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The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells

Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, α/β/ω) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, α/β/ω) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.
In addition to TLR7/9, pDCs express multiple specific receptors that facilitate antigen capture and presentation and, moreover, regulate pDC function, preventing thus abnormal immune responses. These regulatory receptors (RRs), include Fc receptors and lectin-like receptors (11,12), which signal through the B cell receptor (BCR)-like pathway involving spleen tyrosine kinase (SYK) associated with the immunoreceptor tyrosine-based activation motif-containing adapter of RR, Bruton's tyrosine kinase, B-cell linker protein, phospholipase Cγ 2, MEK1/2-ERK, and induction of intracellular Ca2+ mobilization (8,9,12). Among these RRs, blood dendritic cell antigen 2 (BDCA-2, CD303, CLEC4C) is an lectin-like receptor (13), while immunoglobulinlike transcript (ILT7, CD85g) binds to and can be activated by bone marrow stromal cell antigen 2 (BST2, CD317, tetherin, HM1.24) protein, the expression of which is found on cells pre-exposed to IFN-I or on the surface of human cancer cells (14). Signaling via pDC RRs attenuates TLR-induced production of IFN-I and proinflammatory cytokines by an unknown mechanism (8-13, 15, 16). This physiological feedback mechanism of IFN control is hijacked in the pathogenesis of several chronic viral infections and cancers, leading to immune tolerance (10,(17)(18)(19). We have recently shown that hepatitis C virus (HCV) particles inhibit the production of IFN-α via the binding of E2 glycoprotein to RRs BDCA-2 and DCIR (dendritic cell immunoreceptor) and induce a rapid phosphorylation of AKT and ERK, in a manner similar to the cross-linking of BDCA-2 or DCIR (10,17,19).
Here, we addressed the question of whether specific pharmacological targeting of BCR-like signaling can restore functionality to pDCs abrogated by ligation of RRs, and what the underlying mechanism of this abrogation is. In our previous work, we demonstrated that a highly specific inhibitor of SYK blocks both BCR-like and TLR7/9 signaling and, therefore, it is not compatible with restoration of pDC function (15). In this study, we have tested the effects of inhibitors of c-Jun N-terminal kinase (JNK), MEK1/2 kinase, p38 kinase, and calcium-dependent phosphatase calcineurin, acting through a BCR-like signaling pathway, and of NF-κB activating TANK binding kinase 1 (TBK1) on the IFN-I production in pDCs exposed to a TLR9 agonist. Surprisingly, we found that inhibitors of MEK1/2 potentiated IFN-I and IL-6 production in pDC cell line GEN2.2, but not in primary pDCs stimulated by the TLR9 agonist. More importantly, inhibitors of MEK1/2 significantly increased TLR9-mediated production of IFN-I that had been blocked in both GEN2.2 cells and primary pDCs by ligation of RRs with BDCA-2 and ILT7 mAbs, or HCV particles, or with BST2 expressing cells. Moreover, the restauration of IFN-I production by MEK1/2 inhibitor was observed when TLR9 signaling had been blocked by phorbol 12-myristate 13-acetate (PMA), an agonist of protein kinase C (PKC), which stimulates MEK1/2-ERK signaling.
Furthermore, our results show that BCR-like and PKC signaling induced in pDCs the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. c-FOS is known to associate with c-JUN to form activator protein 1 (AP-1) transcription factor and to exert within the cell a pleiotropic effect, including cell differentiation, proliferation, apoptosis, and the immune response (20)(21)(22)(23). While a previous study reported that the c-FOS induced by tumor progression locus 2 (TPL-2) inhibits TLR9-mediated production of IFN-I in mouse macrophages and myeloid DCs, but not in pDCs (24), we show that MEK1/2-ERKinduced c-FOS was involved in the inhibition of TLR9-mediated production of IFN-I in human pDCs. Our results suggest that the MEK1/2-ERK-dependent expression and phosphorylation of c-FOS exerts an intrinsic block of TLR9-mediated production of type I IFN. Pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

