Regulatory Cells by Human Dendritic CellsSupports the Homeostatic Generation of T The SOCS3-Independent Expression of IDO2
Roberto M. LemoliPedro Romero, George C. Prendergast, Antonio Curti and Richard Metz, Cecilia Evangelisti, Lisa Laury-Kleintop,Valentina Salvestrini, Mariangela Lecciso, Camilla Jandus, Sara Trabanelli, Darina Ocadlíková, Marilena Ciciarello,
The SOCS3-Independent Expression of IDO2 Supports theHomeostatic Generation of T Regulatory Cells by HumanDendritic Cells
Sara Trabanelli,*,1 Darina O�cadlıkova,* Marilena Ciciarello,* Valentina Salvestrini,*
Mariangela Lecciso,* Camilla Jandus,† Richard Metz,‡ Cecilia Evangelisti,*
Lisa Laury-Kleintop,x Pedro Romero,† George C. Prendergast,x Antonio Curti,*,2 and
Roberto M. Lemoli*,2,3
Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the
tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans,
different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern
of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in cir-
culating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not
modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and
translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in
patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and
plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas
IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state
conditions and may contribute to the homeostatic tolerogenic capacity of DCs. The Journal of Immunology, 2014, 192: 1231–1240.
Human blood dendritic cells (DCs) represent 0.1–1% oftotal circulating PBMCs and can be divided into twomajor subsets: myeloid DCs (mDCs), expressing either
BDCA1 (CD1c) or BDCA3 (CD141), and plasmacytoid DCs
(pDCs) (1). Because of their rarity, studies of human DC func-tionality were difficult before the availability of protocols for DCdifferentiation from CD34+ stem/progenitor cells (2) and mono-cytes (3). Both circulating and in vitro–differentiated DCs sharethe main functional properties of APCs by priming naive T cellsand inducing immune tolerance (4). The expression of the im-munosuppressive enzyme IDO1 is one of the DC tolerogenicmechanisms. IDO1 catalyzes tryptophan degradation along thekynurenine pathway (5), resulting in the inhibition of T cell ac-tivation (6) and in the expansion of T regulatory cells (Tregs) (7).The tolerogenic role of IDO1 has been described during maternaltolerance toward the allogeneic fetus (8), regulation of autoim-mune disorders (9), suppression of transplant rejection (10), andtumor immune escape (11, 12).Recently, it has been shown that the first and rate-limiting step
of tryptophan degradation is catalyzed not only by IDO1, but alsoby IDO2 (13, 14). IDO2 is expressed in human pancreatic, colon,gastric, and renal cancer, and in cell lines. In pancreatic cancercell lines, IDO2 protein expression is inducible after exposurewith IFN-g (15, 16). However, it is not clear whether IDO2 isfunctional in cancer cells. Although the functional expressionof IDO2 in mouse DCs has been described (14), it is not yetestablished whether different subsets of human DCs expressIDO2, whether that expression is functional, and how IDO2 isregulated. To address these questions, we analyzed the two mainsubsets of circulating DCs from healthy donors for IDO1 andIDO2 expression and compared their induction, degradation, andfunction. Then, we assessed the expression of IDO1 and IDO2in blood DCs of patients suffering from inflammatory arthritis.Taken together, our results suggest that, at variance with inducibleIDO1, IDO2 is a functional immunosuppressive enzyme that maycontribute to human DC constitutive tolerance in homeostaticconditions.
*Department of Specialistic, Diagnostic, and Experimental Medicine, Institute of He-matology “Seragnoli,” University of Bologna, 40138 Bologna, Italy; †Ludwig Centerfor Cancer Research of the University of Lausanne, 1011 Lausanne, Switzerland;‡NewLink Genetics, Ames, IA 50010; and xLankenau Institute for Medical Research,Wynnewood, PA 19096
1Current address: Ludwig Center for Cancer Research of the University of Lausanne,Lausanne, Switzerland.
2A.C. and R.M.L. contributed equally to this work.
3Current address: Department of Medicine, University of Genoa, Genoa, Italy.
Received for publication March 19, 2013. Accepted for publication November 28,2013.
This work was supported by the Italian Leukemia Association and National Institutesof Health Grants CA109542 and CA159337.
S.T. designed and performed the experiments, analyzed the data, and wrote thepaper; D.O. performed flow cytometry and functional tests; M.C. performed West-ern blot assay; V.S. performed nucleofection; M.L. performed DC purification ofhealthy subjects; C.J. performed DC purification of patients; R.M. developed anti-IDO1 and anti-IDO2 Abs and reviewed the manuscript; C.E. performed IDO1 real-time PCR; L.L.-K. developed anti-IDO1 and anti-IDO2 Abs; P.R. provided pa-tients’ samples and critically reviewed the manuscript; G.C.P. critically reviewedthe manuscript; A.C. and R.M.L. designed the experiments, supervised the study,critically reviewed the manuscript, and gave the final approval for submission ofthe manuscript.
Address correspondence and reprint requests to Dr. Sara Trabanelli, Institute ofHematology “Seragnoli,” Via Massarenti 9, 40138 Bologna, Italy. E-mail address:[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; mDC, myeloid DC; Mo-DC,monocyte-derived DC; 1-MT, 1-methyltryptophan; pDC, plasmacytoid DC; siRNA,small interfering RNA; Treg, T regulatory cell.
Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
Cells were obtained from healthy donor buffy coats. PBMCs were separatedby density gradient centrifugation (Ficoll-Hypaque; Amersham Bioscience,Piscataway, NJ). Cells were cultured in RPMI 1640 medium (Lonza, Milan,Italy) supplemented with 10% heat-inactivated FBS (Life Technologies/Invitrogen, Carlsbad, CA), 2 mM L-glutamine, 100 U/ml penicillin, and100 mg/ml streptomycin (MP Biomedicals, Verona, Italy) at 37˚C in 5%CO2. CD14
+, CD3+, and CD4+CD25+ cells were purified by magnetic sepa-ration column (Miltenyi Biotec, Bergisch Gladbach, Germany) according tothe manufacturer’s instructions.
