Kynurenine Pathway Enzymes in Dendritic
Cells Initiate Tolerogenesis in the Absence of
Functional IDO
This information is current as
of June 16, 2017.
Maria L. Belladonna, Ursula Grohmann, Paolo Guidetti,
Claudia Volpi, Roberta Bianchi, Maria C. Fioretti, Robert
Schwarcz, Francesca Fallarino and Paolo Puccetti
J Immunol 2006; 177:130-137; ;
doi: 10.4049/jimmunol.177.1.130
http://www.jimmunol.org/content/177/1/130
Subscription
Permissions
Email Alerts
This article cites 51 articles, 27 of which you can access for free at:
http://www.jimmunol.org/content/177/1/130.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
References
The Journal of Immunology
Kynurenine Pathway Enzymes in Dendritic Cells Initiate
Tolerogenesis in the Absence of Functional IDO1
Maria L. Belladonna,* Ursula Grohmann,* Paolo Guidetti,† Claudia Volpi,* Roberta Bianchi,*
Maria C. Fioretti,* Robert Schwarcz,† Francesca Fallarino,* and Paolo Puccetti2*
T
he tolerogenic pathway of L-tryptophan catabolism has
been implicated, with either protective or disease-promoting roles, in a variety of physiopathologic conditions,
ranging from pregnancy (1) to transplantation (2), and from autoimmunity (3) and inflammation (4 – 6) to infection (7) and neoplasia (8, 9). There is growing evidence that tryptophan catabolism
may be involved in the development of depressive-like behaviors
in both humans and mice (10, 11). In the experimental models of
immune regulation, a central role for IDO has been established
either by using the competitive enzyme inhibitor 1-methyl-tryptophan (1-MT)3 under in vivo or in vitro conditions or by using
dendritic cells (DCs) ex vivo from IDO-deficient mice (12). However, pharmacologic or genetic inhibition of IDO activity may not
be completely informative, because IDO could not be the only
relevant target of 1-MT (13), and IDO-deficient mice, which are
genetically altered hosts, might have abnormal development or differentiation of their DC lineage. Interestingly, these animals manifest compensatory or redundant mechanisms for tolerogenesis in
vivo (14). Also relevant in this regard is the recent and rather
puzzling finding that the D-isomer of 1-MT is apparently more
effective at inhibiting tryptophan catabolism than the universally
*Department of Experimental Medicine, University of Perugia, Perugia, Italy; and
†
Maryland Psychiatric Research Center, University of Maryland School of Medicine,
Baltimore, MD 21228
Received for publication February 22, 2006. Accepted for publication April 12, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Italian Association for Cancer Research.
2
Address correspondence and reprint requests to Dr. Paolo Puccetti, Department of
Experimental Medicine, Section of Pharmacology, University of Perugia, Via del
Giochetto, Perugia 06126, Italy. E-mail address: [email protected]
3
Abbreviations used in this paper: 1-MT, 1-methyl-tryptophan; DC, dendritic cell;
3-HAA, 3-hydroxyanthranilic acid; QUIN, quinolinic acid; siRNA, small interfering
RNA; GC/MS, gas chromatography with electron capture negative ionization mass
spectrometry; KYN, L-kynurenine; PI, propidium iodide; 3-HK, 3-hydroxykynurenine; AA, anthranilic acid; Treg, regulatory T cell.
Copyright © 2006 by The American Association of Immunologists, Inc.
used mixture of D,L-stereoisomers (15, 16), in which the L-isomer
is expected to be the active form of the inhibitor (13).
Two major theories have been proposed to account for tolerance
induction via tryptophan catabolism. One theory posits that tryptophan breakdown suppresses T cell proliferation by critically reducing the availability of this indispensable amino acid in local
tissue microenvironments (17). The other theory assumes that
downstream metabolites of tryptophan catabolism, collectively
known as kynurenines, act to suppress immune reactivity, probably through direct interactions with effector T lymphocytes and
other cell types (10). These are not mutually exclusive possibilities, and each might have a role in the immunobiology of IDO
(16), including unique actions by specific kynurenines such as
3-hydroxyanthranilic acid (3-HAA) (18) and the downstream
product quinolinic acid (QUIN) (19). Clarification of the relative
contributions of the different mechanisms would be crucial to an
improved understanding of the complex role of tryptophan catabolism in health and disease and to the implementation of immunotherapies targeting IDO function (13).
IDO activity is regulated at both transcriptional and posttranslational levels (20), and IFN-␥ represents the principal regulator of
Indo transcription (21). We have previously shown that the default
functional programs of murine splenic CD8⫺ and CD8⫹ DCs in
the presentation in vivo of poorly immunogenic peptides are disparate but flexible (22). IFN-␥, however, fails to modify the default
immunogenic function of CD8⫺ DCs, although it does potentiate
the basic tolerogenic program of CD8⫹ DCs in an IDO-dependent
fashion. The cytokine, in fact, will not confer tryptophan-catabolizing activity on CD8⫺ DCs, although the resultant IDO protein
expression is comparable in the two DC subsets (23).
In this study, we examined the role of kynurenines that are physiologically generated downstream of the initial and rate-limiting
step mediated by IDO in tryptophan degradation. In addition, in
IDO-competent DCs, we compared 1-MT and Indo gene silencing
for modulation of the potent tolerogenic function induced by
IFN-␥ in this subset. We obtained evidence that Indo silencing in
0022-1767/06/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Dendritic cell (DC) tryptophan catabolism has emerged in recent years as a major mechanism of peripheral tolerance. However,
there are features of this mechanism, initiated by IDO, that are still unclear, including the role of enzymes that are downstream
of IDO in the kynurenine pathway and the role of the associated production of kynurenines. In this study, we provide evidence
that 1) murine DCs express all enzymes necessary for synthesis of the downstream product of tryptophan breakdown, quinolinate;
2) IFN-␥ enhances transcriptional expression of all of these enzymes, although posttranslational inactivation of IDO may prevent
metabolic steps that are subsequent and consequent to IDO; 3) overcoming the IDO-dependent blockade by provision of a
downstream quinolinate precursor activates the pathway and leads to the onset of suppressive properties; and 4) tolerogenic DCs
can confer suppressive ability on otherwise immunogenic DCs across a Transwell in an IDO-dependent fashion. Altogether, these
data indicate that kynurenine pathway enzymes downstream of IDO can initiate tolerogenesis by DCs independently of tryptophan
deprivation. The paracrine production of kynurenines might be one mechanism used by IDO-competent cells to convert DCs
lacking functional IDO to a tolerogenic phenotype within an IFN-␥-rich environment. The Journal of Immunology, 2006, 177:
130 –137.
