix, Germany) (DCG4) or IL-3 (50 UI/ml) (R & D Systems Europe, UK) and IFN-␤ (100UI/ ml) (Avonex, Biogène, France) (DCI3). Nonadherent cells were cryopreserved for subsequent functional assays. DC maturation was achieved initially by stimulation with polyinosinic-polycytidylic acid (poly [I:C]) at 10 ␮g/ml (Sigma, Belgium) for 0, 6, 9, 12, and 18 h and culture supernatants were collected for assessment of cytokine levels. In further experiments, poly (I:C) stimulation was performed for 18 h. For immunophenotyping, cells were washed in phosphate-buffered saline supplemented with 0.5% bovine serum albumin (Sigma, Bornem, Belgium) and stained with the following monoclonal antibodies: anti-CD80, anti-CD86 (BD Biosciences, Mountain View, CA), and anti-CD83 (Immunotech, Marseille, France). A total of 2 ⫻ 105 cells were incubated with the relevant mAbs for 20 minutes at 4°C. After washing, acquisition of fluorescence intensity was performed using a FACS-Calibur (Becton-Dickinson, San Jose, CA) running under the Cell Quest software. Purification of CD4ⴙ T cells CD4⫹ T cells were purified by magnetic cell sorting using a commercially available CD4⫹ T-cell isolation kit (MACS, Miltenyi Biotec, Germany). Purity of the resulting CD4⫹ T-cell population was always ⬎90%. In some experiments, CD4⫹ T cells were further depleted of CD25⫹ cells by negative selection using magnetic cell sorting (MACS, Miltenyi Biotec, Germany). The remaining CD25⫹ T cells in the depleted population was always ⬍5%. Mixed Lymphocyte Reaction A total of 2 ⫻ 104 mature DCI3 or DCG4 were cocultured in 96-well round-bottom plates (NunclonTMSurface, Nunc, Denmark) with 2 ⫻ 105 allogeneic total CD4⫹ T cells (CD4⫹ CD25⫹/⫺) or CD4⫹ T cells depleted of CD25⫹ T cells (CD4⫹ CD25⫺ T cells). All MLR experiments involved different responder/stimulator combinations. To assess the role of specific cytokines, blocking experiments were performed. Anti–IFN-␣ (PBL Biomedical Laboratories, New Brunswick, NJ) or anti–IL-6 (R&&D Systems, UK) antibodies were added at 5 ␮g/ml to neutralize the respective cytokines. An irrelevant matched Ab was used at the same concentration as control: rabbit polyclonal IgG for IFN-␣ neutralizing experiments (BD Biosciences Pharmingen, San Diego, CA); murine monoclonal IgG1 for IL-6 neutralizing experiments (R&D Systems, UK). After 6 days of

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culture, supernatants were harvested for determination of cytokine levels. The percentage of inhibition was calculated according to the following formula: %inhibition of IFN ⫺ ␥ release ⫽ 关1⫺共 IFN ⫺ ␥ concentration in the presence of neutralizing Ab/IFN ⫺ ␥ concentration in the presence of isotype control兲兴 ⫻ 100 Determination of Cytokine Levels Enzyme-linked immunosorbent assay kits were purchased from Biosource Europe (Fleurus, Belgium) for the determination of IFN-␥, IFN-␣, IL-12p40, and IL-6 levels and from Endogen (Woburn, MA) for IL-12p70. In some experiments, the Luminex device (Biosource, Fleurus, Belgium) was used to quantify IL-6 and IL12p40 production. DC Gene Profiling Total RNA of matured DC (5 ⫻ 106) was extracted using a commercially available kit (RNeasy, Qiagen Benelux, Venlo, The Netherlands). The RNA concentration was determined by absorbance reading. The integrity of the RNA preparation was checked by ethidium bromide-stained agarose gel electrophoresis. After reverse transcription of 1 ␮g of total cellular RNA, cDNA probes were amplified by linear polymerase reaction (GEArray Ampolabeling-SuperArray Bioscience Corp. Frederick, Maryland) with incorporation of biotin-16-dUTP (Roche, Brussels, Belgium). Afterwards, cDNA probes were hybridized to a human “Dendritic & Antigen Presenting Cell Gene Array” (SuperArray Bioscience Corp.). This membrane contains 192 sequence-verified known marker genes. After hybridization, each gene signal was normalized against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene signal on the same membrane. Only gene signals above background (⬎10% of GAPDH signal) were considered as specific gene signals. These data were collected, stored and analyzed with the Scanalyse software (Stanford University, Stanford, CA). Statistical Analysis Statistical significance was determined using the twotailed paired Wilcoxon test. A p value less than 0.05 was considered as statistically significant. RESULTS Influence of Maturation on Immunophenotype and Cytokine Production The influence of the maturation process on the immunophenotype and cytokine release by both DC types was assessed by using the viral analog poly (I:C) as stimulating agent. Poly (I:C) was chosen because of its ability to efficiently trigger the maturation process on both DCG4