Patients
Cells were obtained from patients suffering either from rheumatoid arthritis,psoriatic arthritis, or ankylosing spondylitis. All patients were in activedisease and signed an informed consent. PBMCs were separated by densitygradient centrifugation (Ficoll-Hypaque) and used for the purification ofcirculating DCs.
DC enrichment and DC generation
Circulating DCs were enriched from PBMCs using a specific immuno-magnetic DC isolation kit (Milteny Biotec), following the manufacturer’sinstructions. For purifying mDCs we used a CD1c (BDCA-1)+ DC iso-lation kit; for purifying pDCs we used the a pDC isolation kit; for puri-fying all circulating DCs we used the blood DC isolation kit II. Whereindicated, mDCs and pDCs were cultured overnight in complete mediumin the absence or presence of IL-1b, TNF-a, and IL-6 with or withoutPGE2.
Owing to the low frequency of circulating DCs, monocyte-derived DCs(Mo-DCs) or bone marrow–derived DCs are often used as a model of DCs.Indeed, Mo-DCs/bone marrow–derived DCs were used to achieve highlevels of infection (17) or of expression of enzymes in DCs (18).
Mo-DCs were generated by a 5-d culture of CD14+ cells in completemedium supplemented with 50 ng/ml GM-CSF (Endogen, Woburn, MA)and 800 U/ml IL-4 (Endogen), at 37˚C in 5% CO2, as previously described(19). For maturation, day 5 Mo-DCs were cultured with GM-CSF and IL-4and incubated for 48 h in the presence of 1) complete medium, 2) 1 mg/mlCD40L (BioLegend, San Diego, CA), 3) 1 mg/ml LPS (Sigma-Aldrich,St. Louise, MO), 4) 1 mg/ml LPS with 100 U/ml IFN-g (Endogen), or5) combinations of a mixture of cytokines made of 10 ng/ml TNF-a(Endogen), 10 ng/ml IL-6 (Endogen), 10 ng/ml IL-1b (Endogen), and 1mg/ml PGE2 (Endogen). Where indicated, protein transcription, proteintranslation, and proteasome activity were inhibited by the addition of ac-tinomycin D (1 mg/ml), cycloheximide (10 mg/ml; both from Sigma-Aldrich), or Velcade (Millennium Pharmaceuticals, Cambridge, MA): 3mM for immature DCs, 20 mM for DCs matured without PGE2, and 30 mMfor DCs matured with PGE2 1 h before DC maturation (20).
DC immunophenotype
Dual-color immunofluorescence was performed to characterize Mo-DCsusing the following panel of mAbs: PE- or FITC-conjugated anti-humanHLA-DR (clone L242; BD Pharmingen); PE- or FITC-conjugated anti-human CD1a (clone HI149; BioLegend); PE- or FITC-conjugated anti-human CD86 (clone IT2.2; BioLegend); FITC-conjugated anti-humanCD40 (clone HB14; BioLegend); PE-conjugated anti-human CD80(clone 2D10; BioLegend); FITC-conjugated anti-human CD83 (cloneHB15e; BioLegend); and PE-conjugated anti-human CCR7 (clone 150503;BD Pharmingen). Negative controls were isotype-matched irrelevant mAbs(BD Pharmingen, BioLegend). Cells were analyzed by using FACScanequipment (Becton Dickinson). A minimum of 10,000 events was collectedin list mode on FACScan software.
IDO1 and IDO2 expression
Total RNA was reverse transcribed using a Promega ImProm II kit andrandom hexamers in a 20 ml final volume according to the manufacturer’sinstruction (Promega, Madison, WI). Quantitative real-time PCR wasperformed using an ABI Prism 7900 sequence detection system (AppliedBiosystems, Foster City, CA). Quantitative real-time PCR data were ana-lyzed using the 22DDCt method. The relative level of a specific mRNA forIDO1 and IDO2 was calculated by subtracting Ct values of the controlgene (GAPDH) from the Ct values of the specific gene. Universal humanRNA (Stratagene/Agilent Technologies, Santa Clara, CA) was used asreference and taken as value of 1. The IDO1 assay ID is Hs00158027_m1;the IDO2 assay ID is Hs00401201_m1 or Hs01589373_m1; and theGAPDH assay ID is Hs00266705_g1.
Western blot analysis
Cell lysates or immunoprecipitation products were separated by SDS gelelectrophoresis, transferred onto nitrocellulose membrane (GE HealthcareUK, Buckinghamshire, U.K.) and subjected to Western blotting. Mem-branes were saturated for 1 h at room temperature in blocking buffer (13PBS, 0.1% Tween 20, 5% nonfat milk) and then incubated overnight at 4˚Cwith the specific primary Abs as described (15). The specificity of the anti-IDO1 and the anti-IDO2 Abs was guaranteed by the method used to de-velop them. Briefly, IDO1 and IDO2 antisera were raised with a mixtureof recombinant murine and human polypeptides. Abs were screened forreactivity against the immunizing Ag by ELISA and Western blotting.Samples with the highest titers were purified by affinity chromatography:the GST-unbound fraction was passed over a GST-IDO1 (or GST-IDO2)column. Then, the unbound material collected from this column was af-finity purified on a GST-IDO2 (or GST-IDO1) column containing bothhuman and mouse recombinant polypeptide. The resulting affinity-purifiedpolyclonal Abs were analyzed by Western blotting and confirmed to beIDO2-specific with no crossreactivity to IDO1 and IDO1-specific with nocrossreactivity to IDO2 (21). The specificity of the two Abs was alsoconfirmed on human cells as shown in Supplemental Fig. 1.