The Journal of Immunology
CD8⫹ DCs had effects comparable to pharmacologic inhibition of
the enzyme. Perhaps of greater interest, initiating the kynurenine
pathway in CD8⫺ DCs downstream of IDO led to the production
of QUIN and imparted tolerogenic properties to those cells in the
absence of tryptophan consumption. Thus, similar to CD8⫹ DCs,
their CD8⫺ counterparts are competent for the portion of the pathway that is subsequent to IDO, and can further metabolize the
initial kynurenine when provided externally. This suggests that the
paracrine production of kynurenines could be one mechanism
whereby IDO-competent cells affect the activity of DCs in which
functional IDO is blocked posttranslationally.
Materials and Methods
Mice and reagents
DC preparation, treatments, and immunization
Splenic DCs were purified by magnetic-activated sorting using CD11c MicroBeads and MidiMacs (Miltenyi Biotec), in the presence of EDTA to
disrupt DC-T cell complexes. Cells were ⬎99% CD11c⫹, ⬎99% MHC
I-A⫹, ⬎98% B7-2⫹, ⬍0.1% CD3⫹, and appeared to consist of 90 –95%
CD8⫺, 5–10% CD8⫹, and 1–5% B220⫹ cells. DC populations were further
separated into CD8⫺ and CD8⫹ fractions by means of CD8␣ MicroBeads
(Miltenyi Biotec) (22, 25). The CD8⫺ fraction was ⬃45% CD4⫹ and typically contained ⬍0.5% contaminating CD8⫹ cells. Less than 1% CD8⫹
and ⬍5% CD8⫺ DCs expressed the B220 marker, respectively (26). DCs
were exposed to 100 ng/ml IL-12 (CD8⫺ DCs) or 200 U/ml IFN-␥ (CD8⫺
or CD8⫹ DCs) for 24 h at 37°C in the presence or absence of 4 ␮M 1-MT
(27). For immunization, cells were washed between and after incubations
before peptide loading (5 ␮M, 2 h at 37°C), irradiation, and i.v. injection
into recipient hosts. A total of 3 ⫻ 105 IL-12-treated CD8⫺ DCs was
injected in combination with 5% IFN-␥-treated CD8⫹ or CD8⫺ DCs.
Small interfering RNA (siRNA) synthesis and transfection
The siRNA sequences specific for murine Indo (sense, 5⬘-GGGCUUCU
UCCUCGUCUCUtt-3⬘; antisense, 5⬘-AGAGACGAGGAAGAAGCCCtt3⬘) were selected, synthesized, and annealed by the manufacturer (Ambion). For transfection, siRNAs (6.7 ␮g) in 30 ␮l 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 ␮g of 1,2-dioleoyl-3-trimethylammonium-propane were mixed with 30 ␮l 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 DCs and incubated for 48 h at 37°C, with
or without IFN-␥ during the last 18 h of incubation. Cells were then recovered, washed, and immediately used for in vitro or in vivo experiments.
Control treatment consisted of Negative Control siRNA (Ambion).
PCR analysis
RT-PCR analysis was performed (30 cycles, annealing temperature of
60°C) using sense and antisense primers, as follows: Indo sense, 5⬘GAAGGATCCTTGAAGACCAC-3⬘; Indo antisense, 5⬘-GAAGCTGCG
ATTTCCACCAA-3⬘; Sod2 sense, 5⬘-AGACCTGCCTTACGACTATG3⬘; Sod2 antisense, 5⬘-AGCGGAATAAGGCCTGTTGT-3⬘.
Real-time PCR were run on a Chromo4 Four-Color Real-Time Detector
(Bio-Rad) using specific primers as follows: Indo sense, 5⬘-GAAGGATC
CTTGAAGACCAC-3⬘; Indo antisense, 5⬘-GAAGCTGCGATTTCCAC
CAA-3⬘; Kynu sense, 5⬘-AAATTCCCTTGGCCTTCAAC-3⬘; Kynu antisense, 5⬘-GCGTTTGCCTACATCATGG-3⬘; Kmo sense, 5⬘-GCCTTG
AAAGCCATTGGTC-3⬘; Kmo antisense, 5⬘-GCACTGTGAGTAC
CCCTTCC-3⬘; Haao sense, 5⬘-ACGCCGTGTGAGAGTGAAGT-3⬘;
Haao antisense, 5⬘-GTAATACCTCAGCCCATCC-3⬘. PCR products were
normalized to murine Gapdh using specific primers (sense, 5⬘-GCCTTC
CGTGTTCCTACCC-3⬘; antisense, 5⬘-CAGTGGGCCCTCAGATGC-3⬘).
Kynurenine and quinolinic acid measurements
These procedures, using gas chromatography with electron capture negative ionization mass spectrometry (GC/MS), have previously been described in detail (28, 29). Briefly, 10 ␮l of culture supernatant was diluted
(1/5, v/v) in water. Fifty microliters of internal standard (100 nM 3,5pyridinedicarboxylic acid and 200 nM homophenylalanine) were added,
and proteins were precipitated with HCl (25 ␮l, 5 N). Fatty compounds
were removed by extraction with chloroform (100 ␮l). After centrifugation
(10 min, 10,000 ⫻ g), 100 ␮l of the acidic supernatant were added to a
glass tube containing 50 ␮l of 62.5 mM tetrabutylammonium hydrogen
sulfate. The mixture was lyophilized and resuspended in 25 ␮l of acetone
containing 7.5% of diisopropylethylamine and 3% pentafluorobenzyl bromide. The samples were incubated in sealed tubes for 15 min at 60 – 65°C.
Fifty microliters of decane and 750 ␮l of water were then added, the samples were thoroughly mixed, and the decane phase was removed. One
microliter of the decane phase was injected into a GC/MS system consisting of a Trace GC coupled with a Trace MS quadrupole mass spectrometer
(ThermoFinnigan). Chromatographic separation was achieved using a
30-m Rtx-5MS capillary column (0.25-mm internal diameter, 0.25-␮m film
thickness; Restek) with helium as a carrier gas. A split/splitless injection port
(1-␮l injection volume) was used. The temperature program was as follows:
155°C for 1.25 min, 40°C/min to 275°C, 10°C/min to 320°C, 1 min at 320°C,
injection port 228°C. The GC/MS uses electron capture negative ion chemical
ionization mass spectroscopy with methane as the reagent gas. The ion source
temperature was 220°C. Selected ion monitoring was performed by recording
signals of characteristic (M-PFB)⫺ ions.
Skin test assay
A skin test assay was used for measuring class I-restricted delayed-type
hypersensitivity responses to synthetic peptides as described previously
(22, 30, 31). Results were expressed as the increase in footpad weight of
peptide-injected footpads over that of vehicle-injected counterparts. Values
(mean ⫾ SD) are compiled data from independent experiments with at
least six mice per group per experiment. The statistical analysis was performed using Student’s paired t test by comparing the mean weight of
experimental footpads with that of control counterparts.
