Ligand and cytokine dependence of the immunosuppressive pathway of tryptophan catabolism in plasmacytoid dendritic cells

Francesca Fallarino1, Ciriana Orabona1, Carmine Vacca1, Roberta Bianchi1, Stefania Gizzi1, Carine Asselin-Paturel2, Maria Cristina Fioretti1, Giorgio Trinchieri2, Ursula Grohmann1 and Paolo Puccetti1

1 Department of Experimental Medicine, Section of Pharmacology, University of Perugia, 06126 Perugia, Italy
2 Laboratory for Immunological Research, Schering-Plough Research Institute, 69571 Dardilly, France

Correspondence to: P. Puccetti; E-mail: plopcc{at}tin.it


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Murine plasmacytoid dendritic cells (pDCs) have been credited with a unique ability to express indoleamine 2,3-dioxygenase (IDO) function and mediate immunosuppression in specific settings; yet, the conditions of spontaneous versus induced activity have remained unclear. We have used maneuvers known to up-regulate IDO in different cell types and have examined the relative efficacy and mechanisms of the induced activity in splenic pDCs, namely, after specific receptor engagement by CTLA-4-Ig, CD200-Ig or CD28-Ig, the latter in combination with silenced expression of the suppressor of cytokine signaling 3 (SOCS3) gene. We found that pDCs (CD11c+ mPDCA-1+ 120G8+) do not express IDO and are not tolerogenic under basal conditions. B7-1 engagement by CTLA-4-Ig, CD200R1 engagement by CD200-Ig and B7-1/B7-2 engagement by CD28-Ig in SOCS3-deficient pDCs were each capable of initiating IDO-dependent tolerance via different mechanisms. IFN-{gamma} was the major cytokine responsible for CTLA-4-Ig effects, and type I IFNs for those of CD200-Ig. Immunosuppression by CD28-Ig in the absence of SOCS3 required IFN-{gamma} induction and IFN-like actions of IL-6. Therefore, although pDCs do not mediate IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by pDCs in the mouse.

Keywords: CD28-Ig, CD200-Ig, CTLA-4-Ig, plasmacytoid dendritic cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Human and mouse plasmacytoid dendritic cells (pDCs) represent a specialized cell population that produces large amounts of type I IFNs in response to viruses, the so-called natural IFN-producing cells (1). Mouse pDCs share most of the morphological, phenotypic and functional characteristics of their human counterparts (24) and express several pDC-restricted surface markers (5, 6). The ability of pDCs to secrete type I IFNs depends on cellular sensors that promptly detect the presence of DNA and RNA viruses. Because pDCs produce large amounts of cytokines, particularly type I IFNs, they regulate inflammation and link innate with adaptive immunity. pDCs also function as antigen-presenting cells and have a role in T cell polarization (1). Furthermore, there is growing evidence for a role of human and murine pDCs in tolerance induction. Human CD8+ T cells primed with CD40L-activated pDCs have a regulatory phenotype, with poor proliferative and cytolytic activity and little production of IFN-{gamma} but release of IL-10 (7). Human pDCs have also been reported to induce CD4+ CD25+ T regulatory cells capable of suppressing T cell proliferation in an antigen-independent fashion (8). Stimulation of mouse pDCs with the tolerogenic agents CTLA-4-Ig (9) or CD200-Ig (10) initiates the immunosuppressive pathway of tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO), leading to the onset of antigen-specific tolerance.

There is an increasing appreciation of the unifying role that IDO may have in mediating tolerance under a variety of physiopathologic conditions (11, 12). Modulation of tryptophan catabolism may represent a general mechanism of action of CTLA-4-expressing regulatory T cells (13), and different cell types in addition to pDCs respond to CTLA-4 engagement of B7 with activation of IDO, including CD8+ (14) and CD8 (15) dendritic cells (DCs), CD4+ T cells (16) and polymorphonuclear leukocytes (17). CTLA-4-IgG2a induces IDO expression in a minority population of splenic CD19+ cells (18). IDO contributes to maternal tolerance in pregnancy (19), protection against autoimmunity (20, 21) and allergy (22) and the control of inflammatory pathology (17, 23, 24). In both humans (25) and mice (26), IDO-expressing DCs are found in tumor-draining lymph nodes, possibly resulting in antigen-specific anergy. The bulk of these studies suggests that IDO-expressing DCs may have a general role in regulating T cell homeostasis, and an inhibitory DC population would be of fundamental importance for the control of autoimmunity and immune regulation (27).