After threewashes, blots were incubated for 1 h at room temperaturewithperoxidase-conjugated secondary Ab (1:10,000; Amersham Biosciences,Uppsala, Sweden) and washed as above. Signal intensities in single blotsfrom at least three separate experiments were measured by means ofChemiDoc-It instrument equipped with a dedicated software (LaunchVIsionWorks LS from Euroclone). Protein expression was quantified byband densitometric analysis using ImageJ 1.44p launcher software (Na-tional Institutes of Health, Bethesda, MD). Statistical analysis of dif-ferences among densitometric band intensities was performed on at leastthree separate experiments.
SOCS3 immunoprecipitation
For immunoprecipitation, protein 4B-Sepharose beads (GE HealthcareUK) were coated with SOCS3 Ab (Santa Cruz Biotechnology, Heidelberg,Germany) and incubated with 500 mg whole-cell lysate overnight at 4˚C.Beads were washed three times and centrifuged at 12,000 3 g for 10 min.
Small interfering RNA transfection of DCs
Immature DCs were transfected with 250 nM control siRNA, 250 nM IDO1-specific siRNA, or 125 nM IDO2-specific siRNA using standard nucleo-fection (22). Briefly, 2 3 106 DCs were transfected for each condition byusing the U02 program. A mixture of four gene-specific siRNAs were usedto silence IDO1 and IDO2 (ON-TARGETplus SMARTpool by Dharma-con, Lafayette, CO). For IDO1, the target sequences were: 1) 59-UCAC-CAAAUCCACGAUCAU-39, 2) 59-UUUCAGUGUUCUUCGCAUA-39, 3)59-GUAUGAAGGGUUCUGGGAA-39, 4) 59-GAACGGGACACUUUGCU-AA-39; for IDO2: 1) 59-CAAACUUCCUCAAUUGAUU-39, 2) 59-UUG-GAAAGCUAUCACAUAU-39, 3) 59-GAGUAUGGCUUUCUUCUUC-39, 4)G59-CACCCAGUUGAAGUUUAA-39. Nontargeting pool siRNAs designedand tested for minimal targeting of human genes were used as negativecontrol (by Dharmacon). After an overnight incubation, DCs were maturedfor 48 h.
Enzyme activity of IDO isoforms
The amount of L-kynurenine in culture supernatants was measured witha spectrophotometric analysis, as previously described (23). Briefly, Mo-DCs were washed, resuspended in HBSS supplemented with 500 mML-tryptophan (Sigma-Aldrich) and incubated (where indicated) with orwithout the IDO inhibitor 1-methyltryptophan (1-MT; D- or L-isoform;1 mM; Sigma-Aldrich). Supernatants were harvested after 4 h and mixedwith 30% trichloroacetic acid (2:1), vortexed, and centrifuged at 8000 3 gfor 5 min. Subsequently, this solution was added to Ehrlich reagent (1:1;Sigma-Aldrich) in a 96-well plate. Triplicate samples were run against astandard curve of defined kynurenine concentrations (0–100 mM; Sigma-Aldrich). OD was measured at 490 nm using a Multiskan EX microplatereader (Thermo Electron, Vantaa, Finland).
Induction of Tregs by DCs
DCs were cultured with autologous CD3+ T cells (1:10) for 1 d, with orwithout the IDO inhibitors 1-MT-D or 1-MT-L (1 mM; Sigma-Aldrich)where indicated. For immunophenotype studies, tricolor immunofluores-cence was performed using FITC-conjugated anti-human CD4 (clone RPA-T4), PE-conjugated anti-human Foxp3 (clone 206D), and allophycocyanin-conjugated anti-human CD25 (clone BC96; BioLegend). For cell-surfacestaining, 1 3 105 cells/100 ml were incubated in the dark for 20 min at 4˚C
1232 IDO2 STABLY SUPPORTS HOMEOSTATIC DC TOLERANCE
with mAbs in PBS-1% BSA. Subsequently, for Foxp3 intracellular staining,cells were incubated at room temperature in the dark for 20 min with Fix/Permbuffer followed by 15min with Perm solution and an additional 30 minwiththe mAb. After twowashes, samples were analyzed using a BD FACSCantoII (BD Biosciences). A minimum of 10,000 events was collected in listmode on FACSDiva software. To test their suppressive activity, purifiedCD4+CD25+ T cells (104/well) were irradiated and added to cultures con-sisting of CFSE-labeled CD3+ T cells (105/well) as responders, stimulatedby 1 mg/ml PHA (Sigma-Aldrich). After 5 d, cultures were analyzed usinga BD FACSCanto II and the proliferation index was calculated using FCSExpress 4 software (24).
Statistical analysis
Results are expressed as means6 SEM. Where indicated, differences werecompared using a Student t test. A p value ,0.05 was considered statis-tically significant.
ResultsmDCs and pDCs differentially express IDO enzymes
First, we asked whether IDO enzymes are expressed in humancirculating DCs and how the inflammatory mediator PGE2 regulatesIDO1 and IDO2 mRNA and protein expression. PGE2 is known toconfer to DCs both immunogenic and tolerogenic features (25)and to regulate IDO1 expression (26, 27). Thus, we isolated CD1c+
mDCs and CD303+ pDCs and evaluated IDO1 and IDO2 expressionin steady-state condition (i.e., immediately after purification) andafter overnight incubation in absence of exogenous stimuli or in thepresence of a cytokine mixture including IL-1b, IL-6, and TNF-a,commonly used to mature DCs, with or without PGE2 (28).As shown in Fig. 1A, at the mRNA level, PGE2 enhanced both
IDO1 and IDO2 expression in mDCs. On the contrary, in pDCsPGE2 had no effect on either IDO1 and IDO2 mRNA expression(Fig. 1B). At the protein level, after overnight incubation, onlymDCs expressed IDO1 protein and this expression was dependenton PGE2 (Fig. 1C–F). Conversely, IDO2 protein was expressedin both mDCs and pDCs, either in steady-state conditions orafter incubation with inflammatory stimuli, and its expression wasPGE2-independent (Fig. 1C–F).We concluded that, in contrast to IDO1, IDO2 protein is expressed
by both mDCs and pDCs and that its expression does not correlatewith its mRNA level in a PGE2-independent manner.