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[9, 10] and DCI3 (Mazouz et al., in press). In that setting, both DCI3 and DCG4 upregulated the maturation markers CD80, CD83, and CD86 (Figure 1a). Both DC types released IL-12p40 in a time-dependent manner (Figure 1b). The amounts of IL-12p40 secreted by DCG4 were significantly higher than those secreted by DCI3, and IL-12p70 was only produced by mature DCG4 (Fig-

FIGURE 1 Influence of DCI3 and DCG4 maturation on surface marker expression and interleukin (IL)-12 production. DCI3 and DCG4 were matured in the presence of poly (I:C) or medium alone. (a) After 18 h stimulation, immunophenotyping was performed for the indicated cell surface molecules (CD80, CD83, CD86). Results are those from one representative experiment of four. (b) IL-12p40 and IL-12p70 productions were assessed by enzyme-linked immunosorbent assay in culture supernatants harvested after 0, 6, 9, 12, or 18 stimulation. Results are those from one representative experiment of four. (c) In the next experiments, IL-12 secretion was only assessed at 18 h. Results of six additional independent experiments are represented as mean ⫾ standard error of the mean (*p ⬍ 0.05).

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ure 1b, c). We also tested the possible secretion of IFN-␥ by DC as previously described [11, 12]. This secretion by both type I IFN DC was below the level of sensitivity of the enzyme-linked immunosorbent assay (⬍10 pg/ml, n ⫽ 10). Induction of Th1 Cytokine Production by DC During the MLR The capacity to present alloantigen to T cells was further tested in one-way MLR using either DCI3 or DCG4 as stimulators and purified CD4⫹ T cells as responders. DCI3 were able to induce the production of larger amounts of IFN-␥ than DCG4 (Figure 2) during the allogeneic reaction. DC Gene Profiling After Maturation In trying to identify other IFN-␥–inducing factors than IL-12 that could be produced by DCI3, we performed a profiling of gene expression on both DCG4 and DCI3 after poly (I:C) stimulation, focusing on genes involved in DC activation and maturation. These genes included those encoding cytokines, chemokines and chemokine receptors, signal transduction molecules, and proteins involved in antigen uptake, processing, and presentation. These experiments revealed more than 20 differences. The most relevant differences for the control of T-cell activation were constituted by an overexpression of genes encoding IL1-␤, IFN-␣, and IL-6 (Figure 3a). This overexpression was confirmed at the protein level for IL-6 and IFN-␣, but not for IL-1␤. Moreover, both IL-6 and IFN-␣ were secreted in larger amounts by DCI3 as compared with DCG4 (Figure 3b). We further noted that IL-6 production during the MLR itself was higher when DCI3 were used as stimulators (Figure 4). Influence of Cytokine Blockade on IFN-␥ Release To further define the precise role of IL-6 and IFN-␣ in the induction of IFN-␥ production, we neutralized these cytokines by adding anti–IL-6 or anti–IFN-␣ antibodies at the initiation of the MLR. No inhibition was observed when anti–IFN-␣ Ab was added (data not shown). On the contrary, IL-6 neutralization resulted in a moderate but significant inhibition of IFN-␥ production when DCI3 were used to trigger the allogeneic reaction (Figure 5). Influence of DC-Derived IL-6 on CD4ⴙ CD25ⴙ T Cells During MLR DC-derived IL-6 has been recently described to be involved in the block of suppression exerted by Treg cells [8, 13]. This led us to hypothesize that if IL-6 was responsible for the increased IFN-␥ release by DCI3 through neutralization of Treg cell suppression, then this effect would disappear in the absence of such T cells. For

FIGURE 2 Th1 cytokine production during the mixed leucocyte reaction (MLR) induced by DCI3 as compared with DCG4. DCI3 or DCG4 matured with poly (I:C) were cocultured with allogeneic CD4⫹ T cells at 1:10 DC/T ratio. After 6 days, interferon-␥ sproduction was assessed by enzymelinked immunosorbent assay in MLR supernatants. Results are shown as mean ⫾ standard error of the mean (n ⫽ 14 ; *p ⬍ 0.05).

