IDO2 is critical for IDO1-mediated T cell regulation and exerts a non-redundant function in inflammation.
ABSTRACT IDO2 is implicated in tryptophan catabolism and immunity but its physiological functions are not well established. Here we report the characterization of mice genetically deficient in IDO2, which develop normally but exhibit defects in IDO-mediated T cell regulation and inflammatory responses. Construction of this strain was prompted in part by our discovery that IDO2 function is attenuated in macrophages from Ido1-/- mice due to altered message splicing, generating a functional mosaic with implications for interpreting findings in Ido1-/- mice. No apparent defects were observed in Ido2-/- mice in embryonic development or hematopoietic differentiation, with wild-type profiles documented for kynurenine in blood serum and for immune cells in spleen, lymph nodes, peritoneum, thymus and bone marrow of naïve mice. In contrast, upon immune stimulation we determined that IDO1-dependent T regulatory cell generation was defective in Ido2-/- mice, supporting Ido1-Ido2 genetic interaction and establishing a functional role for Ido2 in immune modulation. Pathophysiologically, both Ido1-/- and Ido2-/- mice displayed reduced skin contact hypersensitivity responses, but mechanistic distinctions were apparent, with only Ido2 deficiency associated with a suppression of immune regulatory cytokines that included GM-CSF, G-CSF, IFN-γ, TNFα, IL-6 and MCP-1/CCL2. Different contributions to inflammation were likewise indicated by the finding that Ido2-/- mice did not phenocopy Ido1-/- mice in the reduced susceptibility of the latter to inflammatory skin cancer. Taken together, our results offer an initial glimpse into immune modulation by IDO2, revealing its genetic interaction with IDO1 and distinguishing its non-redundant contributions to inflammation.
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ABSTRACT: Indoleamine 2,3-dioxygenases (IDOs) are tryptophan-catabolizing enzymes with immunomodulatory functions. However, the biological role of IDO2 and its relationship with IDO1 are unknown. To assess the relationship between IDO2 and IDO1, we investigated the effects of co-expression of human (h) IDO2 on hIDO1 activity. Cells co-expressing hIDO1 and hIDO2 showed reduced tryptophan metabolic activity compared with those expressing hIDO1 only. In a proteomic analysis, hIDO1-expressing cells exhibited enhanced expression of proteins related to the cell cycle and amino acid metabolism, and decreased expression of proteins related to cell survival. However, cells co-expressing hIDO1 and hIDO2 showed enhanced expression of negative regulators of cell apoptosis compared with those expressing hIDO1 only. Co-expression of hIDO1 and hIDO2 rescued the cell death induced by tryptophan-depletion through hIDO1 activity. Cells expressing only hIDO2 exhibited no marked differences in proteome profiles or cell growth compared with mock-transfectants. Cellular tryptophan metabolic activity and cell death were restored by co-expressing the hIDO2 mutant substituting the histidine 360 residue for alanine. These results demonstrate that hIDO2 plays a novel role as a negative regulator of hIDO1 by competing for heme-binding with hIDO1, and provide information useful for development of therapeutic strategies to control cancer and immunological disorders that target IDO molecules.Experimental and Molecular Medicine 11/2014; 46:e121. · 2.46 Impact Factor
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ABSTRACT: IDO2 is a relative of IDO1 implicated in tryptophan catabolism and immune modulation but its specific contributions to normal physiology and pathophysiology are not known. Evolutionary genetic studies suggest that IDO2 has a unique function ancestral to IDO1. In mice, IDO2 gene deletion does not appreciably affect embryonic development or hematopoiesis, but it leads to defects in allergic or autoimmune responses and in the ability of IDO1 to influence the generation of T regulatory cells. Gene expression studies indicate that IDO2 is a basally and more narrowly expressed gene than IDO1 and that IDO2 is uniquely regulated by AhR, which serves as a physiological receptor for the tryptophan catabolite kynurenine. In the established KRN transgenic mouse model of rheumatoid arthritis, where IDO1 gene deletion has no effect, IDO2 deletion selectively blunts responses to autoantigen but has no effect on responses to neoantigen challenge. In human populations, natural variations in IDO2 gene sequence that attenuate enzymatic activity have been reported to influence brain cancer control and adaptive immune responses to the IDO2 protein itself, consistent with the concept that IDO2 is involved in shaping immune tolerance in human beings. Biochemical and pharmacological studies provide further evidence of differences in IDO2 enzymology and function relative to IDO1. We suggest that IDO2 may act in a distinct manner from IDO1 as a set-point for tolerance to "altered-self" antigens along the self-non-self continuum where immune challenges from cancer and autoimmunity may arise.Frontiers in Immunology 11/2014; 5:585.
