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The Journal of Immunology, 2003, 171: 3385-3393.
Copyright © 2003 by The American Association of Immunologists

IFN-{alpha} Skews Monocyte Differentiation into Toll-Like Receptor 7-Expressing Dendritic Cells with Potent Functional Activities1

Mohamad Mohty*, Alexandra Vialle-Castellano*, Jacques A. Nunes{dagger}, Daniel Isnardon*, Daniel Olive*,{dagger} and Béatrice Gaugler2,*,{dagger}

* Laboratoire d’Immunologie des Tumeurs, Institut Paoli-Calmettes, Université de la Méditerranée, and {dagger} Institut National de la Santé et de la Recherche Médicale Unité 119, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN-{alpha} is an important cytokine for the generation of a protective T cell-mediated immune response to viruses. In this study, we asked whether IFN-{alpha} can regulate the functional properties of dendritic cells (DCs). We show that monocytes cultured in the presence of GM-CSF and IFN-{alpha} can differentiate into DCs (IFN-{alpha}-derived DCs (IFN-DCs)). When compared with DCs generated in the presence of GM-CSF and IL-4 (IL-4-derived DCs), IFN-DCs exhibited a typical DC morphology and expressed, in addition to DC markers CD1a and blood DC Ag 4, a similar level of costimulatory and class II MHC molecules, but a significantly higher level of MHC class I molecules. After maturation with CD40 ligand, IFN-DCs up-regulated costimulatory, class I and II MHC molecules and expressed mature DC markers such as CD83 and DC-lysosome-associated membrane protein. IFN-DCs were endowed with potent functional activities. IFN-DCs secreted large amounts of the inflammatory cytokines IL-6, IL-10, TNF-{alpha}, IL-1{beta}, and IL-18, and promoted a Th1 response that was independent of IL-12p70 and IL-18, but substantially inhibited by IFN-{alpha} neutralization. Furthermore, immature IFN-DCs induced a potent autologous Ag-specific immune response, as evaluated by IFN-{gamma} secretion and expansion of CD8+ T cells specific for CMV. Also, IFN-DCs expressed a large number of Toll-like receptors (TLRs), including acquisition of TLR7, which is classically found on the natural type I IFN-producing plasmacytoid DCs. Like plasmacytoid DCs, IFN-DCs could secrete IFN-{alpha} following viral stimulation or TLR7-specific stimulation. Taken together, these results illustrate the critical role of IFN-{alpha} at the early steps of immune response to pathogens or in autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{alpha}, originally considered as a simple antiviral substance, is an important cytokine for the generation of a protective T cell-mediated immune response to virus infections and tumor growth (1). It is present at low levels under normal physiological conditions (2), but can be secreted at high levels soon after cell exposure to viruses or other stimuli. Recently, it has been reported that the natural IFN-producing cells (IPCs),3 also defined as plasmacytoid monocytes (plasmacytoid dendritic cells (PDCs)), are represented by type 2 circulating blood dendritic cells (DCs) (3), which can produce high amounts of IFN-{alpha} after microbial challenge (3, 4, 5). The in vivo effects of type I IFN are associated with promoting an antiviral state involving a broad spectrum of cellular targets. IFN-{alpha} may act by enhancing the cytotoxic activity of NK cells and macrophages, by inducing T cell activation, or by maintaining the survival of activated T cells (6, 7, 8, 9). It has also been shown that IFN-{alpha} can induce Th1 activity in human CD4+ T cells (10). At the pathological level, serum from systemic lupus erythematosus (SLE) patients induced normal monocytes to differentiate into DCs (11). In addition, the capacity of SLE patients’ serum to induce DC differentiation correlated with disease activity and depended on the actions of IFN-{alpha} (11). Taken together, these findings illustrate the critical role of IFN-{alpha} for linking innate and adaptive immunity or driving autoimmune responses.

DCs play a key role in the induction of Ag-specific immune responses to bacteria, viruses, allergens, or tumor Ags. Because they serve as essential constituents of the immune system for triggering immune reactions, they are considered promising targets for immunotherapy (12, 13, 14). In vitro-differentiated DCs show functional and phenotypic characteristics of immature DCs that are able to capture and process Ags. Immature DCs can be further differentiated in vitro into mature DCs with TNF-{alpha}, LPS, or CD40 ligand (CD40L) (12). However, these factors may not represent the earliest physiological signals for DC differentiation in antimicrobial immune responses. Therefore, in this study, we asked whether IFN-{alpha} secreted after a microbial challenge can influence monocyte differentiation into DCs. We also investigated whether IFN-{alpha} alone can regulate the functional properties of DCs and what would be its impact at the early steps of immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Blood samples

PBMC from healthy donors (Etablissement Français du Sang, Marseille, France) were isolated on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradients before cryopreservation.

Cell lines

Murine L cells transfected with human CD40L were kindly provided by Schering-Plough (Laboratory for Immunological Research, Dardilly, France) and used after a 75-Gy irradiation (15).