that purpose, we analyzed the influence of IL-6 neutralization on the allogeneic response triggered by DCI3 and DCG4 on CD4⫹CD25⫺ T cells (Treg-depleted CD4⫹ T cells). As shown in Figure 5, the effect of IL-6 neutralization was totally abrogated when the coculture was depleted of CD25⫹ T cells. DISCUSSION Besides the role for immature DCG4 in the differentiation of Treg cells, our work provides evidence for a negative feedback loop exerted by mature type I IFN DC through IL-6 production. This property came to light by the observation that mature type I IFN DC are able to induce strong IFN-␥ release despite an impairment to produce IL-12 together with the demonstration that this IFN-␥ release was partially inhibited after IL-6 neutralization. This IFN-␥ release could be clearly attributed to T cells taking into account the 2 log–less IFN-␥ secretion by mature type I IFN DC as compared with levels measured in MLR supernatant. These data are reminiscent of the Treg cell regulation that Medzhitov et al. have recently described in an in vitro murine model of T-cell stimulation by TLR-activated-DC. In this model, block

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FIGURE 3 Cytokine gene expression and production by mature DCI3 as compared with mature DCG4. (a) mRNA level: immature monocyte-derived DCI3 and DCG4 were matured for 6 or 18 h in the presence of poly (I:C). Total RNA was extracted, reverse transcripted, and amplified by polymerase chain reaction before hybridization on Gearray membrane. Results are those from one representative experiment of three. (b) protein level: immature DCI3 and DCG4 were matured for 18 h in the presence of poly (I:C) or medium alone. IL-6 and IFN-␣ productions were assessed by enzyme-linked immunosorbent assay in culture supernatants. Data are shown as mean ⫾ standard error of the mean (n ⫽ 13; *p ⬍ 0.05; **p ⬍ 0.001).

Type I IFN DC Control Treg Cells

FIGURE 4 Interleukin (IL)-6 production during the mixed leucocyte reaction (MLR) induced by DCI3 as compared with DCG4. DCI3 or DCG4 matured with poly (I:C) were cocultured with allogeneic CD4⫹ T cells. After 6 days of culture, IL-6 production was assessed in MLR supernatants. Results are shown as mean ⫾ standard error of the mean (n ⫽ 14 ; **p ⬍ 0.001).

of suppression was independent of costimulation, but required IL-6 and possibly other factors [8, 13]. Whether costimulation or other cell-to-cell contact interaction contributes to Treg control by type I IFN DC still needs to be assessed. In the past 5 years, there has been an intense focus on the role of Treg cells in controlling immune responses. Initially defined in the 1970s by Gershon and Kondo [14], Treg cells were subsequently identified as CD4⫹CD25⫹ T cells by virtue of their ability to suppress day 3 thymectomy-induced polyautoimmune syndrome [15, 16]. Treg cells are either naturally occurring, such as CD4⫹CD25⫹ cells [16], or induced in response to specific tolerogenic stimuli. The adaptive Treg cells include CD4⫹CD25⫹ cells, Tr1 cells that owe their suppressive effects to IL-10 secretion [17], and Th3 cells that confer immunosuppressive effects by TGF-␤ secretion [18]. The role of these cells has not been tested in our model because they are not supposed to naturally occur in the course of a primary MLR. They could be efficiently induced only after repetitive DC stimulation or exogenous IL-10 addition [19 –21]. We cannot exclude that, in secondary MLR performed with type I IFN DC as stimulators, induction of Treg cells occurs. This is