In humans, Munn et al. (25) originally described the existence of a subset of DCs that constitutively express IDO and have T-cell-suppressive properties. Recent work, however, indicates that environmental factors may be most important in determining the IDO competence of human DCs and subsets thereof (28). Because of the growing recognition of the role of pDCs in the mechanisms of tolerance, the present study has examined the expression pattern of the IDO mechanism by mouse pDCs under different conditions. Although we found no constitutive expression of IDO, both type I and type II IFNs, released in response to different stimuli, were capable of initiating IDO-dependent tolerance in an autocrine fashion in the pDCs.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and DC purification
Female C57BL/6 (H-2b) mice were obtained from Charles River Breeding Laboratories (Calco, Milan, Italy). B7-deficient C57BL/6 mice homozygous for the CD80 (B7-1–/–) or CD86 (B7-2–/–) targeted mutation were from The Jackson Laboratory (Bar Harbor, ME, USA). Mice deficient for the IFN-{alpha}/ßR–/– on an A129 background were as described (3). All in vivo studies were done in compliance with National and Perugia University Animal Care and Use Committee guidelines. Splenic DCs were purified by magnetic-activated sorting using CD11c MicroBeads and MidiMacs (Miltenyi Biotec, Bergisch Gladbach, Germany), in the presence of EDTA to disrupt DC–T cell complexes (10, 14). Cells were >99% CD11c+, >99% MHC I-A+, >98% B7-2+ and <0.1% CD3+, and appeared to consist of 90–95% CD8, 5–10% CD8+ and ~5% mPDCA-1+ cells. For positive selection of mPDCA-1+ DCs (6), CD11c+ cells were fractioned using mPDCA-1 MicroBeads (Miltenyi Biotec). In accordance with previous data (5, 10), >95% of the mPDCA-1+ cells were stained by 120G8 (Fig. 1). For negative selection of CD8 DCs, CD11c+ cells were fractioned by means of CD8{alpha} MicroBeads (Miltenyi Biotec) (10, 14). The CD8 fraction was ~45% CD4+ and typically contained <0.5% contaminating CD8+ cells. Less than 5% CD8 DCs expressed the 120G8 marker.



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Fig. 1. Expression of 120G8 on CD11c+ mPDCA-1+ cells positively selected from the spleens of DBA/2 mice. CD11c+ mPDCA-1+ cells, isolated from total spleen cells by magnetic-activated cell sorting, were assayed by flow cytometry. (A) Cells were stained with anti-CD11c–FITC and anti-mPDCA-1–PE. The percentages of cells present in each area are indicated. (B) Histogram plot showing cells stained with 120G8–Alexa 488 (thick line) or isotype control within the CD11c+ mPDCA-1+ fraction.

 
FACS analysis
In all FACS analyses, cells were treated with rat anti-CD16/32 (2.4G2) antibody for 30 min at 4°C to block FcRs. Expression of CD11c, mPDCA-1 and 120G8 was analyzed by the respective use of hamster anti-CD11c–FITC, rat anti-mPDCA-1–PE (Miltenyi Biotec) and rat 120G8–Alexa 488. Goat anti-mouse IgG3–PE was from Southern Biotechnology (Birmingham, AL, USA).

DC treatments
CTLA-4-Ig (14), CD200-Ig (10) and CD28-Ig (29) were fusion proteins generated from the extracellular domains of murine CTLA-4, CD200 or CD28, respectively, and the Fc portion of a murine IgG3, with Ig-C{gamma}3 representing the control treatment. Ig-C{gamma}3 is a construct produced by the same cell line as the fusion proteins, and consists of the hinge, CH2, and CH3 regions of murine IgG3 Fc inserted downstream of the IgG3 signal peptide in the absence of CTLA-4, CD200 or CD28 domains (29). Neutralizing XMG1.2 mAb to IFN-{gamma} and mAbs 6B4 (anti-mouse IL-6) and 15A7 (anti-mouse IL-6R) were previously described (29). DCs were exposed to 40 µg ml–1 CTLA-4-Ig, CD28-Ig or Ig-C{gamma}3 or to 5 µg ml–1 CD200-Ig for 24 h at 37°C in the presence or absence of the enzyme inhibitor 1-methyl-D,L-tryptophan (1-MT) at 2 µM. In selected experiments, DCs were treated with 2 µg ml–1 oligodeoxynucleotide 5'-TCCATGACGTTCCTGACGTT-3' (CpG DNA 1826; Invitrogen Life Technologies, San Diego, CA, USA), as described (10). For cytokine neutralization, DCs were exposed to fusion proteins in vitro in the presence of 6B4 and 15A7 mAbs (for IL-6 neutralization; each at 10 µg ml–1) or anti-mouse IFN-{gamma} mAb (10 µg ml–1) as described, using rat IgG or IgG2a as the respective control treatments (29). For immunization in vivo, cells were loaded with the HY peptide in vitro (5 µM, 2 h at 37°C), before irradiation and intravenous injection into recipient hosts. A total of 3 x 105 CD8 DCs were injected either alone or in combination with 9 x 103 pDCs, either untreated or exposed to the different agents (10). The viability of DC cultures for in vitro or in vivo experiments always exceeded 70%.