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ABSTRACT: Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that have independently evolved to catalyze the first step in tryptophan catabolism via the kynurenine pathway (KP). The depletion of tryptophan and formation of KP metabolites modulates the activity of the mammalian immune, reproductive, and central nervous systems. IDO and TDO enzymes can have overlapping or distinct functions depending on their expression patterns. The expression of TDO and IDO enzymes in mammals differs not only by tissue/cellular localization but also by their induction by distinct stimuli. To add to the complexity, these genes also have undergone duplications in some organisms leading to multiple isoforms of IDO or TDO. For example, many vertebrates, including all mammals, have acquired two IDO genes via gene duplication, although the IDO1-like gene has been lost in some lower vertebrate lineages. Gene duplications can allow the homologs to diverge and acquire different properties to the original gene. There is evidence for IDO enzymes having differing enzymatic characteristics, signaling properties, and biological functions. This review analyzes the evolutionary convergence of IDO and TDO enzymes as tryptophan-catabolizing enzymes and the divergent evolution of IDO homologs to generate an enzyme family with diverse characteristics not possessed by TDO enzymes, with an emphasis on the immune system.Frontiers in Immunology 10/2014; 5:485.
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IDO2 is critical for IDO1-mediated T-cell regulation
and exerts a non-redundant function in inflammation
Richard Metz1*, Courtney Smith2*, James B. DuHadaway2, Phillip Chandler3, Babak Baban3,
Lauren M. F. Merlo2, Elizabeth Pigott2, Martin P. Keough2, Sonja Rust1, Andrew L. Mellor3,
Laura Mandik-Nayak2,4, Alexander J. Muller2,5 and George C. Prendergast2,5,6
1New Link Genetics Corporation, Ames, IA 50010, USA
2Lankenau Institute for Medical Research, Wynnewood, PA 19096, USA
3Immunotherapy Center, Georgia Regents University, Augusta, GA 30912, USA
4Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107, USA
5Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA
6Department of Pathology, Anatomy and Cell Biology, Jefferson Medical School, Thomas Jefferson University, Philadelphia, PA
Correspondence to: G. C. Prendergast, Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA.
*These authors contributed equally to this study
Received 9 August 2013, accepted 16 December 2013
IDO2 is implicated in tryptophan catabolism and immunity but its physiological functions are
not well established. Here we report the characterization of mice genetically deficient in IDO2,
which develop normally but exhibit defects in IDO-mediated T-cell regulation and inflammatory
responses. Construction of this strain was prompted in part by our discovery that IDO2 function
is attenuated in macrophages from Ido1−/− mice due to altered message splicing, generating a
functional mosaic with implications for interpreting findings in Ido1–/– mice. No apparent defects
were observed in Ido2–/– mice in embryonic development or hematopoietic differentiation, with
wild-type profiles documented for kynurenine in blood serum and for immune cells in spleen, lymph
nodes, peritoneum, thymus and bone marrow of naive mice. In contrast, upon immune stimulation
we determined that IDO1-dependent T regulatory cell generation was defective in Ido2−/− mice,
supporting Ido1–Ido2 genetic interaction and establishing a functional role for Ido2 in immune
modulation. Pathophysiologically, both Ido1−/− and Ido2−/− mice displayed reduced skin contact
hypersensitivity responses, but mechanistic distinctions were apparent, with only Ido2 deficiency
associated with a suppression of immune regulatory cytokines that included GM-CSF, G-CSF,
IFN-γ, TNF-α, IL-6 and MCP-1/CCL2. Different contributions to inflammation were likewise indicated
by the finding that Ido2−/− mice did not phenocopy Ido1−/− mice in the reduced susceptibility of the
latter to inflammatory skin cancer. Taken together, our results offer an initial glimpse into immune
modulation by IDO2, revealing its genetic interaction with IDO1 and distinguishing its non-
redundant contributions to inflammation.