Cell separation and DC generation

CD14+ peripheral blood monocytes were immunomagnetically purified with CD14 mAb-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of the CD14+ cells by flow cytometry analysis was >98%. Purified monocytes were cultured in RPMI 1640 medium containing 10% FCS (BioWhittaker, Verviers, Belgium), 0.55 mM L-arginine, 0.24 mM L-asparagine, and 2 mM L-glutamine (Life Technologies, Paisley, Scotland) at 0.5 x 106/ml in the presence of 100 ng/ml GM-CSF (Leucomax; Novartis, Rueil-Malmaison, France) and 20 ng/ml IL-4 (kind gift from Dr. F. Brière (Laboratory for Immunological Research, Schering-Plough, Dardilly, France)) for IL-4-derived DC (IL-4-DC) or 500 IU/ml IFN-{alpha}2b (Introna; Schering-Plough, Levallois-Perret, France) for IFN-{alpha}-derived DC (IFN-DC). Final maturation of IL-4-DC or IFN-DC was induced on day 5 by adding 75-Gy-irradiated CD40L-transfected L cells (ratio, 1:10) or 10 µg/ml LPS (LPS from Escherichia coli, serotype O26:B6; Sigma-Aldrich, St. Quentin Fallavier, France) or 15 µg/ml polyriboinosinic polyribocytidylic acid (poly(I:C); Sigma-Aldrich). The medium was replenished with cytokines every 3 days.

Flow cytometry analysis

The following mAbs were used for flow cytometry: anti-CD1a, -CD14, -CD40, -CD54, -CD58, -CD80, -CD83, -HLA-DR, and -HLA-ABC, and isotypic controls mouse IgG1, mouse IgG2a, and mouse IgG2b from Beckman Coulter (Marseille, France). CD86 and CD123 (IL-3R{alpha}) mAbs were purchased from BD PharMingen (San Diego, CA). DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) mAb was purchased from R&D Systems (Abingdon, U.K.). Blood DC Ag (BDCA)2 and -4 (16) mAbs were purchased from Miltenyi Biotec. For intracellular CD83 expression, cells were permeabilized using the CytoStain kit (BD PharMingen) according to the manufacturer’s instruction. All mAbs were used as FITC-, PE-, Cy5-, or allophycocyanin-conjugated mAbs. Samples were analyzed using a FACSCalibur (BD Biosciences, Le Pont de Claix, France). Data were analyzed using CellQuest software (BD Biosciences).

Confocal microscopy

Confocal microscopy analysis for DC-lysosome-associated membrane protein (DC-LAMP) mAb (Beckman Coulter) intracellular expression was performed as previously described in detail (17). Mouse IgG1 isotypic control was included in confocal microscopy experiments.

Primary MLR

CD4+CD45RA+ naive T cells were purified by negative selection of adult blood PBMC using goat anti-mouse Ig-coated magnetic beads (Beckman Coulter) incubated with mAbs against CD8, CD14 (D. Olive (Institut National de la Santé et de la Recherche Médicale Unité 119)), CD56 (kind gift from A. Moretta (University of Genoa, Genoa, Italy)), CD45RO (kind gift from R. Beverly (London, U.K.)), and CD19 (Diaclone, Besançon, France). Purity was >95%, as controlled by FACS analysis. Serial dilutions (3 x 102–3 x 103 cells/well) of stimulating cells were cultured in triplicate with 105 allogenic naive CD4+ T cells in 96-well flat-bottom plates (Costar, Corning, NY). Proliferation of T cells was monitored by measuring methyl-[3H]thymidine (1 µCi/well; Amersham, Little Chalfont, U.K.) incorporation during the last 16 h of a 6-day culture (18). Thymidine uptake was counted on a gas-phase beta counter (Matrix 9600; Packard, Downers Grove, IL).

Determination of cytokine production by DCs

Supernatants of DC cultures were harvested at day 5, and at day 7 after 2 days of maturation with CD40L. Cytokine contents of supernatants were determined by ELISA. IL-6, IL-10, TNF-{alpha}, and IL-12p70 concentrations were measured using OptEIA sets purchased from BD PharMingen. IL-1{beta} and IL-18 immunoassay kits were purchased from Beckman Coulter and Bender MedSystems Diagnostics (Vienna, Austria), respectively.

Analysis of cytokine production by DC-stimulated allogenic CD4+ T cells

CD4+CD45RA+ T cells were cocultured for 7 days with extensively washed IL-4-DCs or IFN-DCs. Neutralization experiments were performed by adding mAbs against IL-12p70 (1 µg/ml; BD PharMingen), IL-18 (clone 52713.11; 10 µg/ml; R&D Systems), IL-18R (1 µg/ml; R&D Systems), IFN-{alpha} (2000 neutralization U/ml; R&D Systems) in the cocultures of DCs and CD4+ T lymphocytes. Cells were then harvested and restimulated with 25 ng/ml PMA (Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) in the presence of 10 µg/ml brefeldin A (Sigma-Aldrich) for 5 h. Intracellular cytokines were detected by flow cytometry using anti-IL-10-PE, anti-TNF-{alpha}-FITC, anti-IFN-{gamma}-allophycocyanin mAbs, and FITC/PE/allophycocyanin-conjugated isotypic controls mAbs (BD PharMingen). Cells were collected, washed, fixed, and permeabilized using the CytoStain kit (BD PharMingen), and stained with 0.5 µg/test cytokine-specific mAbs according to the manufacturer’s instruction. Detection of IFN-{gamma} production by allogenic naive CD4+CD45RA+ T cells cocultured with DCs was also performed by ELISA. Cells were harvested after 6 days, and restimulated in 96-well flat-bottom culture plates at 105 cells/well in triplicate with PMA (25 ng/ml; Sigma-Aldrich) and ionomycin (1 µg/ml; Sigma-Aldrich) (19). After 24 h, supernatants were harvested, and IFN-{gamma} levels analyzed by ELISA using the OptEIA set for IFN-{gamma} (BD Biosciences).