Reverse transcription–PCR analysis of suppressor of cytokine signaling 3 expression
Expression of suppressor of cytokine signaling 3 (SOCS3) (sense, 5'-GCCATGGTCACCCACAGCAAGTT-3'; anti-sense, 5'-AAGTGGAGCATCATACTGATCCAGGA-3') and control SOCS1 (sense, 5'-ATGGTAGCACGCAACCAGG-3'; anti-sense, 5'-AAGACGAGGACGAGGAGGG-3') transcripts was evaluated by reverse transcription (RT)–PCR analysis using specific primers, as described (30). Expression of IFN-{alpha} (sense, 5'-ATGAGCTACTGGCCAACCTG-3'; anti-sense, 5'-CCTGCTGCATCAGACAACCT-3') and IFN-ß (sense, 5'-TTCCTGCTGTGCTTCTCCAC-3'; anti-sense, 5'-GGAGAGCAGTTGAGGACATC-3') was also assessed by RT–PCR.

Small interfering RNA synthesis and transfection
These procedures have been described previously (30). Briefly, the small interfering RNA (siRNA) sequences specific for murine SOCS3 (sense, 5'-GGAGCAAAAGGGUCAGAGGtt-3'; anti-sense, 5'-CCUCUGACCCUUUUGCUCCtt-3') were selected, synthesized and annealed by the manufacturer (Ambion, Austin, TX, USA). For transfection, siRNAs (6.7 µg) in 30 µl of transfection buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were pipetted into a sterile Eppendorf tube. In a separate polystyrene tube, 6.7 µg of 1,2 dioleoyl-3-trimethylammonium-propane was mixed with 30 µl of transfection buffer and then both solutions were mixed gently by pipetting several times. After incubation at room temperature for 20 min, the mixture was added to 1 ml of complete medium containing 106 pDCs and incubated for 24 h at 37°C in the presence of CD28-Ig or control Ig-C{gamma}3. Cells were then recovered, washed and immediately used for in vitro or in vivo experiments. siRNA treatment resulted in the complete disappearance of SOCS3 transcripts at 24–48 h. Control treatments consisted of cells treated with negative control siRNA (Ambion). The binding of CD28-Ig to pDCs was not modified by SOCS3 gene silencing. After siRNA transfection, DC viability always exceeded 70%.

Skin test assay
A skin test assay was used for measuring class I-restricted delayed-type hypersensitivity responses to poorly immunogenic peptides, as previously described (10, 13, 29). The HY (WMHHNMDLI) peptide was obtained from A&A Labs (San Diego, CA, USA). This peptide is recognized in the context of the MHC class I H-2Db molecules and induces tolerance when injected into female mice, resulting in long-term survival of male skin graft (31). Peptide-loaded CD8 DCs, or combinations of CD8 DCs and pDCs, were transferred intravenously into recipient hosts that were assayed at 2 weeks for the development of peptide-specific reactivity in response to intrafootpad challenge with the peptide. Results were expressed as the increase in footpad weight of peptide-injected footpads over that of vehicle-injected counterparts. Data are the mean ± SD for at least six mice per group. The statistical analysis was performed using Student's paired t-test by comparing the mean weight of experimental footpads with that of control counterparts.

IDO expression and functional analysis
IDO expression was investigated by immunoblot with rabbit polyclonal anti-murine IDO antibody, as described (13). IDO functional activity was measured in vitro in terms of the ability of DCs to metabolize tryptophan to kynurenine, whose concentrations were measured by HPLC (10).

ELISA assessments of cytokine production and intracellular cytokine expression
Murine IFN-{alpha} was measured by means of specific ELISA (PBL Biomedical Laboratories, Piscataway, NJ, USA), as described (10). The assay sensitivity was 10–500 pg ml–1. Measurements of IFN-{gamma} and IL-6 were also conducted as described (29). In particular, MP5-20F3 and biotinylated MP5-32C11 were used for IL-6 ELISA, whereas R4-6A2 and biotinylated XMG1.2 were used for IFN-{gamma} (all from PharMingen, San Diego, CA, USA). Data are the means ± SD of triplicate determinations. To evaluate the cytoplasmic IFN-{gamma} and IL-6 contents of pDCs, cells were stained by the PFA–saponin procedure, as described (32). After extensive washing in immunofluorescence buffer (PBS with 3% FCS and 0.02% NaN3), cells were fixed in 2% PFA for 10 min at room temperature, washed again and incubated with PE-labeled anti-IFN-{gamma} or anti-IL-6 antibodies (both from PharMingen) and 120G8–Alexa 488 in immunofluorescence buffer containing 0.3% saponin for 30 min on ice. Isotype-matched irrelevant mAbs were used as controls in the analysis of intracellular IFN-{gamma}/IL-6 expression.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Binding of CTLA-4-Ig, CD28-Ig and CD200-Ig to pDCs
Despite their limited expression of surface B7 molecules, mouse pDCs respond to CTLA-4-Ig with the activation of IDO (9). CTLA-4-Ig and CD28-Ig have opposite effects on IDO function in non-pDCs (29). Strong induction of tryptophan catabolism is induced in pDCs by CD200-Ig engagement of CD200R1 (10). We preliminarily examined binding of CTLA-4-Ig, CD28-Ig and CD200-Ig to purified splenic CD11c+ mPDCA-1+ cells. pDCs were incubated with each fusion protein prior to staining with anti-mouse IgG3–PE (Fig. 2). Specific and significant binding was detected with all three fusion proteins.