resUlTs
MeK1/2 inhibitor Potentiates cpg-a-induced Production of iFn-α in pDc cell line gen2.2 In order to restore TLR7/9-mediated production of IFN-I blocked by ligation of RRs, we first searched for an inhibitor of BCR signaling that does not inhibit signaling triggered by TLR7/9 agonists. To this end, we selected a panel of kinase inhibitors involved in BCR-like, MAPK, NF-ĸB, and calcium signaling, and control inhibitors of TLR7/9 signaling, and tested their effect on the production of IFN-α in a pDC cell line GEN2.2 exposed to TLR9 agonist CpG-A (Figures 1A,B; Figure S1 in Supplementary Material). To facilitate biochemical analyses of cell signaling, which is still difficult to perform in rare and in vitro short living human primary pDCs, we performed our studies in human pDC line GEN2.2, which shares the key features of human primary pDCs (15,(25)(26)(27)(28)(29)(30).
The same results were obtained with MEK1/2 inhibitor U0126 (data not shown). We found that in addition to IFN-α also IL-6 production in CpG-A-stimulated GEN2.2 cells was synergized by MEK1/2 inhibitor PD0325901 (Figures 1C,D), whereas production of TNF-α was inhibited ( Figure 1E), suggesting that the MEK1/2-ERK pathway positively regulates TNF-α expression or secretion (31). The strongest synergistic effects on IFN-α production (synergistic index >3) were observed for combinations of ≥0.01 μM PD0325901 and 4 µg/ml CpG-A. Synergistic effects of these combinations were also demonstrated for the production of IL-6 (synergistic index >2). In contrast to the synergistic effect observed with ≥0.01 μM PD0325901, the combination of 0.001 µM PD0325901 with 4 µg/ml CpG-A had only an additive effect on the production of IL-6 ( Figure 1D). In the control experiment, PMA-induced the production of TNF-α (but not that of IFN-α and IL-6), which was strongly inhibited by   (Figures 1F-H). Collectively, these results show that the CpG-A-induced TLR9-mediated production of IFN-α and IL-6 are potentiated by MEK1/2 inhibitor PD0325901.
As in GEN2.2 cells, exposure of primary pDCs from healthy donors to BDCA-2 mAb suppressed the production of IFN-α induced by CpG-A to 11.5% (N = 9, p = 0.0039, Figure 3D). The major difference observed in primary pDCs compared to GEN2.2 cells consisted in the lack of the potentiation of CpG-A-induced production of IFN-α by PD0325901 in the absence of BDCA-2 mAb (Figures 3B-E). In contrast, a similar restoration effect to the one in GEN2.2 was observed in primary pDCs Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 364 exposed to PD0325901 prior to BDCA-2 mAb (Figures 3D,E). PD0325901 significantly restored the production of IFN-α inhibited by BDCA-2 mAb (2.4-fold, p = 0.0039, Figure 3E). A similar restoration effect was observed with PD0325901 at 10 nM concentration ( Figure S3 in Supplementary Material) and with MEK1/2 inhibitor U0126 using ILT7 mAb for crosslinking RR ( Figure S4 in Supplementary Material). In conclusion, these results show that MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked by ligation of RRs.

MeK1/2 inhibitor restores Tlr9-Mediated iFn-α Production Blocked by PMa
A recent study showed that treatment of pDCs with PMA, an agonist of PKC activating MEK1/2-ERK signaling pathway, has led to a dose-dependent reduction of IFN-α secretion (34). We investigated the capacity of PD0325901 to reverse the inhibitory effect of PMA on TLR9-mediated IFN-α production ( Figure 6A).
c-FOs levels in pDc cell line gen2.2 are higher Than Those in Primary pDcs The implication of MEK1/2 in the crosstalk of BCR-like and TLR7/9 signaling led us to investigate the role of c-FOS, a downstream immediate early response gene (20), in the regulation of TLR7/9 response. To this end, we compared the levels of c-FOS protein in the GEN2.2 cell line with those in primary pDCs (Figures 7A,B). We found that the quantity of c-FOS in GEN2.2 cells cultured in complete medium was approximately double that of primary pDCs ( Figure 7B).  (Figures 8B,C). In addition to the quantification of c-FOS mRNA by qRT-PCR, we determined the c-FOS protein levels in the serum-starved GEN2.2 cells exposed to CpG-A or BDCA-2 mAb by western blot (Figures 8D,E).  (Figures 9A,B). Proliferating GEN2.2 cells were treated with PD0325901, corresponding concentration of DMSO, CpG-A, and BDCA-2 mAb, or starved in serum-free medium, and the impact on their cell cycle was analyzed 16 h later. Cell cycle of a control culture of GEN2.2 cells was analyzed immediately after separation from MS-5 cells. We found that the MEK1/2-ERK pathway inhibitor PD0325901 blocked the cell cycle in proliferating GEN2.2 cells. The cell cycle was also strongly inhibited in the serum-starved GEN2.2 cells, although the impairment of the cell cycle in this cell culture did not permit to calculate residual S phase and G2/M phase cells according to mathematical model used in our analyses. As expected, BDCA-2 crosslinking did not block,  (Figures 7 and 8D,E) and with the potentiation of CpG-A-induced production of IFN-α ( Figure 1C).