Activation of CD8+ T lymphocytes and tetramer staining

DCs were generated from monocytes of HLA-A2 healthy donors and pulsed with 10 µg/ml pp65CMV/HLA-A2 peptide (NLVPMVATV) for 2 h. Autologous CD8+ T lymphocytes were immunomagnetically purified from PBMC by positive selection using CD8-coated microbeads (Miltenyi Biotec). Purity of the CD8+ T cells was determined by flow cytometry and was >98%. CD8+ T lymphocytes (1 x 106) were cocultured with 105 unpulsed or pp65CMV/HLA-A2 peptide pulsed-DC in IMDM containing 10% human AB serum and 10 IU/ml IL-2. Activation of Ag-specific CD8 T cells was monitored at day 7 by cytokine production and class I tetramer staining. For cytokine assay, effector cells were restimulated for 5 h with peptide-pulsed DC in the presence of 10 µg/ml brefeldin A. Intracellular cytokines IFN-{gamma} and TNF-{alpha} were detected by flow cytometry using anti-TNF-{alpha}-FITC and anti-IFN-{gamma}-allophycocyanin mAbs (BD PharMingen). For tetramer binding assay, T cells were labeled for 30 min at room temperature with PE-conjugated iTAG pp65CMV/HLA-A*0201 tetramer (Beckman Coulter), washed, and stained with anti-CD8-FITC (Beckman Coulter) for 30 min at 4°C. Samples were analyzed on a FACSCalibur. Allogenic CD8+ T lymphocytes were stimulated with IL-4-DCs or IFN-DCs, and restimulated at day 7 for 5 h with 25 ng/ml PMA and 1 µg/ml ionomycin in the presence of 10 µg/ml brefeldin A before intracellular cytokine labeling assay by flow cytometry as described above.

IFN-{alpha} secretion by IFN-DCs following viral stimulation

IL-4-DCs and IFN-DCs were plated in 96-well flat-bottom plates at a concentration of 5 x 104 cells/well either with medium alone, 10-5 M imiquimod (Aldara dissolved in DMSO; Laboratoires 3M Sante, Cergy Pontoise, France) or with inactivated HSV1 (kind gift from Dr. C. Zandotti (Laboratoire de Virologie, Hôpital La Timone, Marseille, France)) as described previously (17). Supernatants were collected after 24 and 48 h and tested for their IFN-{alpha} contents by ELISA (ELISA kits; Beckman Coulter). Freshly isolated PDC from healthy donors were obtained as described previously (17) and used as positive control for IFN-{alpha} secretion following stimulation with HSV or imiquimod.

Immunoblot analysis

IL-4-DCs and IFN-DCs were removed from culture, washed twice in RPMI 1640, then pelleted in a microcentrifuge, and lysed in a buffer containing 50 mM HEPES (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 20 mM NaF, 20 mM iodoacetamide, 1 mM PMSF, 1 µg/ml protease inhibitors (leupeptin, pepstatin A, and chymotrypsin), and 1 mM Na3VO4. Proteins from postnuclear cell lysates corresponding to 5 x 105 cells were resolved in 15% SDS-PAGE standard gels. For immunoblotting, membranes were blocked and hybridized with the monoclonal anti-human IL-18 mAb (clone 125-2H; R&D Systems) followed by an HRP-conjugated goat anti-mouse IgG (DAKO, Glostrup, Denmark), or the polyclonal rabbit anti-mitogen-activated protein kinase (extracellular signal-regulated kinase) (Promega, Charbonnières, France) followed by an HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and developed by ECL-Supersignal West Pico (Pierce, Rockford, IL) according to the manufacturer’s instructions.

RT-PCR

Total RNA from IL-4-DCs, IFN-DCs, and PDCs was extracted using RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. First-strand DNA were prepared using oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies). PCR amplification was performed on 2% of the cDNA with 2 U of TaqDNA polymerase (Life Technologies) in a final volume of 25 µl with previously reported Toll-like receptor (TLR) primers for TLR1 to -8 (20) and -9 (21). Reaction mixtures were heated at 94°C for 5 min, and PCR amplification was performed for 30 cycles (1 min at 94°C, 2 min at 65°C, and 3 min at 72°C). Final extension was obtained at 72°C for 15 min.