Induction of apoptosis by IDO⫹ DCs
Conditions for inducing and assaying IDO-dependent apoptosis have been
described previously (32). Briefly, CD8⫹ or CD8⫺ DCs (2 ⫻ 105), treated
overnight with IFN-␥ in the presence or absence of 10 ␮M L-kynurenine
(KYN), were washed and cultured for 48 h with 4 ⫻ 105 F76 cells, a
Th1-type P815AB-specific CD4⫹ T cell clone, in the presence of 5 ␮M
P815AB peptide. At the end of the coculture, the CD11c⫹ cells were removed by the use of CD11c MicroBeads (Miltenyi Biotec), and the remainder of the population was surface stained with anti-CD4-PE and
FITC-labeled annexin V and propidium iodide (PI; BD Pharmingen). For
measurement of apoptosis, a gate was set on CD4⫹ T cells, and the percentage of cells in the very early stages of apoptosis was determined by
annexin V staining excluding PI⫹ cells.
Transwell DC conditioning
DC conditioning by IDO-competent cells was performed using a 6.5-mm
Transwell system (Corning) with a 0.4-␮m pore polycarbonate membrane
insert. CD8⫺ DCs (106) were added to the lower chamber, while CD8⫹
DCs (106) were added to the upper chamber. Cultures were set up in the
presence of 200 U/ml IFN-␥, and, after 18 h, CD8⫺ DCs were recovered,
extensively washed, and used for in vivo sensitization.
Results
Indo silencing and 1-MT have comparable effects on functional
IDO in vitro and in vivo
IDO catalyzes the initial and rate-limiting step in tryptophan degradation to QUIN through the sequential formation of KYN, 3-hydroxykynurenine (3-HK) or alternatively anthranilic acid (AA),
and 3-HAA (Fig. 1). Thus, pharmacologic blockade of IDO by the
use of 1-MT results in impaired formation of KYN and the downstream metabolites including QUIN. siRNA technology is a means
of posttranscriptional gene silencing that can be used to modulate
gene expression in murine DCs (25, 27). To comparatively examine the effects of 1-MT and posttranscriptional Indo silencing,
splenic DCs were fractionated and the CD8⫹ fraction, treated or
not with IFN-␥, was transfected with Indo-specific or control
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Female DBA/2J (H-2d) mice were obtained from Charles River Breeding
Laboratories. Synthetic kynurenines (used in vitro at 10 or 100 ␮M according to the experimental design) and racemic 1-MT (D,L-isomers) were
purchased from Sigma-Aldrich. The kynurenine 3-monooxygenase inhibitor PNU 156561 (4,5-dichlorobenzoylalanine; synthesized in the Pharmacia and Upjohn Research Laboratories of Nerviano, Italy) has been characterized previously (24). The P815AB peptide (amino acid sequence
LPYLGWLVF) was synthesized and purified as described previously (22,
25). All in vivo studies were performed in compliance with National and
Perugia University Animal Care and Use Committee guidelines.
131
132
FIGURE 1. Kynurenine pathway of tryptophan catabolism in mammalian cells, in which IDO catalyzes the initial and rate-limiting step under
transcriptional regulation by IFN-␥. Indicated in the figure are the enzyme
activities, and the corresponding genes, leading to the production of immunologically relevant metabolites.
DC populations in the spleens of DBA/2 mice consist of CD8⫺
(⬃90%) and CD8⫹ (⬃10%) fractions that mediate the respective
immunogenic and tolerogenic presentation of the synthetic tumor/
self nonapeptide P815AB. Upon transfer into recipient hosts, peptide-loaded CD8⫺ DCs initiate immunity, and CD8⫹ DCs initiate
anergy, when Ag-specific skin test reactivity is measured at 2 wk
after cell transfer (30, 31). On cotransferring cytokine-conditioned
DCs of the two subsets, the IDO-inducing tolerogenic potential of
IFN-␥ acting on CD8⫹ DCs enables these cells to prevail over the
adjuvant properties of IL-12 acting on CD8⫺ DCs, resulting in
lack of skin test reactivity (22, 34 –36). P815AB-pulsed, IL-12treated CD8⫺ DCs were injected in combination with 5% CD8⫹
DCs, either untreated or treated with IFN-␥, with or without concomitant 1-MT or Indo siRNA treatment (Fig. 2D). The results
showed that IFN-␥ was an absolute requirement for the occurrence
of suppressive effects by CD8⫹ DCs cotransferred with IL-12primed CD8⫺ DCs. However, the effect was equally ablated by
treatment of the tolerogenic fraction with 1-MT or Indo siRNA
before DC cotransfer. This confirms that IDO is a major pharmacologic target of racemic 1-MT in our experimental setting of
CD8⫹ DC conditioning in vitro with IFN-␥.
Detection of transcriptional but not functional expression of
kynurenine pathway enzymes in CD8⫺ DCs treated with IFN-␥
Using RT-PCR, we previously showed that IFN-␥ activates Indo
transcription in both CD8⫹ and CD8⫺ DCs, resulting in comparable expressions of IDO protein in the two subsets (21, 23). In this
FIGURE 2. Indo silencing has effects comparable to 1-MT on functions mediated by IDO in vitro and in vivo. A, Kinetic RT-PCR analysis of Indo
expression in CD8⫹ DCs treated with Indo-specific siRNA in the presence or absence of IFN-␥. Control cells were treated with negative control (nc) siRNA.
Expression of the IFN-␥-inducible Sod2 gene was also assayed as a specificity control. B, Effect of IFN-␥ on tryptophan (100 ␮M) conversion to KYN
in CD8⫹ DCs previously subjected to 1-MT treatment or Indo silencing by siRNA for 48 h. KYN levels in 5-h supernatants were measured by GC/MS,
and the results are mean ⫾ SD of three different experiments, each performed in triplicate. ⴱ, p ⬍ 0.005 (IFN-␥ vs medium treatment). C, Effect of Indo
silencing on IDO-dependent apoptosis mediated by IFN-␥-treated CD8⫹ DCs. Apoptosis was measured in Ag-specific CD4⫹ T cells cultured with CD8⫹
DCs pre-exposed to IFN-␥ with or without Indo siRNA or 1-MT. Representative dot plots are shown of annexin V staining of gated CD4⫹ PI⫺ cells.