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Fig. 2. Cytofluorimetric analysis of CTLA-4-Ig, CD28-Ig and CD200-Ig binding to splenic pDCs. CD11c+ mPDCA-1+ cells were treated with 1 µg ml–1 of each fusion protein or control Ig-C{gamma}3 for 30 min on ice. The secondary antibody was anti-mouse IgG3–PE, and controls consisted of cells treated with the secondary reagent alone (thin lines).

 
IDO induction by CTLA-4-Ig and CD200-Ig but not CD28-Ig in pDCs
CTLA-4-Ig and CD28-Ig are both agonist ligands of B7 molecules on conventional DCs but they bias the downstream response in opposite directions, and CTLA-4-Ig promotes tolerance (14) whereas CD28-Ig favors the onset of immunity (29). Although B7 engagement by either ligand leads to a mixed cytokine response, a dominant IL-6 production in response to CD28-Ig will prevent the IFN-{gamma}-driven induction of IDO (29). We examined IDO expression and function in pDCs after 24 h exposure to CTLA-4-Ig, CD28-Ig, CD200-Ig or control Ig-C{gamma}3. Significant and comparable levels of IDO protein expression (Fig. 3A) and IDO-mediated conversion of tryptophan to kynurenine (Fig. 3B) were observed in response to CTLA-4-Ig and CD200-Ig, but not CD28-Ig or Ig-C{gamma}3.



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Fig. 3. Ability of CTLA-4-Ig and CD200-Ig to induce IDO expression and function in pDCs. (A) IDO expression was assessed by western blot analysis. pDCs were treated in vitro with CTLA-4-Ig, CD28-Ig or CD200-Ig, Ig-C{gamma}3 representing the control treatment. IDO expression was investigated with an IDO-specific antibody. The positive control consisted of IDO-expressing MC24 transfectants and the negative control consisted of mock-transfected MC22 cells. Loading controls consisted of samples re-probed with ß-tubulin-specific antibody. One experiment of three. (B) Functional IDO activity in response to fusion proteins was measured in vitro in terms of the ability to metabolize tryptophan to kynurenine using pDCs treated as indicated above. Kynurenine levels in supernatants were measured by HPLC, and results are the mean ± SD of triplicate samples in one of three experiments.

 
Modulation of SOCS3 in pDCs by CTLA-4-Ig and CD28-Ig
Recent studies with non-pDCs indicate that a major discriminator of function in response CTLA-4-Ig versus CD28-Ig engagement of B7 receptor molecules is represented by the activity of SOCS3, which impairs IFN-{gamma} signaling and prevents STAT1-dependent activation of IDO (29). Although SOCS3 expression is typically induced by IL-6 to attenuate its own signaling (33), up-regulation of SOCS3 by IL-6 may be responsible for inhibition of STAT-dependent signaling by IFNs (34). In the case of conventional CD8+ DCs, CTLA-4-Ig and CD28-Ig appear to induce early changes in SOCS3 expression, with CTLA-4-Ig decreasing and CD28-Ig increasing transcription of the SOCS3 gene (30). We examined SOCS3 transcriptional expression in pDCs treated with CTLA-4-Ig, CD28-Ig or CD200-Ig (Fig. 4). Semi-quantitative RT–PCR analysis revealed that as early as 30 min of pDC exposure to a fusion protein, marked down-regulation and up-regulation of SOCS3 transcripts were induced by CTLA-4-Ig and CD28-Ig, respectively. In contrast, treatment with CD200-Ig had no effect on SOCS3 expression.



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Fig. 4. Ability of CTLA-4-Ig and CD28-Ig to modulate SOCS3 transcriptional expression in pDCs in opposite directions. Semi-quantitative RT–PCR analysis of SOCS3 expression was performed in pDCs treated with CTLA-4-Ig, CD28-Ig or CD200-Ig for 30 min. Control treatment consisted of Ig-C{gamma}3. Each reaction contained decreasing amounts of cDNA (indicated). One experiment is representative of three.