Statistical analysis

The significance of differences between the indicated values was assessed by two-tailed Student’s t test for paired and unpaired data as appropriate. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of DCs in the presence of IFN-{alpha}

To investigate the effect of IFN-{alpha} on DC differentiation from human monocytes, we cultured purified CD14+ monocytes with either the classical treatment with GM-CSF and IL-4 (IL-4-DC) currently used for obtaining immature DCs from monocytes (22) or clinical grade GM-CSF and IFN-{alpha}. After 5 days, monocytes cultured in the presence of IFN-{alpha} acquired a typical DC morphology (Fig. 1, A and B). These cells will be referred to as IFN-DCs hereinafter. We next characterized IFN-DCs in comparison with IL-4-DCs. On the phenotypic level, the presence of IFN-{alpha} instead of IL-4 at the beginning of culture did not impair DC differentiation from monocytes. The monocytic marker CD14 was down-regulated, and CD1a, a lineage marker of DC, was expressed on IFN-DCs (Fig. 2A). We next analyzed the expression of the adhesion molecules (CD54 and CD58), MHC molecules (class I, HLA-DR), costimulatory molecules (CD40, CD80, and CD86), and the maturation marker CD83. IFN-DCs showed a similar expression profile of these molecules as compared with IL-4-DCs. However, IFN-DCs had significantly higher surface levels of MHC class I molecules (Fig. 2, A and B). IFN-DCs did not express extracellular CD83, but expressed significant levels of intracellular CD83 (Fig. 2C). We also examined the maturation capacities of IFN-DCs after culture for 5 days followed by CD40L for 2 days. Phenotypic analysis indicated that IFN-DCs could acquire the expression of extracellular CD83 and up-regulated the expression of costimulatory molecules CD80 and CD86 (Fig. 3, A and B). The expression of CD40, CD54, CD58, HLA-DR, and especially class I MHC molecules was also up-regulated (Fig. 3B). Furthermore, as for IL-4-DCs, confocal staining of IFN-DCs indicated that, after maturation in culture, they expressed DC-LAMP (Fig. 1, C and D), which is a DC-specific lysosomal marker specifically induced in maturing DCs (23).



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  		FIGURE 1. IFN-{alpha} induces DC differentiation from monocytes. A and B, CD14+ monocytes were cultured either with GM-CSF and IL-4 (IL-4-DC) or with GM-CSF and IFN-{alpha} (IFN-DC) as described in Materials and Methods. After culture, they acquired typical DC morphology (IL-4-DC (A) and IFN-DC (B)) (representative of three experiments). C and D, Mature IFN-DCs express intracellular DC-LAMP similarly to mature IL-4-DCs (mature IL-4-DC (C) and mature IFN-DC (D)) (confocal microscopy; field, 130 x 100 µm; mouse IgG1 was used as isotypic control; data not shown) (representative of three experiments).

 


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  		FIGURE 2. Phenotype of immature IFN-DCs. A and B, IFN-DCs acquire CD1a, lose CD14, are HLA-DR+, and express HLA-class I molecules, CD54, CD58, CD40, and CD86 similarly to immature IL-4-DCs (representative of six independent experiments). C, Intracellular expression of CD83 by immature IFN-DCs (representative of two independent experiments). Empty histograms show the background staining with isotype control mAbs, and solid histograms represent specific staining of the indicated cell surface markers. The mean fluorescence intensities and SEMs are provided. *, p = 0.01 for IFN-DC vs IL-4-DC.

 


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  		FIGURE 3. Phenotype of mature IFN-DCs. A and B, IFN-DCs acquire a mature DC phenotype (CD83+, CD40high, CD80high, CD86high, and high levels of HLA-class I molecules and adhesion molecules CD54 and CD58) when cultured with CD40L for 2 days similarly to mature IL-4-DCs (representative of four independent experiments). Empty histograms show the background staining with isotype control mAbs, and solid histograms represent specific staining of the indicated cell surface markers. The mean fluorescence intensities and SEMs are provided. *, p = 0.01 for IFN-DC vs IL-4-DC.

 
Functional properties of IFN-DCs

To investigate the function of IFN-DCs as stimulators of naive CD4+ T cells, their ability to stimulate an allogenic MLR was compared with that of immature and mature IL-4-DCs. At all stimulator:responder ratios, immature and mature IFN-DCs were found to be as potent stimulators of allogenic MLR as immature and mature IL-4-DCs (Fig. 4A). We next examined the profile of primary allogenic T cell responses induced by immature IFN-DCs as compared with immature and mature IL-4-DCs. Naive CD4+CD45RA+ T cells isolated from human peripheral blood were cocultured with immature IFN-DCs, immature IL-4-DCs, mature IFN-DCs, and mature IL-4-DCs. As expected, T cells originally cultured with immature IL-4-DCs secreted small amounts of IFN-{gamma} as compared with mature IL-4-DCs (Fig. 4B). Surprisingly, T cells originally cultured with phenotypically immature IFN-DCs secreted significant amounts of IFN-{gamma} without induction of IL-10 or IL-4 (Fig. 4, B and C, and data not shown). Although they are at a phenotypically immature state when compared with mature IL-4-DCs, this polarization profile suggested a potent Th1 response associated with IFN-DCs in the absence of any maturation stimulus.