The role of PGE2 in regulating IDO1 and IDO2 expression inMo-DCs
To better investigate the mechanisms underlying IDO1 and IDO2expression in human DCs during maturation with inflammatorycytokines/mediators, we used Mo-DCs as a model (17, 18). Inparticular, we analyzed IDO1 and IDO2 expression before and afterDC maturation with combinations of three out of four cytokines ofthe mixture. As expected, we found that in absence of TNF-a andPGE2, DCs were not induced to full maturation as evaluated phe-notypically (Fig. 2A). Immature DCs were always taken as controlsamples and expressed HLA-DR, 71.5 6 5.8%; CD1a, 74.5 64.9%; CD86, 29.5 6 6.2%; CD40, 56.3 6 5.1%; CD80, 53.7 65.8%; CD83, 4.5 6 3.2%; and CCR7, 11.2 6 3.2% (data notshown). At the mRNA level, TNF-a and, more significantly, PGE2,contributed to IDO1 upregulation, and only PGE2 was required forIDO2 upregulation (Fig. 2B). As shown in Fig. 2C, the effect ofPGE2 on IDO1 and IDO2 induction was dose-dependent. Similarto circulating DCs (see Fig. 1C–F), IDO1 protein level followed itstranscriptional level, whereas IDO2 protein expression was inde-pendent from any stimuli and did not parallel its transcriptionallevel (Fig. 2D–G). In fact, whereas the removal of PGE2 inhibitedalso the upregulation of IDO1 protein (Fig. 2D–F), which was PGE2
dose-dependent (Fig. 2E–G), the expression level of IDO2 proteinremained unchanged also in the absence of PGE2 (Fig. 2D–G).
Taken together, these data indicate that PGE2 upregulatesboth IDO1 and IDO2 transcription but not IDO2 protein ex-pression also in the Mo-DC experimental model. Therefore,Mo-DCs were found suitable for further investigating IDO1and IDO2 regulation.
In human DCs IDO2 is differentially regulated than IDO1
To further elucidate how IDO2 and IDO1 are regulated in humanDCs, we added either actinomycin D or cycloheximide as specificinhibitors of protein transcription or translation, respectively, before
FIGURE 1. Expression of IDO1 and IDO2 in blood DCs. Human mDCs
and pDCs were isolated from healthy donors’ PBMCs by using magnetic
beads. DCs were tested freshly purified or after an overnight (o.n.) incu-
bation in RPMI 1640 alone or in the presence of IL-1b (10 ng/ml), IL-6
(10 ng/ml), and TNF-a (10 ng/ml) with or without (w/o) PGE2 (1 mg/ml).
Cells were lysed and RNA and protein were extracted. mRNA expression
of IDO1 and IDO2 (normalized to GAPDH) was evaluated by real-time
RT-PCR in mDCs (A) and pDCs (B). Universal human RNA was used as
reference and taken as value of 1. Data are expressed as the means 6 SEM
of 12 independent experiments. *p , 0.05, **p , 0.01 versus o.n. or
versus o.n. w/o PGE2, where indicated. (C–F) IDO1, IDO2, and b-actin
(bACT) protein expression in fresh or cultured mDCs (C, E) and pDCs (D,
F) was determined by Western blotting. (C and D) Data shown are repre-
sentative results from one of four independent experiments, each per-
formed by bulking protein lysate of either mDCs or pDCs of the same
three donors. (E and F) Protein expression was quantified by band densi-
tometric analysis and expressed as the ratio between IDO1 (or IDO2) and
b-actin band intensity. Data are expressed as the means 6 SEM of four
independent experiments. *p , 0.05, **p , 0.01 versus o.n. or versus o.n.
DC maturation was induced with or without PGE2. As shown inFig. 3, both inhibitors were effective in blocking the expression ofIDO1, but not IDO2. These results indicate that the expression ofIDO1 protein, but not IDO2, relies on its continuous synthesis,thereby suggesting that only IDO1 is continuously transcribed,translated, and degraded, in contrast to IDO2, which, oncesynthesized, appears to be stable. We then asked whether IDO2has a different regulation of protein degradation in comparisonwith IDO1. It is known that IDO1 contains two tyrosines withintwo canonical ITIM sequences that direct protein turnover throughSOCS3-mediated ubiquitination and proteasomal degradation (29).Thus, we asked whether human IDO2 could bind SOCS3-likeIDO1. Immature DCs and DCs matured with or without PGE2
were lysed and SOCS3 was immunoprecipitated with an anti-SOCS3 Ab, followed by sequential immunoblotting with eitherIDO1- or IDO2-specific Abs. As shown in Fig. 4A (left panel)and 4B (open bars), after SOCS3 immunoprecipitation, IDO1protein was detectable only in PGE2-treated DCs, whereas IDO2protein was always absent. Because we previously observed that the
highest expression of IDO1 occurred in the presence of PGE2 (seeFig. 2B–G), the finding that, under this condition, IDO1 bound toSOCS3 was unexpected, because this would drive IDO1 to protea-somal degradation. Therefore, we hypothesized that in the absence ofinflammatory stimuli, such as PGE2, IDO1 turnover would be sorapid that we could not find IDO1 bound to SOCS3 because it wouldbe already degraded by the proteasome. To confirm this hypothesis,we used bortezomib (Velcade) as a specific proteasome inhibitorbefore DC maturation (20). As shown in Fig. 4A (right panel) and4B (filled bars), after SOCS3 immunoprecipitation, IDO1 was de-tectable in immature DCs or DCs matured in the absence of PGE2,but its expression increased after exposure to PGE2. On the contrary,IDO2 protein was not detectable. Because SOCS3 is also subjectedto proteasomal degradation, we verified the effective enhancement ofIDO1, and not IDO2, expression after proteasome inhibition byquantifying IDO1 (Fig. 4C) and IDO2 (Fig. 4Ci) protein expressionin whole-cell lysates before SOCS3 immunoprecipitation.Taken together, these results indicated that human IDO1, but not
IDO2, is degraded by proteasome as a result of targeting by SOCS3.