 
IDO induction by CD28-Ig in SOCS3-deficient pDCs
Silencing the expression of the SOCS3 gene in conventional DCs by specific RNA interference renders CD28-Ig capable of activating IDO as a result of unrestrained IFN-{gamma} signaling and IFN-{gamma}-like actions of IL-6 (30). We compared IDO expression and function in pDCs after exposure to CTLA-4-Ig or CD28-Ig, the latter used in combination with SOCS3 siRNA treatment. Specific SOCS3 gene silencing was achieved in the pDCs (Fig. 5A). Untreated pDCs exposed to CTLA-4-Ig and SOCS3 siRNA-treated pDCs exposed to CD28-Ig were assayed for IDO expression (Fig. 5B) and function (Fig. 5C). In contrast to pDCs with functional SOCS3, cells with silenced SOCS3 expression exhibited levels of IDO protein and function in response to CD28-Ig comparable to those resulting from CTLA-4-Ig treatment. The effects of CTLA-4-Ig and CD28-Ig on kynurenine productions were also assayed using donors of pDCs genetically deficient in the expression of B7-1 or B7-2 (Fig. 5D). As observed previously with conventional DCs (29), IDO induction by CTLA-4-Ig was strictly dependent on the expression of B7-1 but not B7-2 molecules, whereas both types of B7 molecule were necessary for optimal IDO-dependent effects of CD28-Ig in SOCS3 siRNA-treated pDCs.



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Fig. 5. Ability of SOCS3 gene silencing to activate IDO in pDCs in response to CD28-Ig and requirement for B7 molecules. (A) RT–PCR analysis of SOCS3 expression in pDCs treated with SOCS3-specific siRNA for different times. Control cells were treated with negative control siRNA. SOCS1 gene expression was assayed as a specificity control. (B) IDO expression as assessed by western blot analysis. pDCs were treated in vitro with CTLA-4-Ig, CD28-Ig or Ig-C{gamma}3 and IDO expression was investigated with an IDO-specific antibody. pDCs were either untreated (CTLA-4-Ig) or treated with control or SOCS3-specific siRNA (Ig-C{gamma}3 and CD28-Ig). (C) Tryptophan conversion to kynurenine (mean ± SD) by pDCs treated as indicated above. (D) Kynurenine production in response to CTLA-4-Ig or CD28-Ig by pDCs deficient in B7-1 or B7-2 expression. Each experiment is representative of at least three independent experiments.

 
Cytokine production by pDCs in response to fusion proteins
The IDO promoter contains a single IFN-{gamma}-activated site specific for IFN-{gamma} as well as two non-specific IFN-stimulated response elements which can respond to IFN-{alpha} and IFN-ß as well as IFN-{gamma}. Depending on the cell type being cultured, IFN-{gamma} has been described as being up to 100 times more potent in inducing IDO expression than IFN-{alpha} or IFN-ß (11). In non-pDCs, IDO activation by CTLA-4-Ig requires IFN-{gamma} (14, 21). In pDCs treated with a combination of CD200-Ig and CpG oligodeoxynucleotide, type I IFNs appear to be required for IDO activation (10). In CD8+ DCs with silenced SOCS3 expression, IL-6 exerts IFN-like actions that lead to IDO activation (30). We measured IFN-{gamma}, IFN-{alpha} and IL-6 production in pDCs exposed to CTLA-4-Ig or CD200-Ig for 24 h as well as in SOCS3 siRNA-treated pDCs exposed to CD28-Ig (Fig. 6A). The results showed that IFN-{gamma} was the major cytokine released in response to CTLA-4-Ig, that measurable levels of IFN-{alpha} were produced in response to CD200-Ig and that IL-6 and IFN-{gamma} were released by SOCS3-deficient pDCs in response to CD28-Ig. In contrast, in control cells treated with CD28-Ig in the absence of SOCS3 silencing, the fusion protein would only induce IL-6. The production of IFN-{gamma} and IL-6 in response to fusion proteins by pDCs either intact or subjected to SOCS3 gene silencing was confirmed by intracellular cytokine staining (Fig. 6A, insets). The possible contribution of IFN-ß to IDO activation in our model system with CD200-Ig was investigated by comparative RT–PCR analysis of IFN-{alpha} and IFN-ß message expression in pDCs treated with CD200-Ig. CpG was also used as a control treatment (Fig. 6B). The results showed that CpG induced high-level expression of both IFN-{alpha} and IFN-ß. IFN-{alpha} induction by CD200-Ig was considerably lower than that by CpG but the amount of IFN-ß transcripts induced by the fusion protein was similar to that resulting from CpG treatment. These data suggest that both IFN-{alpha} and IFN-ß might contribute to IDO activation by CD200-Ig.