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  		FIGURE 4. Immature IFN-DCs induce IL-12-independent IFN-{gamma}-secreting CD4+ T cells. A, Proliferation of allogenic CD4+CD45RA+ naive T lymphocytes was measured in response to various numbers of irradiated stimulating cells. The mean results obtained from six independent experiments are indicated. B, Th1-polarizing capacity of immature IFN-DCs. Naive CD4+CD45RA+ T cells cocultured for 6 days with immature IL-4-DC, mature IL-4-DC, and immature IFN-DC were restimulated with PMA and ionomycin for 5 h in the presence of brefeldin A. IFN-{gamma} and IL-10 cytokines were measured by intracellular staining. A representative experiment and percentage of IFN-{gamma}-secreting cells (gated on CD3+CD4+ T cells) measured by intracellular staining obtained from six independent experiments are presented. C, IFN-{gamma} content in the supernatant of coculture of T cells with the indicated stimulating cells was also measured by ELISA. Results are represented as the mean and SEM obtained from four independent experiments. D, Addition of anti-IL-12 neutralizing Ab significantly diminished CD4+ T cell IFN-{gamma} induction by mature IL-4-DCs, whereas there was no significant effect of anti-IL-12, anti-IL-18, and anti-IL-18R neutralizing Abs on induction of CD4+ T cell IFN-{gamma} by immature IFN-DCs (representative of four independent experiments). Interestingly, addition of anti-IFN-{alpha} neutralizing Ab could partially inhibit CD4+ T cell IFN-{gamma} induction by immature IFN-DCs (representative of two independent experiments).

 
Cytokine production by IFN-DC

We investigated the cytokine secretion profile by immature and mature IFN-DCs as compared with immature and mature IL-4-DCs. Supernatants were quantified for IL-10, IL-12p70, TNF-{alpha}, IL-6, IL-1{beta}, and IL-18. In comparison to IL-4-DCs, immature IFN-DCs spontaneously secreted significant amounts of IL-18, which was up-regulated following maturation with CD40L (Fig. 5A). The specific synthesis of IL-18 by IFN-DCs was further confirmed by Western blot analysis, which showed the presence of the pro-IL-18 protein only in IFN-DCs, but not in IL-4-DCs (Fig. 5B). Immature IFN-DCs did not secrete detectable levels of IL-12p70. In response to CD40L, mature IFN-DCs could secrete significant amounts of IL-12p70 (Fig. 5A). In contrast, compared with IL-4-DCs, in response to CD40L, IFN-DCs produced higher levels of the inflammatory cytokines IL-1{beta}, IL-6, IL-10, and TNF-{alpha} (Fig. 5A).



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  		FIGURE 5. Cytokine secretion profile of IFN-DCs. A, IL-10, IL-12p70, TNF-{alpha}, IL-6, IL-1{beta}, and IL-18 secretion was tested by ELISA from culture supernatants of 105 immature and mature IFN-DC or control immature and mature IL-4-DC. Results are represented as the means and SEMs obtained from four to seven independent experiments. B, The specific synthesis of the pro-IL-18 protein only in IFN-DCs but not in IL-4-DCs was confirmed by Western blot analysis as described in Materials and Methods (representative of two independent experiments). Mitogen-activated protein kinase Western blots were used as controls for protein loading. Positions of the molecular mass standards are indicated.

 
Induction of CD4+ T lymphocytes IFN-{gamma} by immature IFN-DCs requires no IL-12p70 and no IL-18

In view of the above cytokine secretion profile, we looked for factors inducing CD4+ T lymphocytes IFN-{gamma} secretion after coculture of naive CD4+ T cells with immature IFN-DCs. Addition of anti-IL-12 neutralizing Ab significantly diminished CD4+ T cell IFN-{gamma} induction by mature IL-4-DCs as expected, whereas there was no significant effect on induction of CD4+ T cell IFN-{gamma} by immature IFN-DCs (Fig. 4D). Similarly, the addition of IL-18 and IL-18R neutralizing Abs did not significantly reduce CD4+ T lymphocyte IFN-{gamma} secretion after coculture of naive CD4+ T cells with immature IFN-DCs. Interestingly, addition of anti-IFN-{alpha} neutralizing Ab could substantially inhibit CD4+ T cell IFN-{gamma} induction by phenotypically immature IFN-DCs (Fig. 4D).

IFN-DCs activate Ag-specific CD8+ T lymphocytes

In addition to the evaluation of the effects of IFN-DCs on CD4+ T lymphocytes, we also asked whether IFN-DCs could activate Ag-specific CD8+ T lymphocytes. When compared with immature or mature IL-4-DCs, as early as 7 days of stimulation, immature IFN-DCs from HLA-A2 donors pulsed with the HLA-A2-restricted pp65 CMV peptide and cocultured with autologous CD8+ T lymphocytes, could induce a higher proportion (1.5- to 2-fold) of Ag-specific autologous CD8+ CTLs as ascertained by pp65-specific tetramer staining (Fig. 6A). The latter correlated with the higher number of autologous IFN-{gamma}- and TNF-{alpha}-secreting CD8+ T cells induced by both immature and mature IFN-DCs as compared with immature and mature IL-4-DCs (Fig. 6B and data not shown). In the allogenic setting, after 7 days of culture, immature IFN-DCs cocultured with allogenic CD8+ T lymphocytes induced slightly higher or comparable amounts of IFN-{gamma}-secreting CD8+ T lymphocytes (Fig. 6C). When matured with CD40L, mature IFN-DCs could induce a ~2-fold higher number of allogenic IFN-{gamma}-secreting CD8+ T lymphocytes as compared with mature IL-4-DCs (Fig. 6C). Thus, IFN-DCs are capable APCs that are more efficient than IL-4-DCs in inducing CD8+ T cell responses.