Keywords: adaptive immunity, contact hypersensitivity, inflammation, tolerance
IDO2 is the most recently discovered of the four tryptophan
catabolic enzymes in mammals (1, 2). IDO2 is most closely
related to IDO1, which like the other enzymes in this group
has been implicated in inflammation and immune control
(2–5). Compared with the other enzymes, IDO2 has a rather
restricted pattern of expression, confined mainly to antigen-
presenting immune cells, liver, kidney, brain and placenta,
suggesting non-redundant functions (2, 6). The IDO2 gene
is located immediately downstream of IDO1 in a region of
human chromosome 8p21 that was annotated poorly until
recently. Given their structural, chromosomal and evolution-
ary relationships (7), one question is how IDO2 may bear on
the immunobiology related or ascribed to IDO1. Initial stud-
ies of IDO1 highlighted a role in the maternal–fetal interface,
following the discovery that the IDO1 inhibitor d,l-1-methyl-
tryptophan (1MT) could trigger rejection of hemiallogeneic
International Immunology Advance Access published January 13, 2014
at Thomas Jefferson University on February 10, 2014
Page 2 of 11 IDO2 supports inflammatory signaling
murine concepti (8, 9). However, this finding preceded the
discovery of IDO2, which under various conditions can also
be inhibited by the d and l racemers of 1MT (2, 7, 10, 11).
1MT has been used widely to implicate IDO1 activation in
numerous pathologies, including cancer, chronic infection,
allergy and autoimmunity (4). The likelihood of some overlap
in the function of these enzymes is suggested by evidence
that Ido1 genetic deficiency in mice leads to compensatory
up-regulation of IDO2 in the epididymis, where IDO1 is nor-
mally highly expressed (12). Elevated IDO1 has been associ-
ated with poor prognosis in a wide variety of human cancers
(13) and genetic studies in mouse models have confirmed
the importance of IDO1 in tumor and metastasis development
(14–17), while the potential contributions of IDO2 in these set-
tings have yet to be explored.
Tryptophan depletion by either enzyme generating kynure-
nine would activate the stress kinase GCN2 and repress
the growth regulatory kinase mTOR, reflecting cellular star-
vation for an essential amino acid (18, 19). However, while
both IDO1 and IDO2 may activate the GCN2 pathway, this
effector mechanism can be reversed by tryptophan restora-
tion only in the case of IDO1, implying that IDO2 generates
a unique tryptophan-independent signal (2). IDO2 can also
blunt T-cell activation, but 1MT racemers cannot stanch this
effect as demonstrated for IDO1 (11). Studies of fungal IDO
homologs also support the notion of functional differences
(20). Lastly, the unique presence in the IDO2 promoter of a
binding site for the transcription factor IRF-7, a master regula-
tor of the maturation of dendritic cells (DC), suggests a dis-
tinct role in these professional antigen-presenting cells (APC)
(S. Trabanelli, unpublished results).
To begin to discern the physiological and pathophysiologi-
cal functions of IDO2, we generated mice that are geneti-
cally deficient in the Ido2 gene. Our initial characterization
of these animals suggests some similarities to IDO1, in that
IDO2 was found to be dispensable for overall development
and hematopoietic cell differentiation, while in the context of
CpG-elicited immune stimulation, we present evidence that
IDO2 is essential for IDO1-dependent induction of T regula-
tory cells (Treg). However, we also document some important
differences between IDO1 and IDO2, in that IDO2 was found
to be non-essential for inflammatory skin carcinogenesis
where IDO1 is critical (21). Further, while deletion of either
IDO1 or IDO2 resulted in attenuated contact hypersensitiv-
ity (CHS) responses, mechanistic differences were apparent,
highlighting their distinct roles in inflammation and immunity.
Overall, our results suggest that IDO2 is non-redundant with
IDO1 and they provide the first direct evidence that it con-
tributes to the control of inflammation and adaptive immunity.