FIGURE 2. Expression of IDO1 and IDO2 in Mo-DCs.
Human CD14+ monocytes were purified form healthy
donors’ PBMCs by using magnetic beads and cultured 5 d
with 50 ng/ml GM-CSF and 800 U/ml IL-4 to obtain im-
mature Mo-DCs. DC phenotype, mRNA expression of
IDO1 and IDO2 (normalized to GAPDH), and protein
expression of IDO1, IDO2, and b-actin (bACT) were
evaluated by flow cytometry, real-time RT-PCR, and
Western blotting, respectively. (A, B, D, and F) Immature
Mo-DCs were cultured for 48 h with the complete mixture
or with combinations of three of four cytokines of the
mixture (w/o indicates without) and evaluated for pheno-
type (A) and IDO1 and IDO2 expression (B, D, and F).
(C, E, and G) Mo-DCs were matured with 10 ng/ml IL-1b,
10 ng/ml IL-6, and 10 ng/ml TNF-a in the presence of de-
creasing doses of PGE2: 1 mg/ml (the normal concentration
in the mixture), 0.02 mg/ml (1:50 of the normal concen-
tration), 0.002 mg/ml (1:500 of the normal concentration),
0.0004 mg/ml (1:2500 of the normal concentration), or
0 mg/ml (without [w/o] PGE2) and evaluated for IDO1 and
IDO2 expression. Universal human RNA was used as ref-
erence and taken as a value of 1. Data are expressed as the
means 6 SEM of 12 independent experiments. *p , 0.05,
**p , 0.01 versus Mo-DCs matured with the complete
mixture. (D and E) Western blotting data show representa-
tive results from five independent experiments, and (F, G)
protein expression was quantified by band densitometric
analysis and expressed as the ratio between IDO1 (or IDO2)
and b-actin band intensity. Data are expressed as the
means 6 SEM of five independent experiments. *p ,0.05, **p , 0.01 versus complete mixture.
1234 IDO2 STABLY SUPPORTS HOMEOSTATIC DC TOLERANCE
Human blood DCs generate a population of Tregs by means ofIDO enzymes
Given the stable expression of IDO2 under homeostatic conditions,we hypothesized a stable functionality for IDO2 expression in DCtolerance. To test this hypothesis, we cocultured mDCs and pDCswith T cells, in the presence or absence of either the L isoform orD isoform of the IDO-specific inhibitor 1-MT. Coculture of T cellswith blood DCs expressing both IDO isoforms increased thepercentage of CD4+CD25+Foxp3+ cells, as compared with base-line (Fig. 5A, 5B). The addition of 1-MT to these cocultures de-creased the population of CD4+CD25+Foxp3+ cells to basal level(Fig. 5A, 5B). The 1-MT–dependent induction of this phenotypicpattern suggested that CD4+ T cells, by means of IDO enzymeexpression in blood DCs, acquire a CD4+CD25+Foxp3+ Tregphenotype. To validate their Treg nature, they were coculturedwith T cells stimulated by PHA. As shown in Fig. 5C, T cellproliferation was significantly reduced when either mDC- orpDC-obtained CD4+CD25+ T cells were added to cell cultures.These data support the hypothesis that CD4+CD25+Foxp3+
T cells induced by blood DCs expressing both IDO isoforms canretain their immunosuppressive activity and be considered bonafide Tregs. As the L isoform and the D isoform of the compound1-MT preferentially block IDO1 and IDO2 (14, 30, 31), re-spectively, our findings confirm the role of IDO1 in Treg induction byhuman DCs (32), but they also strongly suggest, to our knowledge forthe first time, a similar role for IDO2. However, because 1-MT-Linhibited the capacity to generate Tregs also in the absence ofIDO1, we tested the specificity of its inhibition in absence of IDO1expression (and the specificity of 1-MT-D in the absence of IDO2expression), such as after its silencing by a specific siRNA. Wefound that in the absence of one of the two IDO enzymes, theinhibition caused by 1-MT acted indifferently on the only enzymeexpressed (Supplemental Fig. 2).
IDO2 also is functionally active in DCs
To obtain further evidence of IDO2 functionality in Treg inductionby human DCs, a further set of experiments was performed withMo-DCs. First, we assessed whether the addition of 1-MT-L or
FIGURE 3. IDO1 and IDO2 regulation in Mo-DCs. Protein expression of IDO1, IDO2, and b-actin (bACT) was evaluated by Western blotting (A) and
quantified by densitometric analysis (B) in immature Mo-DCs and in Mo-DCs treated with 1 mg/ml actinomycin d (Act-d), to inhibit gene transcription, or
10 mg/ml cycloheximide (Chx), to inhibit protein translation, 30 min before maturation with the cytokine mixture either without (w/o) or with PGE2 (1 mg/
ml). (B) Protein expression was quantified by band densitometric analysis and expressed as the ratio between IDO1 (or IDO2) and b-actin band intensity.
Data are expressed as the means 6 SEM of four independent experiments. **p , 0.01 versus immature.