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  		FIGURE 6. IFN-DCs activate Ag-specific CD8+ T cells. A, IFN-DCs are more efficient to expand CMV-specific CD8+ T cells as shown by tetramer staining. CD8+ T lymphocytes were cocultured with unpulsed or pp65CMV peptide-pulsed DC. Activation of autologous Ag-specific CD8+ T cells was monitored at day 7 by pp65-PE-tetramer staining (representative of four independent experiments). B, Activation of autologous Ag-specific CD8+ T cells was monitored at day 7 by intracellular cytokine secretion assay. Effector cells were restimulated for 5 h with peptide-pulsed DCs in the presence of brefeldin A. Intracellular IFN-{gamma} was detected by flow cytometry using anti-IFN-{gamma}-allophycocyanin mAbs (representative of four independent experiments). C, Allogenic CD8+ T lymphocytes were stimulated with IL-4-DCs or IFN-DCs for 7 days and restimulated with PMA and ionomycin in the presence of brefeldin A. Intracellular IL-10 and IFN-{gamma} were detected by flow cytometry using anti-IFN-{gamma}-allophycocyanin and anti IL-10-PE mAbs (representative of five independent experiments).

 
IFN-DCs express different TLRs and secrete IFN-{alpha} following viral stimulation

TLRs are a family of innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens and induce antimicrobial immune responses. Thus, we investigated the pattern of TLRs expressed by IFN-DCs as compared with IL-4-DCs. As shown in Fig. 7A, immature IL-4-DCs expressed high levels of TLR1, -2, -3, -4, -6, and -8, very low levels of TLR5, but undetectable levels of TLR7 and -9. In marked contrast to IL-4-DCs, immature IFN-DCs expressed, in addition to TLR1, -2, -3, -4, -5, -6, and -8, high levels of TLR7, but no TLR9. After maturation with CD40L, as described previously (21), IL-4-DCs down-regulated the expression of TLR1, -2, -3, -4, and -6. The same held true for IFN-DCs, but to a lesser extent as for TLR1, -2, -4, and -6 (Fig. 7A). The expression of TLR7 but not TLR9 by IFN-DCs was intriguing, because TLR7 and -9 are specific for the natural type I IPCs or PDCs (20, 21). Thus, we investigated, whether IFN-DCs expressed other phenotypic markers specific of PDCs. IFN-DCs expressed high surface levels of the IL-3R{alpha} (CD123) and BDCA4 that is present on PDCs, but also up-regulated on cultured myeloid DCs (16). However, IFN-DCs failed to express BDCA2 (Fig. 7B), which is a PDC-specific marker that was not yet described on any other DC subset (16). Like PDCs, IFN-DCs expressed very small amounts of DC-SIGN, a C-type lectin, described on a small subset of peripheral blood PDC precursors (24). In view of the specific expression of TLR7 by IFN-DCs, we assessed whether immature IFN-DCs could secrete IFN-{alpha} when stimulated with HSV. PDC from healthy donors used as positive control produced large amounts of IFN-{alpha} as expected (Fig. 7C). IL-4-DCs did not produce IFN-{alpha} (mean, 6 pg/ml, at the limit of detection level). However, immature IFN-DCs could secrete a significant amount of IFN-{alpha} following stimulation with HSV compared with that produced by IL-4-DCs (mean, 265 pg/ml) (Fig. 7C). In view of the pattern of expression of TLRs by IFN-DCs, we also assessed whether IFN-DCs can respond to TLR agonists such as LPS, poly(I:C), or imiquimod. IFN-DCs stimulated with LPS showed a mature phenotype through expression of CD83, up-regulation of CD80, CD86, and HLA-DR. This mature phenotype could be also achieved to a lesser extent following stimulation with poly(I:C) (Fig. 8A). In addition, IFN-DCs could secrete significant amounts of IL-12p70 when stimulated with LPS (Fig. 8B). As for HSV stimulation, when stimulated with imiquimod that is a specific agonist of TLR7, IFN-DCs could secrete significant amounts of IFN-{alpha} (Fig. 8C). In these experiments, the supernatant of IFN-DCs that were not stimulated with HSV or imiquimod, did not contain any detectable amounts of IFN-{alpha} (Fig. 7C and 8C), thus excluding a possible contamination with IFN-{alpha} that was used for the generation of IFN-DCs and confirming that the production of IFN-{alpha} is another prominent feature of IFN-DCs in response to viral stimulation or TLR7 agonists.



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  		FIGURE 7. IFN-DCs express different TLRs and secrete IFN-{alpha} following viral stimulation. A, mRNA expression of TLR-1, -2, -3, -4, -5, -6, -7, -8 and -9 was examined in IL-4-DCs and IFN-DCs by RT-PCR as described in Materials and Methods (representative of three independent experiments). B, CD123, BDCA2, BDCA4, and DC-SIGN molecule expression pattern by immature IL-4-DCs and immature IFN-DCs. Empty histograms show the background staining with isotype control mAbs, and solid histograms represent specific staining of the indicated cell surface markers. The mean fluorescence intensities are provided for this representative experiment of four independent experiments. C, IFN-{alpha} secretion by IFN-DCs. After 5 days of culture, IL-4-DCs and IFN-DCs were intensively washed and stimulated with HSV without any additional cytokine. Supernatants from 5 x 104 IL-4-DCs, IFN-DCs, or control PDCs were harvested after 48 h of stimulation with HSV. IFN-{alpha} secretion was analyzed by ELISA. The supernatant of IFN-DCs that were not stimulated with HSV did not contain any detectable amounts of IFN-{alpha}. Results are represented as the mean of IFN-{alpha} concentration obtained from four independent experiments.