Numbers within boxes indicate percentages of CD4⫹ apoptotic cells in one experiment representative of three. The fold increase values in apoptosis rate
induced by IFN-␥ in the remainder experiments were 2.9 and 2.5, respectively, and these values decreased to ⬍1.1 upon Indo silencing. D, Indo silencing
ablates the tolerogenic potential of CD8⫹ DCs. Combinations of IL-12-treated CD8⫺ and CD8⫹ DCs subjected to various treatments (indicated) were
loaded with P815AB and injected into recipient mice to be assayed at 2 wk for skin test reactivity to the eliciting peptide. ⴱ, p ⬍ 0.0001 (experimental
vs control footpads). Compiled data from three independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
siRNA, resulting in efficient and selective inhibition of Indo transcript expression at 48 h of transfection (Fig. 2A). This was accompanied by inhibition of IDO function as measured by the production of KYN by IFN-␥-treated CD8⫹ DCs (Fig. 2B) as well as
by their ability to induce apoptosis of a Th1 clone, as documented
previously (32, 33) (Fig. 2C). All of these functional effects in
vitro were mimicked by the use of 1-MT in place of Indo siRNA.
KYNURENINE PATHWAY ENZYMES IN TOLERANCE
The Journal of Immunology
study, we used real-time PCR for quantitative and comparative
assessment of Indo expression in CD8⫺ and CD8⫹ DCs and extended the examination to a series of genes coding for enzymes
downstream of IDO, namely Kmo (coding for kynurenine 3-monooxygenase), Kynu (kynureninase), and Haao (3-hydroxyanthranilate 3,4-dioxygenase) (Fig. 3A). The results confirmed enhanced
transcriptional activity of Indo by IFN-␥ in CD8⫺ DCs, to an extent comparable to CD8⫹ counterparts. Similar to Indo albeit to a
lesser extent, the expressions of Kmo, Kynu, and Haao were all
enhanced by IFN-␥ in both DC subsets. Despite the increase in
gene transcription, IFN-␥ induced little or no production of KYN
and QUIN in CD8⫺ DCs (Fig. 3B), possibly accounting for the
failure of the recombinant cytokine to promote tolerogenic properties in this subset (21, 23, 35). In contrast, in CD8⫹ DCs, IFN-␥
resulted in early (5 h) production of KYN, followed by later (18 h)
detection of high levels of QUIN. Collectively, these data indicated that DCs are endowed with the whole enzymatic machinery
necessary for the production of one of the end products of the
kynurenine pathway, QUIN. However, these data do not clarify
whether the inability of IFN-␥ to activate the pathway in the CD8⫺
subset of DCs is due solely to an inefficient rate-limiting step (i.e.,
the one mediated by IDO) or to a combination of the latter with
posttranscriptional regulation of downstream enzymes.
KYN conversion into QUIN occurs in CD8⫺ DCs, resulting in
tolerogenic properties
We investigated whether the enzyme response induced by IFN-␥
downstream of IDO in CD8⫺ DCs is associated with the production of QUIN when the precursor KYN is added to the culture
medium. We first validated the model by adding KYN to a culture
of Indo siRNA-treated CD8⫹ DCs and found that the addition of
the external precursor was indeed associated with the production
of QUIN when the cells had been exposed to IFN-␥ (Fig. 4A).
Similar to the latter cells, CD8⫺ DCs produced high levels of
QUIN if cotreated with IFN-␥ and KYN. The ability of CD8⫺ DCs
to release QUIN in response to IFN-␥ and the externally added
precursor prompted us to investigate whether synthesis of QUIN
was associated with the acquisition of properties typical of the
tolerogenic CD8⫹ DC subset. In the experimental setting illustrated above, we measured apoptosis of CD4⫹ cells cultured with
CD8⫺ DCs either untreated or treated with IFN-␥ and KYN, singly
or in combination (Fig. 4B). The results revealed an ability of the
CD8⫺ DCs to induce apoptosis in ⬃25% of Th1 target cells upon
coexposure to IFN-␥ and KYN. We also asked whether the CD8⫺
DCs conditioned by KYN and IFN-␥ treatment would be capable
of suppressive properties in vivo when cotransferred with otherwise immunogenic DCs. In an experimental model system similar
to that of Fig. 2D, P815AB-pulsed and IL-12-primed CD8⫺ DCs
were injected in combination with 5% CD8⫺ DCs cotreated with
a mixture of IFN-␥ and KYN, either alone or together with PNU
156561, a selective inhibitor of kynurenine 3-monooxygenase
(Fig. 5A). The results showed that the combined effects of IFN-␥
and KYN turned CD8⫺ DCs from immunogenic to suppressive,
thus mimicking the behavior of the CD8⫹ DC subset. However,
inhibiting the conversion of KYN into 3-HK, and thus preventing
synthesis of QUIN, abolished the onset of suppressive properties in
the CD8⫺ DC fraction. Of interest, the mice sensitized with the DC
population containing 5% IFN-␥/KYN-treated CD8⫺ DCs were
not amenable to subsequent immunogenic priming to the peptide
when performed 1 mo after the first exposure (Fig. 5B). Again, this
effect is similar to that observed with IFN-␥-treated CD8⫹ DCs
(35) and indicates the onset of an Ag-specific tolerant state. Taken
together, these data suggest that even in the absence of functional
IDO, kynurenine pathways enzymes, if activated by IFN-␥ in the
presence of an appropriate precursor, catalyze the synthesis of
QUIN, and the effect is associated with DC acquisition of tolerogenic properties.
IDO-competent DCs transfer tolerogenic potential across a
cell-impermeable membrane
The finding that kynurenine pathway enzymes downstream of IDO
can initiate tolerogenesis in DCs lacking functional IDO prompted
us to investigate whether IDO-competent cells might turn otherwise immunogenic DCs into tolerogenic cells via soluble molecules. CD8⫹ DCs were exposed overnight to IFN-␥, and CD8⫺
DCs were added to the lower chamber of a Transwell system. The
CD8⫹ cells had been treated with either negative control or Indospecific siRNAs. The CD8⫺ DCs were then recovered and assayed
in vivo for their ability to suppress induction of skin test reactivity
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 3. CD8⫺ DCs express kynurenine pathway enzymes but produce little or no KYN and QUIN. A, Induction of Indo, Kmo, Kynu, and
Haao transcripts in CD8⫹ and CD8⫺ DCs by IFN-␥. Sorted CD8⫹ and
CD8⫺ DCs were exposed to rIFN-␥ for 4 h. At the end of the culture,
mRNA levels of genes related to the kynurenine pathway were quantified
by real-time PCR using Gapdh normalization. Data (means ⫾ SD of three
experiments using triplicate samples) are presented as normalized transcript expression in the samples relative to normalized expression in the
respective control cultures, i.e., cells unexposed to IFN-␥ but maintained in
medium alone (fold change ⫽ 1; dotted line). B, Conversion of tryptophan
(100 ␮M) to KYN and QUIN by CD8⫹ vs CD8⫺ DCs in response to
exposure to IFN-␥ for 5 or 18 h. Values are means ⫾ SD of three independent experiments performed in triplicate. ⴱ, Undetectable levels.