BDca-2 crosslinking induces Phosphorylation of c-FOs
It was reported that ERK1/2-mediated post-translational phosphorylation enhances c-FOS stability and transcriptional activity (20,22,23). We assessed the phosphorylation of ERK1/2 at T202/ Y204 and c-FOS at T325 in serum-starved GEN2.2 cells treated with RR agonist BDCA-2 mAb, TLR9 agonist CpG-A, and PKC agonist PMA (Figure 10A). c-FOS phosphorylation was analyzed using Western blotting with the P(T325)-c-FOS antibody. In the control experiment, 15 or 60 min exposure of GEN2.2 cells to PMA-induced strong phosphorylation of ERK1/2 at T202/ Y204 and the c-FOS at T325, which was efficiently inhibited by PD0325901 ( Figure 10B). The levels of total c-FOS and ERK1/2 remained unchanged in GEN2.2 cells stimulated with PMA for 15 or 60 min ( Figure S6 in Supplementary Material). Stimulation with BDCA-2 mAb induced strong phosphorylation of ERK1/2 at T202/Y204 and the c-FOS phosphorylation at T325, which was abrogated by pretreatment with MEK1/2 inhibitor PD0325901 ( Figure 10C; Figure S7 in Supplementary Material). In contrast to BDCA-2 mAb or PMA, CpG-A-induced ERK-1/2 T202/Y204 phosphorylation without inducing the phosphorylation of c-FOS T325 ( Figure 10D). In conclusion, all three agonists induced phosphorylation of ERK-1/2, which was inhibited by 1 µM PD0325901. BDCA-2 mAb and PMA induced phosphorylation of c-FOS while CpG-A did not. The phosphorylation of c-FOS was inhibited by PD0325901, which is consistent with the regulation of c-FOS by MEK1/2-ERK signaling.

BDca2 crosslinking in gen2.2 cells and Primary pDcs induces Upregulation of c-FOs
A recent study reported that BDCA-2 crosslinking and internalization result in up to 16 hr-lasting resistance of pDCs to TLR7/9mediated stimulation suggesting a stability of the IFN-I inhibitory signal (35). Although c-FOS expression is usually rapid and transient, c-FOS stability is enhanced by phosphorylation (20,22,23). These observations led us to investigate the stability of c-FOS levels after stimulation of the BCR-like or TLR9 pathways. We analysed the quantity of c-FOS in the GEN2.2 cell line 16 h after stimulation with the control PMA, BDCA-2 mAb, and CpG-A by flow cytometry in the presence or absence of PD0325901 ( Figure 11A). The results show that stimulation with PMA and  Figure 11C). While To assess whether stimulation of BDCA-2 in primary pDCs also upregulates the expression of c-FOS, we exposed PBMCs from three healthy donors to BDCA-2 mAb and determined the level of c-FOS in a rapidly dying population of primary pDCs 4 hr later. Because the low proportion of pDCs in PBMCs makes their biochemical analyses difficult, we used flow cytometry for this purpose (Figures 11A, D-F). The MFI of c-FOS induced by BDCA-2 mAb increased 2.19 ± 0.85 times compared to isotypic IgG1 control in pDCs ( Figure 11E). These results show that the stimulation of RRs of pDCs results in a sustained increase of the c-FOS level not only in the GEN2.2 cell line but also in primary pDCs.