 


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  		FIGURE 8. IFN-DCs respond to different TLR agonists. A, IFN-DCs acquire a mature DC phenotype when stimulated with TLR agonists LPS and poly(I:C) (representative of four independent experiments). Empty histograms show the background staining with isotype control mAbs, and solid histograms represent specific staining of the indicated cell surface markers. The mean fluorescence intensities of this representative experiment are provided. B, IL-12p70 secretion by IFN-DCs following stimulation with LPS. After 5 days of culture, IL-4-DCs and IFN-DCs were intensively washed and stimulated with LPS. Supernatants were harvested after 48 h, and IL-12p70 secretion was analyzed by ELISA. Results are represented as the mean of IL-12p70 concentration obtained from four independent experiments. C, IFN-{alpha} secretion by IFN-DCs following stimulation with imiquimod. After 5 days of culture, IL-4-DCs and IFN-DCs were intensively washed and stimulated with imiquimod without any additional cytokine. Supernatants were harvested after 48 h of stimulation. IFN-{alpha} secretion was analyzed by ELISA. The supernatant of IFN-DCs that were not stimulated with imiquimod did not contain any detectable amounts of IFN-{alpha}. Representative of four independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Monocytes are circulating myeloid DC precursor cells. In vitro, they can differentiate into monocyte-derived DCs following culture with GM-CSF and IL-4 (22). Different factors may influence the phenotype of the differentiated DCs (25, 26). Monocytes exposed to PGE2 in addition to GM-CSF and IL-4 will differentiate into DCs, but are impaired in IL-12 secretion (27). Exposure to IL-10 can inhibit DC maturation and IL-12 secretion (27). Addition of type I IFN to monocytes differentiating in the presence of GM-CSF and IL-4 significantly impairs their ability to differentiate into IL-12-secreting, IFN-{gamma}-inducing DCs (28). In contrast, it was also shown that IFN-{beta} has the ability to enhance the maturation of IL-4-DCs (29) or induce DC differentiation if associated with IL-3 (30). Moreover, previous reports suggested that monocytes cultured with GM-CSF plus IFN-{alpha} can be induced toward the DC lineage (31, 32). However, little is yet known about the functional properties of these cells. In the present study, we show that peripheral blood monocytes cultured in the presence of clinical grade GM-CSF and IFN-{alpha} alone can differentiate into DCs (IFN-DC). When compared with the classical IL-4-DCs, IFN-DCs exhibited a typical DC morphology and expressed, in addition to DC markers CD1a and BDCA4, a similar level of costimulatory and class II MHC molecules, but a significantly higher level of MHC class I molecules. After maturation, IFN-DCs up-regulated costimulatory and class I and II MHC molecules, and expressed specific DC markers, such as DC-LAMP (23). Depending on their maturation stage, IFN-DCs secreted large amounts of inflammatory cytokines such as IL-1{beta}, IL-6, IL-10, TNF-{alpha}, and especially IL-18, which could be detected at both maturational stages. The latter finding is of particular interest. To our knowledge, this is the first report directly depicting secretion of a significant amount of IL-18 in the supernatant of a DC culture. Despite its extracellular role, IL-18 is not a classical secretory protein in that it lacks a secretory signal sequence (33). The reported biological activities of IL-18 include stimulation of activated T cell proliferation, NK cell activation, induction of IFN-{gamma} and GM-CSF by activated T lymphocytes and macrophages, and regulation of cytokine production during the early phases of microbial infections (34, 35, 36, 37). Thus, IFN-DCs, through IL-18 and secretion of other inflammatory cytokines, might play an important role in regulating the immune response, especially at the early steps of cell-mediated immunity. Although they did not secrete IL-12p70, immature IFN-DCs promoted a strong Th1 response. These data represent one of the few demonstrations of an IL-12-independent T cell IFN-{gamma} induction by a DC subset (38). Addition of anti-IFN-{alpha} neutralizing Ab could partially inhibit CD4+ T cell IFN-{gamma} induction by phenotypically immature IFN-DCs, suggesting a possible autocrine loop regulating this phenomenon. However, inability to completely inhibit IFN-{gamma} through neutralization of IFN-{alpha} suggests that other factors might interfere in Th1 induction by immature IFN-DCs. In this regard, some {beta}-chemokines were shown to be Th1 polarizing or associated with Th1-polarized immunity in some instances (39). Thus, in addition to IFN-{alpha}, induction of Th1 responses by immature IFN-DCs might also rely upon chemokines or other yet-unknown cytokines directly involved in the induction of Th1 responses. In this regard, recent evidence from different experimental systems has demonstrated that a novel set of cytokines from the IL-12 family members, such as IL-23 and IL-27, might represent key immunoregulatory cytokines, with their effects encompassing actions on T cells and enhancement of the Th1 costimulatory functions of APCs (40, 41, 42, 43). Further studies are needed to characterize the role of these cytokines in the orchestration of DC-induced Th1 responses. In contrast, PDCs, which are not reported to secrete IL-12 (4), likewise induce Th1 responses following viral infection (44, 45). Although the clear mechanism of this IFN-{gamma} induction has not yet been reported, some data suggested that it might rely to some extent upon IFN-{alpha} secretion (46), further supporting our findings. In this respect, IFN-DCs up-regulated IL-3R{alpha}, a classical but nonspecific PDC marker (4). IFN-DCs also expressed a broad spectrum of TLRs, including acquisition of TLR7, which is, despite some conflicting data (46), specific to the natural type I IPCs or PDCs (20, 21). TLR7 play an important role in the synthesis of IFN-{alpha} and other inflammatory cytokines in a variety of cell types, including DCs (46, 47). IFN-DCs could respond to different TLRs agonists. Like PDCs, IFN-DCs could also secrete IFN-{alpha} following viral or specific TLR7 stimulation. Likewise, the physiological factors involved in the regulation of DC differentiation from monocytes are still unknown. In an in vivo model, Randolph et al. (48) showed that DC differentiation and maturation can occur directly from monocytes during transendothelial migration. In a pathological situation, Blanco et al. (11) showed that the serum of SLE patients can induce DC differentiation, and this mainly depended on the actions of IFN-{alpha}. Similarly, our results suggest that IL-4-DCs might not be the earliest professional APCs present at the first steps of the immune response. Although the direct putative role of viral replication within IFN-DCs remains to be analyzed, one could hypothesize that following viral or other microbial infection, the natural type I IPCs will secrete large amounts of type I IFN (3, 4, 5), thus favoring differentiation of DCs from monocytes (IFN-DCs). IFN-DCs will secrete inflammatory cytokines that can be beneficial for an efficient host immune response against infections. In addition, through secretion of IFN-{gamma}-inducing factors, IFN-DCs will promote a strong Th1-biased CD4+ T cell response. Although it is not possible from our experiments to know whether IFN-DCs can activate genuine naive CD8+ T cells, they still might represent an important event in the adjuvant activity for the in vivo expansion of Ag-specific CD8+ T cells. Interestingly, IFN-DCs could elicit a potent Ag-specific CD8+ response. In contrast to IL-4 DCs, and as described previously for PDCs (24), IFN-DCs expressed low levels of the adhesion molecule DC-SIGN. Different studies reported that DC-SIGN is a key molecule mediating virus entry into target cells (49, 50). Thus, low expression of DC-SIGN by IFN-DCs might protect them from virus-induced cell death, providing an additional advantage toward an efficient host immune response. IFN-DCs have the ability to secrete IFN-{alpha} through their specific pattern of TLR7 expression, determining a possible natural loop, and thus relaying, at least in part, PDCs for IFN-{alpha} production. Consistent with this, depletion of PDCs from the blood of SLE patients resulted only in a partial reduction of IFN-{alpha} release upon viral triggering (11), further confirming that other circulating cells, different from PDCs, are capable of IFN-{alpha} production, at least in pathological situations. Thus, the maintenance of IFN-{alpha} production by IFN-DCs in a pathological situation may represent a feedback mechanism to further promote DC differentiation, and to drive the host immune response against pathogens or to perpetuate an autoimmune response. Taken together, these results illustrate the critical role of IFN-{alpha} through cooperation with the DC system at the early steps of the immune response to pathogens or in autoimmune diseases.