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even probable taking into account the higher capacity of these cells to secrete IL-10 as compared with myeloid DC (data not shown) and could constitute an additional level of T-cell control. Although IL-6 can act systemically, production of high levels of IL-6 during infection will not normally result in a nonspecific block of suppression, because activation of antigen-specific T cells still requires the costimulatory signals provided in a cognate T cell–APC interaction. However, it is tempting to speculate that locally, in the immunobiologic synapse, conditions may arise that lead to IL-6 –mediated release and suppression of self-reactive T cells, which has been suggested to explain the link between infection and some autoimmune diseases [22]. By contrast, during self-damaging inflammatory reactions to viruses or transplanted tissue, or in the setting of inflammatory autoimmune diseases, adaptive Treg cells might be induced to suppress the pathologic immune responses. Although this functional activity might also require cell-to-cell contact, the resulting suppression is amplified by the secretion of inhibitory cytokines [23]. In that context, immune intervention using type I IFN DC could help to abrogate this negative feedback when undesired, as in cancer. IL-12 is commonly considered as one of the major Th1 driving factor. However, other factors have been described, including IFN-␣, IL-15, IL-18, IL-23, and IL-27 [24 –29]. Neutralization of IFN-␣, IL-15, and IL-18 did not influence IFN-␥ release in our model (data not shown). Mohty et al. recently reported that IFN-␣ neutralization resulted in a partial inhibition of CD4⫹ T-cell– derived IFN-␥ production during the primary MLR induced by type I IFN DC [30]. However, these authors used immature type I IFN DC as stimulators and naive CD45 RA⫹ T-helper cells as responders. The different role of IFN-␣ for T-cell activation in this model compared with ours could thus be explained by different cytokines requirements of naive T cells as compared with memory cells and by a restricted panel of growth factors that can be produced by immature DC as compared with mature DC. The influence of IL-23 and IL-27 could not be tested because of the lack of neutralizing antibodies available. IL-23 is a recently discovered heterodimeric cytokine that shares biological properties with proinflammatory cytokines. The biologically active heterodimer consists of p19 and p40 subunit of IL-12. IL-23 has been shown to possess biological activities on T cells that share similarities with IL-12 [31]. IL-27, a product of activated APCs, is formed by the association of EBI3, an IL-12p40 –related polypeptide, and p28, a protein related to IL-12p35. IL-27 promotes the growth of naive CD4⫹ T cells and was suggested to play a role in the differentiation of Th1 cells in vitro [27]. Because IL-6

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FIGURE 5 Influence of interleukin (IL)-6 neutralization and T reg cell depletion on DCI3- versus DCG4-induced interferon (IFN)-␥ release during the mixed leucocyte reaction (MLR). DCI3 or DCG4 were cocultured with allogeneic CD4⫹CD25⫹/⫺ or CD4⫹CD25⫺ T cells with or without anti–IL-6 or isotype control antibodies. After 6 days, IFN-␥ production was assessed by enzyme-linked immunosorbent assay in MLR supernatants. Results of six independent experiments are summarized as mean ⫾ standard error of the mean % inhibition of IFN-␥ production as compared to the isotype control condition in Figure 5a (*p ⬍ 0.05). Concentrations of IFN-␥ in two representative experiments are shown in Figure 5b.

neutralization only yielded a partial inhibition of IFN-␥ release, these cytokines remain valuable candidates that could synergize with IL-6 to optimally induce IFN-␥ during type I IFN DC-induced T-cell activation. They could constitute the synergistic TLR-induced cytokine(s) necessary for efficient blockade of Treg cell function suggested by some authors [8]. DC are promising natural adjuvants for immunotherapy. They have been mainly tested in cancer. Despite encouraging results in pioneer pilot trials, DC-based cancer immunotherapy has yielded limited clinical efficacy. One possible explanation is the negative effect of regulatory T cells on the generation or expansion of antitumor effector cells. To circumvent this deleterious action of Treg cells, some investigators have initiated

clinical studies exploiting anti-CD25 antibodies to remove CD25⫹ Treg cells before DC vaccination. However, the risk of such an approach is to induce uncontrolled autoimmune manifestations related to the nonspecific depletion of Treg cells. The intrinsic capacity of type I IFN DC to block the suppressive effect of Treg cells could avoid the need for further in vivo Treg depletion. Moreover, one can speculate that by doing so, the action of type I IFN DC could target only Treg cells controlling the immune response elicited against the presented antigen specifically boosting the expected immune response. Indeed, Treg cells bearing some degree of specificity have been described. Antigen-pulsed DC induce CD8⫹ Treg cells specific for the influenza matrix peptide [32], and these CD25⫹ Treg cells clearly divide

Type I IFN DC Control Treg Cells

after challenge [33]. Likewise, Treg cells can prevent graft-versus-host disease and still allow for antitumor or graft-versus-leukemia effect [34 –36]. This targeting of specific Treg could occur locally as proposed by Bluestone and Abbas [23]. In conclusion, the present study shows that the innate immune response triggered through TLR recognition by type I IFN DC contributes to the control of the adaptive immune response through high IL-6 production. Besides providing new insights on the mechanisms of T-cell control, these data could constitute a basis for using type I IFN DC as natural adjuvant in immunotherapy. ACKNOWLEDGMENT

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We thank Dr. D. Egrise (Nuclear Medicine Department— Erasme Hospital, Brussels, Belgium) for helpful discussion and assistance for genes profiling experiments. 14.

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