133
134
KYNURENINE PATHWAY ENZYMES IN TOLERANCE
FIGURE 4. Synthesis of QUIN by CD8⫺ DCs is associated with ability
to induce apoptosis. A, Effective conversion of KYN into QUIN by IFN␥-treated CD8⫺ DCs. CD8⫺ DCs and CD8⫹ DCs treated with negative
control (nc) or Indo siRNA were compared for ability to catabolize KYN
to QUIN on activating kynurenine pathway enzymes with IFN-␥. Cells
were cultured for 18 h in the presence of 200 U/ml IFN-␥ and 100 ␮M
KYN before measurement of QUIN concentrations in supernatants. Data
are the means ⫾ SD of three experiments with triplicate samples. ⴱ, p ⬍
0.01 (IFN-␥ vs medium treatment). B, Induction of potential for apoptosis
by IFN-␥ and KYN (10 ␮M) in CD8⫺ DCs. Apoptosis of Ag-specific
CD4⫹ T cells was measured as in Fig. 2C. Numbers within boxes indicate
percentages of CD4⫹ apoptotic cells in one experiment representative of
three. The fold increase in apoptosis rate induced by KYN ⫹ IFN-␥ in the
other two experiments was 4.0 and 6.8, respectively.
to P815AB (Fig. 6). According to the experimental model described above, a minority fraction of conditioned CD8⫺ DCs was
added to an IL-12-treated counterpart, and the mixture was transferred into recipients to be assayed at 2 wk for the development of
skin test reactivity. The results showed that otherwise nontolerogenic CD8⫺ DCs became suppressive as a result of coculture in the
Transwell system with CD8⫹ DCs treated with negative control
but not Indo siRNA. This result demonstrates that IDO-competent
DCs can confer tolerogenic potential on cells lacking functional
IDO by means of tryptophan catabolism and the production of
soluble diffusible molecules.
Discussion
The kynurenine pathway is a major route of L-tryptophan catabolism, resulting in the production of nicotinamide adenine dinucleotide and other neuroactive (37, 38) and immunomodulatory (10)
intermediates. Although all kynurenines are found in high concentration in urine, none of them has so far been assigned an important
physiological function in peripheral organs. In the brain, the
kynurenine pathway yields several intermediates, including the
free radical generator 3-HK, the excitotoxic N-methyl-D-aspartate
receptor agonist QUIN as well as its antagonist, kynurenic acid.
Astrocytes, neurons, and microglia all express IDO, but only microglial cells are capable of producing substantial quantities of
QUIN (39). Although KYN can be produced in the brain to a
FIGURE 6. IDO-competent DCs added across a Transwell to a culture
of nontolerogenic cells induce suppressive properties in the latter via IDOdependent mechanisms. CD8⫹ DCs (106) were added to the upper chamber
of a Transwell system, the bottom chambers of which contained an equal
number of CD8⫺ DCs. rIFN-␥ was present in the cultures at 200 U/ml, and
the CD8⫹ DCs had been transfected with Indo siRNA or negative control
(nc) siRNA. After 18 h, the recovered CD8⫺ DCs were pulsed with
P815AB and added to a majority fraction (95%) of peptide-pulsed, IL-12treated DCs. The cells were then transferred into recipient mice that were
assayed at 2 wk for P815AB-specific skin test reactivity. The contents of
the lower and upper chambers of the Transwell are indicated in the figure.
Compiled data from three experiments. ⴱ, p ⬍ 0.0001 (experimental vs
control footpads).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. The combined effects of IFN-␥ and KYN confer tolerogenic
potential in vivo to CD8⫺ DCs. Combinations of IL-12-treated CD8⫺ DCs
and a minority fraction (5%) of the same cells not treated with IL-12 but
exposed to 200 U/ml IFN-␥ and 10 ␮M KYN were loaded with P815AB.
A group of IFN-␥/KYN-treated CD8⫺ DCs was also exposed to 20 ␮M
PNU 156561, a selective inhibitor of kynurenine 3-monooxygenase. The
cells were then transferred into recipient hosts. Mice were assayed for skin
test reactivity on day 15 after cell transfer (A) or, alternatively, were treated
on day 30 with an otherwise effective priming consisting of P815ABpulsed CD8⫺ DCs, to be assayed for skin test reactivity on day 45 (B).
Compiled data from three experiments. ⴱ, p ⬍ 0.0001 (experimental vs
control footpads).
The Journal of Immunology
synthetic 3-HAA is also taken up by DCs, resulting in the production of QUIN (data not shown).
DCs have several modes at their disposal to mediate tolerogenic
functions, and metabolic pathways of immune regulation are but
one possibility (48). A distinctive feature of tryptophan catabolism
as an effector mechanism of suppression is that its function is compatible with a directive role of Tregs in orchestrating peripheral
tolerance (30). We have previously shown that the default immunogenic or tolerogenic program of Ag presentation by DC subsets
is highly flexible, and that otherwise immunogenic DCs can be
rendered tolerogenic by the actions of inhibitory ligands expressed
by T cells (2, 22, 26, 27). Unlike soluble or cell-bound CTLA-4,
IFN-␥ is unable per se to induce functional IDO expression in
CD8⫺ DCs (21). Our current data demonstrate that, in the presence
of IFN-␥, CD8⫺ DCs can be rendered tolerogenic by the nearby
production of kynurenines that are taken up by the cells and then
metabolized to downstream products of the pathway. This mechanism is qualitatively different from that of CTLA-4, which acts on
CD8⫺ DCs to activate IDO, making it functionally responsive to
IFN-␥ up-regulation (21). On the one hand, the bulk of these data
demonstrates that tryptophan consumption may not always be an
absolute requirement for the DCs to exert suppressive properties.
On the other hand, it is possible that, to meet the needs of flexibility and redundancy, the paracrine production of kynurenines is
exploited by IDO-competent DCs to convert DCs that are not competent to a tolerogenic phenotype.
A model of cross-talk of DCs and T cells in tryptophan catabolism within an IFN-␥-rich environment is illustrated in Fig. 7.