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Fig. 6. Expression of type I and type II IFNs as well as IL-6 in pDCs. (A) Production of IFN-{gamma}, IFN-{alpha} and IL-6 by pDCs treated with fusion proteins. CD11c+ mPDCA-1+ DCs, either as such or with silenced SOCS3 gene expression, were exposed for 24 h to CTLA-4-Ig, CD28-Ig, CD200-Ig or Ig-C{gamma}3. Culture supernatants were assayed for cytokine contents by ELISA. Insets depict the expression of 120G8 and intracellular IFN-{gamma} or IL-6 as determined by flow cytometric analysis in pDCs stimulated for 8 h with CTLA-4-Ig (for IFN-{gamma}, upper panel) and in SOCS3 siRNA-treated pDCs stimulated with CD28-Ig (for IL-6 and IFN-{gamma}, lower panels). Control cells, either untreated or treated with control siRNA or SOCS3 siRNA, were stimulated with Ig-C{gamma}3, and the percentage of cells expressing either cytokine was in all instances <0.5%. Isotype-matched irrelevant mAbs were used as controls in the analysis of intracellular cytokine expression, resulting in <1% positive cells. Data are representative of three independent experiments. (B) Expression of IFN-{alpha} and IFN-ß transcripts in pDCs exposed to CpG or CD200-Ig. CD11c+ mPDCA-1+ cells were exposed to CpG or CD200-Ig (or control Ig-C{gamma}3) for 6 h to be assayed for IFN-{alpha} and IFN-ß message expression by RT–PCR. One experiment is representative of three.

 
Cytokine requirement of IDO-dependent effects of fusion proteins on pDCs
In mouse, purified CD8 DC fractions mediate the immunogenic presentation of poorly immunogenic peptides, including the HY peptide in female hosts (13, 29, 31). Upon transfer into recipient hosts, peptide-loaded CD8 DCs initiate immunity, leading to the detection of antigen-specific skin test reactivity at 2 weeks after cell transfer. However, the addition of as few as 3% conventional DCs treated with CTLA-4-Ig (15) or pDCs treated with CD200-Ig (10) to a population of CD8 DCs inhibits priming by the latter cells through IDO-dependent mechanisms. Because of the ability of fusion proteins to activate IDO in pDCs under different conditions, we examined the possible requirement for IFN-{gamma}, type I IFNs or IL-6 in the actions of the fusion proteins associated with IDO induction. Splenic DCs were fractionated and SOCS3 gene silencing was achieved in the pDCs with siRNA technology. HY-pulsed CD8 DCs were injected either alone or in combination with 3% pDCs, either untreated or treated with CTLA-4-Ig, CD200-Ig or CD28-Ig, with or without concomitant SOCS3 siRNA treatment. CTLA-4-Ig treatment of pDCs was conducted in the presence or absence of anti-IFN-{gamma} antibody. For CD200-Ig, pDCs were recovered from wild-type or IFN-{alpha}/ßR–/– mice. SOCS3 siRNA-treated pDCs were instead exposed to CD28-Ig with or without concomitant neutralization of IFN-{gamma} or IL-6. All pDC preparations were then assayed for IDO-dependent ability to block host priming to HY by CD8 DCs, as measured by skin test assay (Fig. 7). The results showed that unconditioned pDCs (as well as CD28-Ig-treated pDCs in the absence of SOCS3 gene silencing, data not shown) failed to inhibit priming. In contrast, CTLA-4-Ig and CD200-Ig, as well as CD28-Ig in SOCS3 siRNA-treated pDCs, were all capable of inhibitory effects, which were dependent on an intact IDO function in that they were reversed by the enzyme inhibitor 1-MT. Similar to 1-MT, IFN-{gamma} neutralization ablated the inhibitory activity of CTLA-4-Ig, and so did the genetic deficiency of IFN-{alpha}/ßR in pDCs treated with CD200-Ig. For CD28-Ig, neutralization of IFN-{gamma} and blockade of IL-6 activity were each capable of negating the induction of a tolerogenic phenotype by the fusion protein in pDCs lacking SOCS3. Therefore, while autocrine type I and type II IFNs are the major cytokines involved in the respective actions of the inhibitory ligands CD200-Ig and CTLA-4-Ig, the effects of IFN-{gamma} and IL-6 may combine in SOCS3-deficient pDCs to activate IDO in response to CD28-Ig according to a pattern previously observed with non-pDCs (30).