   Acknowledgments
 
We thank L. Leserman (Centre d’Immunologie Luminy, Marseille, France), C. Mawas (Institut National de la Santé et de la Recherche Médicale Unité 119), and L. Galibert (Immunex, Seattle, WA) for their critical reading of the manuscript. We thank Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, and GEFLUC for their support of our ongoing basic research work. We thank S. Just-Landi and N. Baratier for excellent technical assistance. We also thank Dr. Didier Blaise (Institut Paoli-Calmettes) for his continuous support and helpful discussions.


   Footnotes
 
1 This study was supported by grants from Fondation de France, Société Française de Greffe de Moelle et de Thérapie Cellulaire, Fondation pour la Recherche Médicale (Paris, France), Association Mediterraneene pour le Developement de la Transplantation (Marseille, France), and La Ligue Départementale Contre le Cancer du Gard (Nimes, France) (to M.M.). A.V.-C. was supported by a grant from the Ligue Nationale Contre le Cancer (Paris, France). Back

2 Address correspondence and reprint requests to Dr. Béatrice Gaugler, Institut de Cancérologie et d’Immunologie de Marseille (IFR57), Institut National de la Santé et de la Recherche Médicale Unité 119, Laboratoire d’Immunologie des Tumeurs, Institut Paoli-Calmettes, 232 Boulevard Sainte Marguerite, 13273 Marseille Cedex 09, France. E-mail addresses: gauglerb{at}marseille.fnclcc.fr or mohtym{at}marseille.fnclcc.fr Back

3 Abbreviations used in this paper: IPC, IFN-producing cell; DC, dendritic cell; PDC, plasmacytoid DC; SLE, systemic lupus erythematosus; CD40L, CD40 ligand; IL-4-DC, IL-4-derived DC; IFN-DC, IFN-{alpha}-derived DC; poly(I:C), polyriboinosinic polyribocytidylic acid; DC-SIGN, DC-specific ICAM-3-grabbing nonintegrin; BDCA, blood DC Ag; DC-LAMP, DC-lysosome-associated membrane protein; TLR, Toll-like receptor. Back

Received for publication May 12, 2003. Accepted for publication July 29, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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