The model, far from detracting from the role of tryptophan starvation in mediating specific effects of the IDO mechanism (16),
reinforces the concept that tryptophan deprivation and tryptophan
FIGURE 7. A model of cross-talk of DCs and T cells in tryptophan
catabolism. In local tissue microenvironments, the activity of IDO competent DCs (IDO⫹) driven by IFN-␥ will result in the sustained production
of kynurenines (Kyn). These, in turn, could recruit other cell types to the
regulatory response, including DCs in which the function of IDO, but not
other kynurenine pathway enzymes, is inhibited posttranslationally
(IDO⫺). The combined effects of tryptophan (Trp) starvation (16), caused
by IDO⫹ DCs, and the high kynurenine production, resulting from the
actions of IDO⫹ and IDO⫺ DCs, is expected to induce a great variety of
effects on target T cells (18, 33, 44) and other cell types (42, 43). Tregs
would have a crucial role in establishing an IFN-␥-rich environment, either
via CTLA-4/B7-dependent interaction with DCs (30) or by direct production of the cytokine (49). Because the combined effects of tryptophan starvation and tryptophan catabolites include the induction of a regulatory
phenotype in naive CD4⫹ T cells (51), an IDO-based positive feedback
loop might be locally operative, resulting in Treg generation by Tregs
(dotted line).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
moderate degree, the cerebral pathway is driven mainly by bloodborne KYN, which enters from the circulation using the large neutral amino acid transporter. In the brain, KYN is rapidly taken up
by astrocytes and microglial cells, to be converted into end products that are then released extracellularly (37).
In the periphery, the immunosuppressive pathway of tryptophan
catabolism has been shown to contribute to successful pregnancy
(1) and transplantation (2), as well as to effective control of autoimmunity (3). We have proposed a model of transplantation tolerance that might apply to both transplantation and autoimmunity
(10). In this model, IFN-␥ acts on tolerogenic DCs to activate IDO,
resulting in the onset of specific tolerance through the induction of
tryptophan starvation and the production of immunomodulatory
kynurenines. Extensive work in our as well as others’ laboratories
has subsequently shown that this model 1) accommodates the activity of regulatory T cells (Tregs) that, expressing surface
CTLA-4, engage B7 coreceptor molecules on DCs and initiate the
production of autocrine IFN-␥ (12, 30); 2) applies to different subsets of DCs, including plasmacytoid DCs (15, 26); and 3) suggests
that the IDO mechanism is quite general in nature, in that, for
example, not only DCs but also CD4⫹ T cells (40) and neutrophils
(7) respond to CTLA-4 engagement of their surface B7 molecules
with the activation of IDO, depending on the release of autocrine
IFN-␥.
Despite much evidence for such a general role of tryptophan
catabolism as a mechanism of immunosurveillance (17), there are
several mechanistic features of the pathway that await further clarification (19). Among these are the relative contributions of IDO
and the remainder pathway enzymes to immunosuppression (41),
as well as the possible function of specific kynurenines as inducers
or mediators of immunoregulation (18, 33, 42– 44). In this study,
we have provided evidence that 1) IDO is the major relevant target
of pharmacologic inhibition of the kynurenine pathway when racemic 1-MT is used as an enzyme inhibitor; 2) DCs express the
entire metabolic machinery necessary for synthesis of QUIN; 3)
IFN-␥ enhances the transcriptional expression of all these enzymes, which may in fact respond as a coordinately regulated cluster of genes (45); yet, posttranslational inactivation of IDO occurs
in nontolerogenic CD8⫺ DCs, and this prevents the action of enzymes consequent to IDO function; and 4) bypassing the IDOassociated blockade by the addition of a downstream QUIN precursor initiates the pathway and leads to the onset of suppressive
properties in CD8⫺ DCs treated with IFN-␥.
Human macrophages are endowed with the ability to convert
tryptophan to QUIN (46, 47); yet, no information was previously
available regarding the functional expression of kynurenine pathway enzymes other than IDO in human or murine DCs. We found
that the splenic CD8⫹ subset of murine DCs could be induced by
IFN-␥ to release high levels of QUIN. Despite functional lack of
the pathway regulatory enzyme IDO in CD8⫺ DCs, these cells
could nevertheless be induced by IFN-␥ to produce QUIN in the
presence of KYN, a downstream precursor of the end product.
Activation of the pathway was accompanied by the appearance of
tolerogenic properties in vivo. Thus, the current study may be the
first to demonstrate the presence of a functional kynurenine pathway in murine DCs and, interestingly, to specifically associate the
tolerogenic phenotype with the function of a coordinately regulated set of enzymes, as observed upon treatment with IFN-␥. In
addition, similar to what was observed with astrocytes and microglial cells (37), our current data demonstrate that KYN is taken up
by DCs. The kynurenine 3-monooxygenase-dependent conversion
of KYN into QUIN by DCs further demonstrates that specific intracellular metabolism of kynurenines occurs in those cells. Of
interest in this regard, we have also found that similar to KYN,
135
136
catabolites may act singly or in combination to establish a regulatory environment most suitable to the control of local inflammatory or systemic T cell-mediated responses. The model also emphasizes the likely dual role of tryptophan catabolism both as an
effector system (30, 49) and an inducer of Treg activity (7, 44, 50,
51). Because 1-MT has been shown to act synergistically with
cytoreductive chemotherapy in an experimental tumor setting (9),
the identification of proper enzyme targets for pharmacologic inhibition of the kynurenine pathway may pave the way to improved
strategies of antitumor chemotherapy (13). More generally, understanding the molecular mechanisms that preside over control of
IDO expression by DCs may provide new opportunities for designing ways to induce or abrogate tolerance in physiopathologic
conditions (21, 25, 52).
KYNURENINE PATHWAY ENZYMES IN TOLERANCE
19.
20.
21.
22.
23.
24.
Disclosures
The authors have no financial conflict of interest.
25.
References
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
1. Munn, D. H., M. Zhou, J. T. Attwood, I. Bondarev, S. J. Conway, B. Marshall,
C. Brown, and A. L. Mellor. 1998. Prevention of allogeneic fetal rejection by
tryptophan catabolism. Science 281: 1191–1193.
2. Grohmann, U., C. Orabona, F. Fallarino, C. Vacca, F. Calcinaro, A. Falorni,
P. Candeloro, M. L. Belladonna, R. Bianchi, M. C. Fioretti, and P. Puccetti. 2002.
CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3:
1097–1101.
3. Grohmann, U., F. Fallarino, R. Bianchi, C. Orabona, C. Vacca, M. C. Fioretti, and
P. Puccetti. 2003. A defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice. J. Exp. Med. 198: 153–160.
4. Gurtner, G. J., R. D. Newberry, S. R. Schloemann, K. G. McDonald, and
W. F. Stenson. 2003. Inhibition of indoleamine 2,3-dioxygenase augments trinitrobenzene sulfonic acid colitis in mice. Gastroenterology 125: 1762–1773.