FIGURE 4. IDO1 and IDO2 degradation in Mo-DCs. (A and B) Expression of IDO1, IDO2, and SOCS3 was evaluated by Western blotting after SOCS3
immunoprecipitation. (A) Immature Mo-DCs were treated for 24 h with RPMI 1640 (immature) or with the cytokine mixture either with or without PGE2,
then DCs were lysed and SOCS3 was immunoprecipitated with an anti-SOCS3 Ab (right) 1 h before that treatment. Velcade (3 mM for immature DCs,
20 mM for DCs matured without PGE2, and 30 mM for DCs matured with PGE2) was added to immature Mo-DC cultures to inhibit proteasomal activity,
then DCs were treated as above (i.e., cultured 24 h either with RPMI 1640 or the cytokine mixture either with or without PGE2), lysed, and SOCS3 was
immunoprecipitated with an anti-SOCS3 Ab. Data shown are representative of results from one of three independent experiments. (B) Protein expression was
quantified by band densitometric analysis and expressed as the ratio between IDO1 (or IDO2) and SOCS3 band intensity. Before SOCS3 immunoprecip-
itation, IDO1 (C) and IDO2 (Ci) protein expression was quantified by band densitometric analysis in whole-cell lysates and expressed as the ratio between
IDO1 (or IDO2) and b-actin band intensity. Data are expressed as the means 6 SEM of three independent experiments. **p , 0.01 versus without Velcade.
1-MT-D had an effect on IDO1 or IDO2 activity. As shown in Fig.6A and 6B, either the enzymatic activity (Fig. 6A) or the capacityof generating CD4+CD25+Foxp3+ T cells (Fig. 6B) was affectedby the addition of 1-MT-L and 1-MT-D, suggesting, also in Mo-DCs, an active role for IDO2 in kynurenine formation and Treggeneration. Thus, to deeply investigate the activity of IDO2,because Mo-DCs, differently from circulating DCs, allow theintroduction of siRNAs through nucleofection, we silenced IDO1and IDO2 by siRNAs for the second set of experiments. Beforematuration with PGE2, DCs were treated with siRNA specific forIDO1 or IDO2 and then tested for kynurenine production andTreg generation. Our results showed that these targeted siRNAsstrongly downregulated IDO1 and IDO2 mRNA and protein, asexpected (Fig. 6C–E). Accordingly, kynurenine concentrationdecreased after silencing either enzyme as compared with thecontrol siRNA (Fig. 6F), thus demonstrating the active functionof both IDO1 and IDO2 in this setting. Moreover, silencing theseenzymes reduced the number of CD4+CD25highFoxp3+ T cellsafter culture of DCs with CD3+ T cells (Fig. 6G, 6H). How-ever, the obtained CD4+CD25highFoxp3+ T cells, purified andcocultured with T cells stimulated by PHA, retained their ca-pacity to suppress T cell proliferation (Fig. 6I), thus validatingtheir Treg nature and demonstrating to our knowledge for the firsttime a role not only for IDO1 but also for IDO2 in Treg gener-ation.
Taken together, our findings show that, in addition to IDO1,also IDO2 is enzymatically and biologically active in DCs.
IDO1 and IDO2 are expressed during in vivo inflammation
Because our data indicated that IDO2 is present in homeostaticconditions, whereas IDO1 expression requires the addition in vitro
of proinflammatory mediators, we hypothesized that IDO2 isa mechanism always active in DCs to maintain a basal level oftolerance, whereas IDO1 is a further mechanism induced byinflammation. To demonstrate our hypothesis we purified bloodDCs from healthy subjects and from subjects suffering frominflammatory arthritis and evaluated IDO1 and IDO2 expression.As shown in Fig. 7A, at the mRNA level, the addition ofproinflammatory mediators to healthy blood DCs resulted in theupregulation of either IDO1 of IDO2. Patients suffering frominflammatory arthritis, specifically rheumatoid arthritis, psoriaticarthritis, or ankylosing spondylitis expressed higher mRNAlevels of both IDO1 and IDO2 in comparison with healthysubjects (Fig. 7B), strongly suggesting that the in vitro manip-ulation of DCs with proinflammatory mediators actually mimicsthe in vivo situation during inflammatory diseases. However,despite that in vivo inflammation enhanced both IDO1 and IDO2mRNA with respect to steady-state conditions (Fig. 7B), theprotein analysis revealed that IDO2 protein expression did notfollow its transcriptional level both in vitro and, more impor-tantly, in vivo (Fig. 7C, 7D). Indeed, IDO2 protein is expressedat the same level in healthy subjects and in arthritis patients (Fig.7C, 7D), who may be considered a patient population with sig-nificantly increased inflammatory conditions. Moreover, at theprotein level, IDO2 is expressed in healthy subjects and its ex-pression persists at the same level in arthritis patients, whereasIDO1 is absent in the steady-state condition (i.e., in healthysubjects) but is expressed during an inflammatory disease (Fig.7C, 7D).Taken together, our data suggest that in humans IDO2 protein
may represent a constitutive mechanism of DC tolerance, whichis active both in steady-state and inflammatory conditions, whereas
FIGURE 5. Treg generation by blood DCs. Circulating
mDCs and pDCs were cultured with autologous CD3+
T cells (1:10) for 24 h in the presence or absence of 1-MT-L
or 1-MT-D (1 mM). At the end of culture, cells were stained
for CD4, CD25, and Foxp3. Foxp3+ T cells were gated as
CD4+CD25+. (A) Representative results for Foxp3+ T cells
after coculture with mDCs (upper) or pDCs in the presence
or absence of 1-MT-L or 1-MT-D. (B) Histograms represent
the means 6 SEM of the percentage of T cells coex-
pressing CD4, CD25, and Foxp3 of three independent
experiments. *p , 0.05 versus the percentage of CD4+
CD25+Foxp3+ cells cultured without DCs (CD3 alone) and
versus RPMI 1640 within the condition mDCs or pDCs. (C)
To test their immunosuppressive activity, CD4+CD25+
T cells obtained at the end of the coculture with mDCs
or pDCs were purified by using magnetic beads and added
(1:10) to CD3+ T cells stimulated with PHA (1 mg/ml).
Histograms represent the means 6 SEM of three independent
experiments. *p , 0.05 versus CD3+ T cells alone (CD3).
1236 IDO2 STABLY SUPPORTS HOMEOSTATIC DC TOLERANCE
IDO1 is an inducible mechanism of DC tolerance, absent inhealthy condition, but is elicitable by inflammation.