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Fig. 7. Ligand and cytokine dependence of the suppressive activity of pDCs in the presentation of the HY peptide to female hosts by otherwise immunogenic conventional CD8 DCs. Splenic DCs were fractionated according to CD8/mPDCA-1 expression, and the CD8 fraction was pulsed with HY and transferred into recipient mice to be assayed for skin test reactivity to the eliciting peptide. The CD8 DCs were used in combination with 3% pDCs from wild-type or IFN-{alpha}/ßR–/– mice. The pDCs from wild-type mice were treated with a variety of reagents, used either singly or in combination (indicated). Control treatments for cytokine neutralization (see Methods) have been omitted for clarity. *P < 0.001, experimental versus control footpads. One experiment is representative of three.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In phenotyping IDO+ splenocytes following treatment of mice with CTLA-4-Ig, Mellor et al. (9) found that the CD11c+ B220+ CD19 pDCs contained cells intensely stained by anti-IDO antibody, as did non-plasmacytoid CD11c+ CD8{alpha}+ B220 cells, which are known to activate IDO in response to CTLA-4-Ig through autocrine IFN-{gamma} (14, 21). We have previously shown that splenic CD11c+ B220+ 120G8+ pDCs respond to CD200-Ig with increased IDO expression and function, the effect which is potentiated by CpG and requires type I IFNR signaling (10). Although these findings suggest that modulation of tryptophan catabolism may represent an important component of the tolerogenic activity of pDCs in the mouse (1), the qualitative and quantitative conditions, and the underlying mechanisms, of the induction of IDO in pDCs remain unclear.

Here we demonstrate that CD11c+ mPDCA-1+ 120G8+ cells do not express functional IDO constitutively; yet, tryptophan catabolism can be readily activated by tolerogenic ligands acting through IFN-dependent actions. Although this pattern is shared by conventional CD8+ DCs, there are both differences and similarities between pDCs and non-pDCs in this regard. The differences include a basal tolerogenic activity in the CD8+ DCs, which can be demonstrated by co-transfer with otherwise immunogenic CD8 DCs (15), the occurrence of multiple mechanisms besides IDO activation in the tolerogenic effects of CD200-Ig acting on CD8+ DCs (10) and the selective induction of IL-6 by CD28-Ig in pDCs with an intact SOCS3 function, as opposed to the mixed (IL-6 + IFN-{gamma}) response observed in CD8+ DCs (29). The similarities include co-expression of IL-6 and IFN-{gamma} by SOCS3-deficient pDCs that will contribute to IDO activation in response to CD28-Ig (30).

The modalities of IDO activation by CTLA-4-Ig [and CTLA-4 transfectants as well (13), data not shown] are intriguing for at least two different reasons. Freshly isolated pDCs express low levels of co-stimulatory molecules, including B7-1 and B7-2 (3), yet both CTLA-4-Ig and CD28-Ig were found to bind pDCs on flow cytometric analysis, and tryptophan conversion to kynurenine by CTLA-4-Ig required B7-1, similar to that observed with conventional DCs (29). pDCs represent in vivo a specialized type I IFN-producing cell (5), and owing to this production they induce an IFN-{gamma} response in different cell types, including NK and T cells (1). However, there are no major immunomodulatory effects that can be ascribed to the direct release of IFN-{gamma} from pDCs. We found that although the baseline production of this cytokine by pDCs was negligible, CTLA-4-Ig induced significant levels of IFN-{gamma} in vitro, and autocrine IFN-{gamma} was required in vivo for the tolerogenic effect of the fusion protein on the pDCs. It has been proposed that engagement of B7 on DCs by CTLA-4-expressing cells may represent an important mechanism of suppression by regulatory T cells (1114). The functional relationship between pDCs and DCs as regards induction of immunity versus tolerance is still unclear. Our current data suggest that, to meet the needs of flexibility and redundancy, both conventional DCs and pDCs can respond to B7 engagement by regulatory T cells with the production of IFN-{gamma} and initiation of the immunosuppressive pathway of tryptophan catabolism. Of interest, the IDO-inducing effect of CTLA-4-Ig is not confined to DCs and pDCs, but can also be demonstrated with CD4+ T cells (16) and neutrophils (17).

CD200 (OX2) is a broadly distributed cell-surface glycoprotein that is expressed in high amounts by DCs and interacts with an inhibitory receptor, CD200R, which is mechanistically related to immunoreceptor tyrosine-based inhibitory motif-bearing receptors (3538). In addition to CD200R (now also termed CD200R1), several cell types express a whole family of immunoregulatory molecules collectively known as CD200R-like receptors, including CD200R3 and CD200R4, which represent activating receptors that associate with immunoreceptor tyrosine-based activation motif-bearing adapter molecules (39). Both conventional DCs and pDCs possess CD200R1, but the former cells also express CD200R4 (our unpublished results). Paired receptors that consist of highly related activating and inhibitory receptors are critical in the functional modulation of several cell types, and cross-regulation of CD200R1 and CD200R4 signaling has recently been described in mast cells (38). We have previously shown that engagement of CD200R1 by CD200-Ig in both DCs and pDCs activates tolerogenic programs, with a predominance of IDO-dependent effects in the pDCs (10). In addition, our earlier studies have indicated that the induction of autocrine type I IFNs might be necessary, albeit not sufficient, for IDO activation by CD200-Ig, and CpG oligodeoxynucleotide was found to potentiate the action of CD200-Ig. Our current data demonstrate that, in the absence of CpG, autocrine type I IFNs remain the key element in the induction of a tolerogenic phenotype by CD200-Ig in pDCs. Given the limited production of IFN-{alpha} observed in vitro and the ability of CD200-Ig to induce IFN-ß message expression to an extent comparable to CpG, it is possible that both types of type I IFN contribute to IDO activation by CD200-Ig. However, because of the limited ability of type I IFNs to activate IDO as compared with IFN-{gamma}, it is possible that signaling through CD200R1 in pDCs initiates a multiplicity of effects that synergize in initiating tryptophan catabolism. It is known that CD200R1 associates with the SH2-containing inositol phosphatase (40). At variance with conventional DCs, CD200R1 signaling in pDCs may not be restrained by counter-regulatory signals involving phosphorylation cascades (41) and activating receptors such as CD200R4 (36).