DiscUssiOn
Our results demonstrate the important role of MEK1/2-ERK signaling in the RR-mediated inhibition of IFN-α and IL-6 production in pDCs. We showed that MEK1/2 inhibitors PD0325901 and U0126 were the only constituents of the panel of inhibitors of BCR-like signaling that not only did not abrogate, but even stimulated TLR9 signaling in GEN2.2 cells. Pharmacological targeting of MEK1/2 in GEN2.2 cells or primary pDCs significantly abrogated inhibition of the TLR9-mediated production of IFN-I induced by BCR-like or PKC signaling. Both BCR-like and PKC signaling activated MEK1/2-ERK pathway.
The molecular mechanism by which the ligation of the RRs antagonizes TLR7/9 signaling in pDCs remains elusive despite years of intense research in many laboratories (8-10, 12-14, 16, 35). We show here that MEK1/2-ERK signaling upregulated the production and phosphorylation of c-FOS. Thus, the potentiation of IFN-I by PD0325901 treatment of GEN2.  pDCs represents a major difference between these cell types and is consistent with the different outcome of MEK1/2-ERK inhibition. The demonstration of the synergistic effect of MEK1/2 inhibitors on the CpG-A-induced production of IFN-α suggests that under steady-state conditions a natural intrinsic block regulated by MEK1/2 controls the IFN-α level in GEN2.2 cells to a higher level than that in primary pDCs. Release of this block could be a part of the restoration mechanism of IFN-α by MEK1/2 inhibitors in pDCs exposed to RR agonists. The levels of inhibition of IFN-I production by crosslinking of RR and their restoration by MEK1/2-ERK inhibitors varied depending on the RR ligand. This could be related to differences in the cell-surface distribution of targeted receptors (BDCA-2, ILT7, DCIR) and avidity of tested ligands (BDCA-2 and ILT7 mAbs, HCV particles, or BST2 expressing cells). Among them, BDCA-2 mAb was the most potent inhibitor of IFN-I production. Surprisingly the relative levels of inhibition and restoration of IFN-I production were similar in GEN2.2 cell line and primary pDCs. In addition to differences in receptor/ligand interactions, the levels of inhibition and restoration of IFN-I production were dependent on the mechanism of stimulation of MEK1/2-ERK pathway by BCR-like or PKC signaling. While pretreatment with PD0325901 led to almost complete inhibition of c-FOS expression induced by PMA, c-FOS expression induced by BDCA-2 mAb was only partially inhibited. This suggests that expression of c-FOS induced by BDCA-2 crosslinking and internalization could be partially MEK1/2-ERK independent.
MEK1/2 inhibitor PD0325901 potentiated production of IFN-α in pDC cell line GEN2.2 stimulated by both synthetic (CpG-A and CpG-B) and natural (HSV-1 and HCMV) agonists. In the absence of PD0325901, exposure of pDCs to HSV-1 and HCMV results in a non-permissive infection and TLR9-mediated production of IFN-α (36,37). Interestingly, the quantity of IFN-α produced by murine pDCs exposed to murine CMV (MCMV) is down-modulated by MCMV-induced stimulation of DAP12, an adaptor molecule of murine RR (38). Recent study demonstrated that EBV and double-stranded DNA viruses induce TRIM29 leading to suppression of IFN-α production (39). The potential role of TRIM29 in HSV-1 and HCVM-mediated inhibition of IFN-α production in pDCs needs to be clarified.
A previous report implicated c-FOS induced by MAP3-kinase TPL-2 in the negative regulation of TLR9-mediated production of IFN-β in mouse macrophages and myeloid (mDCs), but not in mouse pDCs (24). In contrast, we show here that c-FOS induced by MEK1/2-ERK signaling is involved in the regulation of TLR9 signaling in human pDCs. It is possible that TPL-2 and MEK1/2-ERK signaling are interpreted differently in mouse and human pDCs compared with macrophages and mDCs as a consequence of an interaction of ERK activation with other signaling pathways triggered by TLR9 (18). Several cell type-specific studies have shown that the interaction of TLR7/9 with BCR-like signaling may be regulated in a different way in human pDCs (7,12,14,16,35,40).
Activation of Ras/MEK1/2/ERK downregulates expression of IFN-I also in human epithelial cancer cells (41). Together with our experiments, these results suggest that MEK1/2-ERK signaling can play a general role in regulation of IFN-I. Another recent study demonstrated that MEK1/2-ERK-mediated phosphorylation of c-FOS in HCV-infected hepatocytes induced miR-21, which targeted MyD88 and IRAK1 and contributed to the suppression of IFN-I production (42). We did not detect a significant increase of miR-21 level in GEN2.2 cells exposed to BDCA-2 mAb or CpG-A (not shown).
We have demonstrated that inhibitors of MEK1/2 restore the production of IFN-I inhibited by ligation of RRs with HCV particles or with BST2 expressing cancer cells. These results suggest that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and reestablish their immunogenic activity. This finding complements our previous results showing that an inhibitor of SYK, a protein kinase involved in both TLR7/9 and BCR-like pathways, could be a useful tool to suppress the overproduction of IFN-I and to re-establish tolerogenic homeostatic functions of pDCs (15). The role of IFN-I in the pathogenesis of chronic viral infections and cancer is unclear and ambivalent. IFN-I responses are critical in the early phases of immune response to infections, but the chronic and systemic activation of pDCs can paradoxically lead to deleterious consequences for the immune system (43,44). It is likely that an intense signaling occurs in the mucosa, involving a local accumulation of pDCs producing IFN-I early during HIV-1 infection, which is associated with the chronic activation of the immune system (45,46). While in this era of great success of direct-acting antivirals against HIV and HCV the stimulation of IFN response might represent an adjuvant therapy, important namely in the case of virus escape, the induction of IFN-I in combination with existing antivirals may cure HBV infection (47)(48)(49). IFN-I also plays an important role in antitumor immunity (3,50). The addition of exogenous IFN-α reverts the immunotolerance of tumor-associated pDCs in breast and ovarian carcinoma (4,51). Pharmacological targeting of MEK1/2 signaling may constitute an attractive new approach to study mechanisms of modulation of pDC activation in pathophysiological conditions such as chronic viral infections and cancer.