DiscussionThe main finding of the present study is that IDO2 is stablyexpressed and functionally active in human DCs. IDO2 expressionconfers to DC tolerogenic features, such as the capacity of starvingkynurenine and of generating Tregs.A large body of evidence shows that IDO1-expressing DCs
mediate immune tolerance mainly by degrading tryptophan,inhibiting T cell proliferation, and inducing a Treg population (23,33). However, IDO1 expression is not a constitutive feature ofhuman DCs under homeostatic immunologic conditions, butit requires induction. Among the multiple mediators of IDO1induction, PGE2 plays a prominent role (26, 27). Indeed, PGE2 hasgenerally been considered a proinflammatory mediator that en-hances the migration toward lymph nodes, the capacity to primenaive T cells, and the improvement of DC maturation (34, 35). Onthe contrary, PGE2 is also required to prevent hyperinflammationby enhancing the release of IL-10 and the recruitment of Tregs,
besides IDO1 expression (25, 36). In fact, PGE2 has an activatoryand/or tolerogenic activity also depending on different DC subsets(37–39).In DCs, it is well known that IDO1 expression parallels specific
maturation markers, such as CD83, and costimulatory molecules,such as CD80 and CD86 (40, 41). Our data confirm these findingson IDO1 expression (see Fig. 2A, 2B). However, we observeda different expression pattern of IDO2 protein, which is inde-pendent from the maturation status of DCs. Indeed, althoughproinflammatory stimuli, which are strictly required for completematuration of DCs, did enhance both IDO1 and IDO2 transcrip-tion, only IDO1, but not IDO2, protein expression was inducibleby the presence of such stimuli in the culture medium. Thus, ourdata in humans support the notion that IDO1 expression mayrepresent a dynamic mechanism of tolerance induced by envi-ronmental factors (42), whereas IDO2 may act as a mechanism ofintrinsic immune privilege causing a nonimmunogenic response toantigenic stimuli (43, 44). Indeed, effector signaling experimentsconducted with IDO1 and IDO2 support the concept that IDO2induction is associated with stable rather than transient effector
FIGURE 6. IDO1 and IDO2 activity in Mo-DCs. (A and B) DCs were matured with the cytokine mixture and cultured with or without 1-MT-L or 1-MT-
D (1 mM) (A) in the presence of 500 mM L-tryptophan for 4 h or (B) with CD3+ T cells (1:10) for 24 h. (A) IDO1 and IDO2 enzymatic activity was evaluated
with a spectrophotometric analysis as the production of kynurenine in the supernatants. Data are expressed as the means 6 SEM of seven independent
experiments. *p , 0.05, **p , 0.01 versus RPMI 1640. (B) IDO1 and IDO2 biologic activity was evaluated by quantifying the percentage of T cells
coexpressing CD4, CD25, and Foxp3 of seven independent experiments. *p , 0.05, **p , 0.01 versus RPMI 1640. To directly inhibit IDO1 or IDO2
expression, immature DCs were nucleofected with a control (CTR) siRNA or with IDO1- or IDO2-specific siRNA and after 24 h matured with the cytokine
mixture for 48 h. (C–E) The expression of IDO1 and IDO2 after siRNA treatment was evaluated by (C) real-time RT-PCR and (D) Western blotting. (E)
Histograms represent the means 6 SEM of mRNA expression (n = 5) of IDO1 and IDO2 (normalized to GAPDH). Universal human RNA was used as
reference and taken as a value of 1. **p , 0.01 versus CTR-siRNA. (D) Western blotting expression of IDO1, IDO2, and b-actin (bACT) is representative
of results from five independent experiments. (E) Protein expression was quantified by band densitometric analysis and expressed as the ratio between IDO1
(or IDO2) and b-actin band intensity. Data are expressed as the means 6 SEM of five independent experiments. **p , 0.01 versus siRNA CTR. (F) DCs
were cultured in the presence of 500 mM L-tryptophan for 4 h and supernatants were collected. IDO1 and IDO2 enzymatic activity was evaluated with
a spectrophotometric analysis as the production of kynurenine in the supernatants. Data are expressed as the means 6 SEM of three independent
experiments. *p , 0.05, **p , 0.01 versus siRNA CTR. (G and H) DCs were cultured with CD3+ T cells (1:10) for 24 h. At the end of culture, cells were
stained for CD4, CD25, and Foxp3. Foxp3+ T cells were gated as CD4+CD25+. (G) Representative results for Foxp3+ T cells after coculture with DCs
treated with CTR-siRNA or IDO1- or IDO2-specific siRNA. (H) Histograms represent the means 6 SEM of the percentage of T cells coexpressing CD4,
CD25, and Foxp3 of three independent experiments. *p , 0.05, **p , 0.01 versus siRNA CTR. (I) To validate their immunosuppressive activity, CD4+
CD25+ T cells obtained at the end of the coculture with DCs treated with CTR-siRNA (Treg siRNA CTR), IDO1-specific siRNA (Treg siRNA IDO1), or
IDO2-specific siRNA (Treg siRNA IDO2) were purified by using magnetic beads and added (1:10) to CD3+ T cells stimulated with PHA (1 mg/ml).