pDCs are critical for the generation of plasma cells and antibody responses through their coordinate release of type I IFNs and IL-6 (42), and a subset of pDCs in the mouse, the Ly49Q+ pDCs, produce high levels of IL-6 upon CpG stimulation (43). We found that exposure of pDCs to CD28-Ig resulted in significant and selective production of IL-6. In conventional DCs treated with CD28-Ig, both IFN-{gamma} and IL-6 are released, but an early and preponderant production of IL-6 prevents the IFN-{gamma}-driven expression of tryptophan catabolism (29). Via direct effects and autocrine IL-6 (29, 30), CD28-Ig up-regulates SOCS3, which may be responsible for the inhibition of STAT-dependent signaling by IFNs (34). In addition to modulation of IFN-{gamma}-inducible genes, a series of recent studies with various experimental approaches have demonstrated that SOCS3 is involved in the prevention of IFN-{gamma}-like responses in hepatocytes and macrophages stimulated with IL-6 (4446). It has been suggested that, in the absence of SOCS3, IL-6 may become immunosuppressive, activating genes typically induced by IFNs (47). In line with this hypothesis, we have previously shown that silencing SOCS3 expression in conventional DCs renders CD28-Ig immunosuppressive because of strong activation of IDO by unopposed IFN-{gamma} signaling and IFN-like actions of IL-6 (30). Our current studies demonstrate that the absence of SOCS3 in pDCs allows CD28-Ig to promote the release of IFN-{gamma}, the activation of IDO and the emergence of a tolerogenic phenotype that is sustained by both IFN-{gamma} and IL-6. This might represent the first demonstration of the role of SOCS proteins, specifically SOCS3, in the modulation of cytokine activity in pDCs. In addition, the current data support the concept that, both in DCs and pDCs with dysfunctional SOCS3, the consequence of prolonged IL-6 signaling is not simply unrestrained induction of IL-6-responsive genes but the activation of a different set of genes. Because pDCs are involved in regulating the balance between tolerance and immunity (1), the finding that SOCS3 influences IL-6 transcriptional program in these cells may be relevant to the recognition of physiopathologic conditions in which SOCS3 could be poorly expressed (47). Finally, our current data extend to pDCs, the paradigm of bidirectional signaling along the B7-CD28 co-receptor pathway, confirming that both B7-1 and B7-2 are required for optimal effects of CD28 as a ligand of B7 (29). Although both CD28 and CTLA-4 interact with B7 by virtue of the MYPPPY sequence, each CTLA-4 dimer can bind two different B7 homodimers, thereby forming a stable zipper-like complex not seen with CD28, which can bind only one B7 homodimer at a time. This difference might contribute to the differential B7-1/B7-2 requirements for CTLA-4 and CD28 effects as ligands of B7 molecules and regulators of SOCS3 expression (30).

In conclusion, our current findings reinforce the concept that modulation of tryptophan catabolism in response to CTLA-4 engagement of B7 is an effector mechanism of pDCs as mediators of tolerance but, at the same time, suggest that this mechanism is only one of several possible ways of IDO regulation by ligands and cytokines. Novel and thus far unexplored roles are demonstrated for autocrine IFN-{gamma} and IL-6, as well as SOCS proteins, in pDCs. The bulk of these studies may be useful to a better understanding of the immunobiology of pDCs and to the implementation of immunotherapy protocols targeting the CD28/B7 co-stimulatory axis in these cells.


    Acknowledgements
 
This work was supported by a grant from the Italian Association for Cancer Research (to P.P.).


    Abbreviations
 
DC   dendritic cell
IDO   indoleamine 2,3-dioxygenase
1-MT   1-methyl-D,L-tryptophan
pDC   plasmacytoid dendritic cell
RT   reverse transcription
siRNA   small interfering RNA
SOCS   suppressor of cytokine signaling

    Notes
 
Transmitting editor: K. M. Murphy

Received 6 June 2005, accepted 18 August 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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