5. Seo, S. K., J. H. Choi, Y. H. Kim, W. J. Kang, H. Y. Park, J. H. Suh, B. K. Choi,
D. S. Vinay, and B. S. Kwon. 2004. 4 –1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med. 10: 1088 –1094.
6. Odemuyiwa, S. O., A. Ghahary, Y. Li, L. Puttagunta, J. E. Lee, S. Musat-Marcu,
A. Ghahary, and R. Moqbel. 2004. Cutting edge: human eosinophils regulate T
cell subset selection through indoleamine 2,3-dioxygenase. J. Immunol. 173:
5909 –5913.
7. Bozza, S., F. Fallarino, L. Pitzurra, T. Zelante, C. Montagnoli, S. Bellocchio,
P. Mosci, C. Vacca, P. Puccetti, and L. Romani. 2005. A crucial role for tryptophan catabolism at the host/Candida albicans interface. J. Immunol. 174:
2910 –2918.
8. Uyttenhove, C., L. Pilotte, I. Theate, V. Stroobant, D. Colau, N. Parmentier,
T. Boon, and B. J. Van den Eynde. 2003. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat. Med. 9: 1269 –1274.
9. Muller, A. J., J. B. DuHadaway, P. S. Donover, E. Sutanto-Ward, and
G. C. Prendergast. 2005. Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Nat. Med. 11: 312–319.
10. Grohmann, U., F. Fallarino, and P. Puccetti. 2003. Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol. 24: 242–248.
11. Moreau, M., J. Lestage, D. Verrier, C. Mormede, K. W. Kelley, R. Dantzer, and
N. Castanon. 2005. Bacille Calmette-Guérin inoculation induces chronic activation of peripheral and brain indoleamine 2,3-dioxygenase in mice. J. Infect. Dis.
192: 537–544.
12. Mellor, A. L., B. Baban, P. Chandler, B. Marshall, K. Jhaver, A. Hansen,
P. A. Koni, M. Iwashima, and D. H. Munn. 2003. Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell
clonal expansion. J. Immunol. 171: 1652–1655.
13. Muller, A. J., W. P. Malachowski, and G. C. Prendergast. 2005. Indoleamine
2,3-dioxygenase in cancer: targeting pathological immune tolerance with smallmolecule inhibitors. Expert Opin. Ther. Targets 9: 831– 849.
14. Baban, B., P. Chandler, D. McCool, B. Marshall, D. H. Munn, and A. L. Mellor.
2004. Indoleamine 2,3-dioxygenase expression is restricted to fetal trophoblast
giant cells during murine gestation and is maternal genome specific. J. Reprod.
Immunol. 61: 67–77.
15. Munn, D. H., M. D. Sharma, D. Hou, B. Baban, J. R. Lee, S. J. Antonia,
J. L. Messina, P. Chandler, P. A. Koni, and A. L. Mellor. 2004. Expression of
indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining
lymph nodes. J. Clin. Invest. 114: 280 –290.
16. Munn, D. H., M. D. Sharma, B. Baban, H. P. Harding, Y. Zhang, D. Ron, and
A. L. Mellor. 2005. GCN2 kinase in T cells mediates proliferative arrest and
anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 22:
633– 642.
17. Mellor, A. L., and D. H. Munn. 2004. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat. Rev. Immunol. 4: 762–774.
18. Brusko, T. M., C. H. Wasserfall, A. Agarwal, M. H. Kapturczak, and
M. A. Atkinson. 2005. An integral role for heme oxygenase-1 and carbon mon-
oxide in maintaining peripheral tolerance by CD4⫹CD25⫹ regulatory T cells.
J. Immunol. 174: 5181–5186.
Moffett, J. R., and M. A. Namboodiri. 2003. Tryptophan and the immune response. Immunol. Cell. Biol. 81: 247–265.
Braun, D., R. S. Longman, and M. L. Albert. 2005. A two-step induction of
indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation.
Blood 106: 2375–2381.
Orabona, C., P. Puccetti, C. Vacca, S. Bicciato, A. Luchini, F. Fallarino,
R. Bianchi, E. Velardi, K. Perruccio, A. Velardi, et al. 2006. Toward the identification of a tolerogenic signature in IDO-competent dendritic cells. Blood 107:
2846 –2854.
Grohmann, U., R. Bianchi, C. Orabona, F. Fallarino, C. Vacca, A. Micheletti,
M. C. Fioretti, and P. Puccetti. 2003. Functional plasticity of dendritic cell subsets
as mediated by CD40 versus B7 activation. J. Immunol. 171: 2581–2587.
Fallarino, F., C. Vacca, C. Orabona, M. L. Belladonna, R. Bianchi, B. Marshall,
D. B. Keskin, A. L. Mellor, M. C. Fioretti, U. Grohmann, and P. Puccetti. 2002.
Functional expression of indoleamine 2,3-dioxygenase by murine CD8␣⫹ dendritic cells. Int. Immunol. 14: 65– 68.
Wu, H. Q., P. Guidetti, J. H. Goodman, M. Varasi, G. Ceresoli-Borroni,
C. Speciale, H. E. Scharfman, and R. Schwarcz. 2000. Kynurenergic manipulations influence excitatory synaptic function and excitotoxic vulnerability in the rat
hippocampus in vivo. Neuroscience 97: 243–251.
Orabona, C., M. L. Belladonna, C. Vacca, R. Bianchi, F. Fallarino, C. Volpi,
S. Gizzi, M. C. Fioretti, U. Grohmann, and P. Puccetti. 2005. Cutting edge:
silencing suppressor of cytokine signaling 3 expression in dendritic cells turns
CD28-Ig from immune adjuvant to suppressant. J. Immunol. 174: 6582– 6586.
Fallarino, F., C. Asselin-Paturel, C. Vacca, R. Bianchi, S. Gizzi, M. C. Fioretti,
G. Trinchieri, U. Grohmann, and P. Puccetti. 2004. Murine plasmacytoid dendritic cells initiate the immunosuppressive pathway of tryptophan catabolism in
response to CD200 receptor engagement. J. Immunol. 173: 3748 –3754.
Fallarino, F., R. Bianchi, C. Orabona, C. Vacca, M. L. Belladonna, M. C. Fioretti,
D. V. Serreze, U. Grohmann, and P. Puccetti. 2004. CTLA-4-Ig activates forkhead transcription factors and protects dendritic cells from oxidative stress in
nonobese diabetic mice. J. Exp. Med. 200: 1051–1062.
Guidetti, P., R. E. Luthi-Carter, S. J. Augood, and R. Schwarcz. 2004. Neostriatal
and cortical quinolinate levels are increased in early grade Huntington’s disease.
Neurobiol. Dis. 17: 455– 461.
Sapko, M. T., P. Guidetti, P. Yu, D. A. Tagle, R. Pellicciari, and R. Schwarcz.