MaTerials anD MeThODs isolation and culture of Primary pDcs
Peripheral blood mononuclear cells (PBMCs) from healthy anonymous donors were obtained from the national blood services (Etablissement Francais du Sang, Marseille, France). Blood samples were obtained after written consent following the approval of the EFS, Marseille, France, and the Center de Recherche en Cancérologie de Marseille (CRCM) in accordance to the convention signed the 20th May 2014. pDCs purified from PBMCs as described previously were 75-95% pure, with a contamination of less than 5% mDCs (32,33,52,53). Isolated pDCs were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS). To optimize viability in overnight experiments, recombinant IL-3 (R&D Systems Europe, Ltd., Abingdon, UK) was added to a final concentration of 10 ng/mL.

pDc line gen2.2
Human pDC line GEN2.2 (25) was grown in a RMPI 1640 medium supplemented with L-glutamine, 10% FCS, 1% sodium pyruvate, and 1% MEM nonessential amino acids, on a monolayer of the murine stromal feeder cell line MS-5 grown in RPMI 1640 supplemented with L-glutamine, 10% FCS, and 1% sodium pyruvate. For the measurement of cytokine production, the dynamic flow cytometry and the Western blot experiments, GEN2.2 cells were separated from the MS-5 feeder cells.
inhibitors, antibodies, and reagents MEK-1/2 inhibitor PD0325901 obtained from InvivoGen (Toulouse, France) and U0126 obtained from Sigma (Sigma-Aldrich, Lyon, France) were used as recommended by supplier. PD0325901 is a selective non-ATP-competitive allosteric MEK1/2 inhibitor with in vitro IC50 0.33 nM, which was shown to be specific against a panel of 70 different kinases at 10 µM range (54

Preparation of BsT2 expressing heK293T cells
The BST2 sequence from pCMV-Sport6-BST2 was cloned into the pRRL.PPT.SF.i2GFPp expression vector to produce a lentiviral vector pRRL-BST2-GFP. HEK293T cells were transduced by the resulting lentivirus construct at MOI = 10 and GFP-positive cells were selected by FACSAria (BD Biosciences). The expression of GFP and BST2 in transduced cells was determined by flow cytometry by LSRII (BD Biosciences).