Histograms represent the means 6 SEM of three independent experiments. *p , 0.05 versus CD3+ T cells alone (CD3).
signals (14). Moreover, a large body of evidence from studies ofinfectious disease indicates that IDO1 acts as a potent regulatorymechanism. Such a mechanism is known to act by counteractinghyperinflammation, which is likely to produce an unacceptable levelof tissue damage and to prevent the complete elimination of patho-gens, which would impair immune memory (45, 46). These findingssupport our data about IDO1 and IDO2 expression in patients suf-fering from inflammatory arthritis versus healthy subjects. In thesepatients, IDO1 expression is probably due to the inflammatory en-vironment (47). However, although an in vivo inflammatory milieumodulates IDO1 expression, IDO2 protein expression is independentfrom those mediators and it is expressed as in healthy subjects (seeFig. 7C, 7D). From this viewpoint, the homeostatic expression ratherthan the inducible expression of IDO2 may be an important part ofimmunological tolerance. Indeed, the generation and initial charac-terization of mice genetically deficient in IDO2 suggest that IDO2acts downstream or in parallel to IDO1 in Treg controls (G.C. Pre-ndergast, personal communication).Moreover, our data show that IDO2 is independent not only from
inflammatory stimuli and from DC maturation, but also fromcontinuous transcription and translation, because we show noproteasomal degradation through SOCS3 (see Fig. 4). Consis-tent with these findings, analysis of the IDO2 protein sequencerevealed only one putative ITIM (http://prosite.expasy.org)(48), which was not expected to act as a docking site for SOCS3.Indeed, IDO2 was not shown to bind to SOCS3, even after pro-teasome inhibition. This finding is in contrast with the appearancein the human IDO1 protein sequence of at least three consensusdegradation sites, allowing for proteasome targeting and subse-quent protein degradation in a cell cycle–dependent manner,compared with the human IDO2 protein sequence, which didnot contain any degradation sequence (http://prosite.expasy.org).Thus, although IDO2 protein may be targeted by other proteindegradation systems, the turnover of IDO2 protein appears to bemechanistically distinct from IDO1. In fact, also IDO2 mRNAmay be targeted by systems of degradation. Indeed, the TargetS-can analysis showed that IDO2 has a conserved site in the 39untranslated region that may be bound by the small noncodingRNA microRNA-590-3p. As recently reported, this bond can turn
on negative feedback loops (49) that might limit IDO2 expressionin stimulated DCs. Anyway, our results on protein expression anddegradation (see Figs. 3, 4) support the concept that IDO1 is areadily tuned system to generate tolerance, because it is tran-scriptionally induced by inflammatory stimuli and then promptlydegraded to avoid dangerous effects, in contrast to IDO2, which isa very stable protein perhaps maintaining steady-state tolerance.By analogy, IDO1 and IDO2 expression may be similar to othermodifier systems (e.g., heat shock protein 90a and 90b) (50)where one isoform is inducible upon exposure to stress and theother isoform is constitutively active and employed as a house-keeping mechanism.In conclusion, our results strongly suggest that IDO2 protein
expression by human DCs is not influenced by inflammatorystimuli and may have, therefore, a tolerogenic role under steady-state conditions. Of note, our data show a lower activity of IDO2with respect of IDO1, which may be important if, as we suggest,IDO2 is the key enzyme for supporting a tolerance homeostasis.In such a scenario, IDO2 may represent an ancestral conservedmechanism of tolerance, whereas IDO1 may represent a refinedmechanism for dynamic control that was acquired later duringevolution. Phylogenetic analysis of IDO genes in lower vertebrates(chickens, frogs, and fish) lend support to this hypothesis (51).From this perspective, it would not be surprising that an ancestralmechanism of homeostatic tolerance might be important to or-ganismal health, such that the absence or abnormal accumulationof either IDO isoform might affect pathological conditions orviability. Whereas 1-MT can trigger T cell–mediated rejection ofthe allogeneic fetus (8), IDO1-deficient mice undergo successfulpregnancy, pointing to alternative redundant mechanisms in theprotection of the fetus from maternal immune attack (52). Evi-dence that 1-MT inhibits both IDO1 and IDO2 may indicate thatIDO1 deficiency is insufficient for fetal rejection, if this relies onthe inactivation of both IDO1 and IDO2. In summary, by showingdirect evidence that IDO2 is required for Treg generation and isexpressed either in healthy and in inflammatory conditions, toour knowledge our work provides the first functional definition ofa role for IDO2 in tolerance, possibly in the setting of cancer andpregnancy, where tryptophan catabolism is critical.
FIGURE 7. Expression of IDO1 and IDO2 in blood DCs of inflammatory arthritis. Human total blood DCs were isolated from healthy donors’ PBMCs
and from PBMCs of patients suffering from rheumatoid arthritis (RA), psoriatic arthritis (Pso AR), or ankylosing spondylitis (Ank Spo) by using magnetic
beads. Healthy DCs were tested freshly purified or after an overnight (o.n.) incubation in RPMI 1640 alone or in the presence of IL-1b (10 ng/ml), IL-6 (10
ng/ml), and TNF-a (10 ng/ml) with or without (w/o) PGE2 (1 mg/ml); patients’ DCs were tested freshly purified. Cells were lysed and RNA and protein
were extracted. mRNA expression of IDO1 and IDO2 (normalized to GAPDH) was evaluated by real-time RT-PCR in healthy DCs (A) and patients’ DCs
(B). Universal human RNA was used as reference and taken as a value of 1. Data are expressed as the means 6 SEM of eight independent experiments.
*p , 0.05, **p , 0.01 versus o.n. or versus o.n. w/o PGE2, where indicated (A), or versus healthy DCs (B). (C and D) IDO1, IDO2, and b-actin (bACT)
protein expression in freshly purified DCs of a healthy subject and of a patient suffering from rheumatoid arthritis was determined by Western blotting. Data
shown are representative of results from one of four independent experiments. Rheumatoid arthritis was chosen as representative of the three inflammatory
types of arthritis. (D) Protein expression was quantified by band densitometric analysis and expressed as the ratio between IDO1 (or IDO2) and b-actin band
intensity. Data are expressed as the means 6 SEM of four independent experiments. **p , 0.01 versus healthy subjects.
1238 IDO2 STABLY SUPPORTS HOMEOSTATIC DC TOLERANCE
DisclosuresR.M. and G.C.P. declare a conflict of interest with regard to employer/
consultancy roles with NewLink Genetics Corporation, which is engaged
in the clinical development of IDO inhibitors. The other authors have no
financial conflicts of interest.
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1240 IDO2 STABLY SUPPORTS HOMEOSTATIC DC TOLERANCE