2006. Endogenous kynurenate controls the vulnerability of striatal neurons to
quinolinate: implications for Huntington’s disease. Exp. Neurol. 197: 31– 40.
Fallarino, F., U. Grohmann, K. W. Hwang, C. Orabona, C. Vacca, R. Bianchi,
M. L. Belladonna, M. C. Fioretti, M. L. Alegre, and P. Puccetti. 2003. Modulation
of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206 –1212.
Orabona, C., U. Grohmann, M. L. Belladonna, F. Fallarino, C. Vacca, R. Bianchi,
S. Bozza, C. Volpi, B. L. Salomon, M. C. Fioretti, et al. 2004. CD28 induces
immunostimulatory signals in dendritic cells via CD80 and CD86. Nat. Immunol.
5: 1134 –1142.
Grohmann, U., F. Fallarino, R. Bianchi, M. L. Belladonna, C. Vacca, C. Orabona,
C. Uyttenhove, M. C. Fioretti, and P. Puccetti. 2001. IL-6 inhibits the tolerogenic
function of CD8␣⫹ dendritic cells expressing indoleamine 2,3-dioxygenase.
J. Immunol. 167: 708 –714.
Fallarino, F., U. Grohmann, C. Vacca, R. Bianchi, C. Orabona, A. Spreca,
M. C. Fioretti, and P. Puccetti. 2002. T cell apoptosis by tryptophan catabolism.
Cell Death Differ. 9: 1069 –1077.
Grohmann, U., R. Bianchi, M. L. Belladonna, C. Vacca, S. Silla, E. Ayroldi,
M. C. Fioretti, and P. Puccetti. 1999. IL-12 acts selectively on CD8␣⫺ dendritic
cells to enhance presentation of a tumor peptide in vivo. J. Immunol. 163:
3100 –3105.
Grohmann, U., R. Bianchi, M. L. Belladonna, S. Silla, F. Fallarino, M. C. Fioretti,
and P. Puccetti. 2000. IFN-␥ inhibits presentation of a tumor/self peptide by
CD8a⫺ dendritic cells via potentiation of the CD8␣⫹ subset. J. Immunol. 165:
1357–1363.
Belladonna, M. L., J. C. Renauld, R. Bianchi, C. Vacca, F. Fallarino, C. Orabona,
M. C. Fioretti, U. Grohmann, and P. Puccetti. 2002. IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells. J. Immunol. 168:
5448 –5454.
Schwarcz, R., and R. Pellicciari. 2002. Manipulation of brain kynurenines: glial
targets, neuronal effects, and clinical opportunities. J. Pharmacol. Exp. Ther. 303:
1–10.
Stone, T. W., and L. G. Darlington. 2002. Endogenous kynurenines as targets for
drug discovery and development. Nat. Rev. Drug. Discov. 1: 609 – 620.
Guillemin, G. J., G. Smythe, O. Takikawa, and B. J. Brew. 2005. Expression of
indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia 49: 15–23.
Boasso, A., J. P. Herbeuval, A. W. Hardy, C. Winkler, and G. M. Shearer. 2005.
Regulation of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA-synthetase
by CTLA-4-Fc in human CD4⫹ T cells. Blood 105: 1574 –1581.
Hwang, S. L., N. P. Chung, J. K. Chan, and C. L. Lin. 2005. Indoleamine 2,
3-dioxygenase (IDO) is essential for dendritic cell activation and chemotactic
responsiveness to chemokines. Cell Res. 15: 167–175.
Terness, P., T. M. Bauer, L. Rose, C. Dufter, A. Watzlik, H. Simon, and G. Opelz.
2002. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J. Exp. Med. 196: 447– 457.
Frumento, G., R. Rotondo, M. Tonetti, G. Damonte, U. Benatti, and
G. B. Ferrara. 2002. Tryptophan-derived catabolites are responsible for inhibition
The Journal of Immunology
44.
45.
46.
47.
48.
of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase.
J. Exp. Med. 196: 459 – 468.
Platten, M., P. P. Ho, S. Youssef, P. Fontoura, H. Garren, E. M. Hur, R. Gupta,
L. Y. Lee, B. A. Kidd, W. H. Robinson, et al. 2005. Treatment of autoimmune
neuroinflammation with a synthetic tryptophan metabolite. Science 310:
850 – 855.
McIlroy, D., S. Tanguy-Royer, N. Le Meur, I. Guisle, P. J. Royer, J. Leger,
K. Meflah, and M. Gregoire. 2005. Profiling dendritic cell maturation with dedicated microarrays. J. Leukocyte Biol. 78: 794 – 803.
Heyes, M. P., C. Y. Chen, E. O. Major, and K. Saito. 1997. Different kynurenine
pathway enzymes limit quinolinic acid formation by various human cell types.
Biochem. J. 326(Pt 2): 351–356.
Chiarugi, A., M. Calvani, E. Meli, E. Traggiai, and F. Moroni. 2001. Synthesis
and release of neurotoxic kynurenine metabolites by human monocyte-derived
macrophages. J. Neuroimmunol. 120: 190 –198.
Bronte, V., and P. Zanovello. 2005. Regulation of immune responses by L-arginine metabolism. Nat. Rev. Immunol. 5: 641– 654.
137
49. Sawitzki, B., C. I. Kingsley, V. Oliveira, M. Karim, M. Herber, and K. J. Wood.
2005. IFN-␥ production by alloantigen-reactive regulatory T cells is important for
their regulatory function in vivo. J. Exp. Med. 201: 1925–1935.
50. Montagnoli, C., F. Fallarino, R. Gaziano, S. Bozza, S. Bellocchio, T. Zelante,
W. P. Kurup, L. Pitzurra, P. Puccetti, and L. Romani. 2006. Immunity and tolerance to Aspergillus involve functionally distinct regulatory T cells and tryptophan catabolism. J. Immunol. 176: 1712–1723.
51. Fallarino, F., U. Grohmann, S. You, B. C. McGrath, D. R. Cavener, C. Vacca,
C. Orabona, R. Bianchi, M. L. Belladonna, C. Volpi, et al. 2006. The combined
effects of tryptophan starvation and tryptophan catabolites down-regulate T cell
receptor ␨ chain and induce a regulatory phenotype in naive T cells. J. Immunol.
In press.
52. Orabona, C., E. Tomasello, F. Fallarino, R. Bianchi, C. Volpi, S. Bellocchio,
L. Romani, M. C. Fioretti, E. Vivier, P. Puccetti, and U. Grohmann. 2005. Enhanced tryptophan catabolism in the absence of the molecular adapter DAP12.
Eur. J. Immunol. 35: 3111–3118.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017