Determination of erK and c-FOs by immunobloting
Total c-FOS and ERK in the whole cell lysate of GEN2.2 cells or primary pDCs were determined by Western blotting by means of rabbit polyclonal c-FOS (sc-52) and ERK1/2 (sc-154) Abs (Santa Cruz Biotechnology, Dallas, USA). Phosphorylation of ERK and c-FOS in the whole cell lysate of GEN2.2 cells was analyzed by Western blotting using phospho-c-FOS-T325 Ab from Abcam (Cambridge, UK) and ERK Ab T202/Y204 (Santa Cruz Biotechnology, Dallas, USA) as described previously (15). After incubation with the appropriate horseradish peroxidaseconjugated secondary antibody, the membranes were washed and the protein bands were detected with Super Signal™ enhanced chemoluminiscent substrate detection reagent (ThermoFisher Scientific, Villebon-sur-Yvette, France). Densitometric analyses were performed using Amersham Imager 600 (GE Healthcare Life Science). Band intensities were normalized to GAPDH or Ponceau red.

cell cycle analysis
For analysis of cell cycle, 10 6 GEN2.2 cells/ml of RPMI 1640 medium supplemented with 10% FCS were aliquoted in 1 ml quantities in 6-well flat-bottom culture plates and exposed to 1 µM PD0325901, 4 µg/ml CpG-A, and 10 µg/ml of BDCA-2 mAb for 16 h. The cells were then resuspended in the RPMI 1640 medium containing 6 µg/ml Hoechst 33342 Dye (ThermoFischer Scientific) and incubated at 37°C in 5% CO2 for 30 min and the amount of DNA was determined by flow cytometry. Live/Dead cell discrimination was performed by Zombie Green™ Fixable Viability Kit (BioLegend, San Diego, USA). Samples were analyzed using a BD LSR FORTESSA cytometer (BD Biosciences, San Jose, USA) and data were processed using FLOWJO software (Treestar, San Carlos, USA). Phases of the cell cycle were calculated by Dean-Jett-Fox model.
Determination of secreted iFn-α, TnF-α, and il-6 The quantities of total IFN-α, TNF-α, and IL-6 produced by pDCs or GEN2.2 were measured in cell-free supernatants using human ELISA kits (IFN-α and IL-6 from Mabtech, and TNF-α from BD Biosciences). The index of synergism was determined from the following formula: the level of cytokine production after stimulation with the combination of CpG and PD0325901 divided by the sum of cytokine production level after stimulation with CpG and PD0325901 separately. PD0325901 alone did not induce a detectable quantity of respective cytokines. Combinations resulting in an index of synergism >1.5 were considered to be synergistic. The combinations resulting in an index of synergism ≤1.5 and in a 30% increase in stimulation compared to the stimulation observed with either of the two stimulators were considered to be additive.

statistical analysis
Quantitative variables are expressed as the mean ± SEM (standard error of the mean). To compare the levels of cytokine production and transcription of c-FOS mRNA by pDCs, we used a Mann-Whitney or a Wilcoxon two-tailed non-parametric tests. For flow cytometry analyses, we used two-tailed t-test. Data were analyzed with GraphPad Prism 4 (GraphPad Software, La Jolla, CA). A p value ≤ 0.05 was considered to be significant.

eThics sTaTeMenT
Peripheral blood mononuclear cells (PBMCs) from healthy anonymous donors were obtained from the national blood services (Etablissement Francais du Sang, Marseille, France).
Blood samples were obtained after written consent following the approval of the EFS, Marseille, France and the Centre de Recherche en Cancérologie de Marseille (CRCM) in accordance to the convention signed the 20th May 2014.

aUThOr cOnTriBUTiOns
Contribution: VJ, BA, and AF-H equally performed research, designed research, and analyzed data. TH, KT, and JW performed research and analyzed data. JN, DO, and PD designed research and analyzed data. LC and JP provided essential materials. RS and IH designed research, analyzed data, and wrote the paper. acKnOWleDgMenTs DO is a scholar of the Institut Universitaire de France. We acknowledge the Imaging Methods Core Facility at BIOCEV for their support with obtaining flow cytometry data presented in this paper.

FUnDing
This work was supported by grants from the Grantova Agentura Ceske Republiky (Czech Science Foundation) grant no. 14-32547S (IH) and by Fondation ARC pour la Recherche sur le Cancer. This work was also supported by institutional grants from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique and Aix-Marseille Université to CRCM, and from Charles University