Generation of a transgenic mouse strain genetically deficient
The 5′ homologous arm (4.5 kb), the 3′ homologous arm
(3.5 kb) and the conditionally targeted region (1.7 kb) were
generated by PCR from the murine BAC genomic clone
RP23-339B16 and cloned in the LoxFtNWCD PCR 4.0 vectors
using standard molecular cloning methods. The final vector
included loxP sequences flanking the conditionally targeted
region, Frt sequences flanking a neo expression cassette
and a diphtheria toxin (DTA) expression cassette for reverse
selection. Not I was used to linearize the final vector for elec-
troporation prior to injection into C57BL6/J blastocytes, ulti-
mately generating chimeras that were evaluated for germline
transmission (Taconic, NY, USA). Cre-dependent deletion of
exons 9 and 10 was achieved by interbreeding with EIIA-cre.
B6 transgenic mice (Jackson Laboratories) to generate prog-
eny with a germline disruption of the Ido2 allele. These mice
were bred subsequently for homozygosity of the recombined
Ido2 mutant allele and loss of the EIIA-cre transgene. Primers
designed to flank exons 9 and 10 were used to follow allelic
deletions in generating a 400 bp product in wild-type (WT)
mice and a 500 bp product in Ido2−/− mice.
Livers harvested from euthanized WT, Ido1−/− and
Ido2−/− C57BL6/J mice were passed through a 70 µm
strainer to generate a single-cell suspension. RNA
extracted with TRIzol (Invitrogen) was subjected to first-
strand cDNA synthesis using oligo-dT primer (Promega
GoScript). Ido1 and Ido2 expressions were meas-
ured by quantitative RT–PCR (qRT–PCR) using SYBR
green for detection (Sigma SYBR Green JumpStart Taq
Ready Mix). Expression of target genes was determined
relative to GAPDH and calculated as 2^−(CtTarget gene
−CtGAPDH) as primers had similar efficiencies. Ido1 prim-
ers were 5′-CCCACACTGAGC ACGGACGG-3′ and
5′-TTGCGGGGCA GCACCTTTCG-3′. Two sets of IDO2
primers were used to confirm deletion of IDO2. Primer
set 1 was 5′-GCCCAGAG CTCCGTGCTTCAT-3′ and
5′-TGGGAAGGCGG CATGTAGTCC-3′ and it spanned
exons 9–10. Primer set 2 was 5′-CAATCCAGCCATGCCT
GTGGGG-3′ and 5′-TGGGCTGCACTT CCTCCAGAGT-3′
and was located in exon 9. Primers for the housekeeping
gene GAPDH were 5′-TGCACCACCAACTGCTTAGC-3′ and
5′-GGCATGGA CTGTGGTCATGAG-3′. For B-cell isolation,
resting B cells isolated from WT or Ido1−/− mouse spleno-
cytes with magnetic beads linked to anti-CD43 antibody
were placed in culture and left unstimulated or stimulated
with 25 µg·ml−1 LPS (Sigma cat. no. L3012) with or with-
out 50 ng·ml−1 recombinant mouse IL-4 (R&D Systems cat.
no. 404-ML). Unstimulated B cells were harvested immedi-
ately and stored at −80°C for processing with stimulated B
cells harvested 48 h later. Cellular RNA isolated using the
Invitrogen RNAEasy™ kit was used for qRT–PCR, using the
ThermoScript RT-PCR System (Invitrogen cat. no. 11146-
016) to generate cDNAs and the primers noted above.
IDO2 and IDO1 cDNA cloning
For ectopic expression analyses, the coding region for the
full-length murine IDO1 message and the murine IDO2 WT
and alternate splice isoform described (∆4) were cloned
by standard methods. For IDO2, the primer set used was
mIDO2FLA 5′-CCATGGAGCCTC AAAGTCAG-3′ and mIDO-
2FLB 5′-CTAAGCACCAGGAC ACAGGAG-3′. For IDO1, the
primer set used was mIDO1FLA 5′-ATGGCACTCAGTAAAA
TATCTCCTAC-3′ and mIDO1FLB 5′-CTAAGGCCAACTCAG
at Thomas Jefferson University on February 10, 2014
IDO2 supports inflammatory signaling Page 3 of 11
Murine IDO2 was detected in livers and kidneys isolated from
WT, Ido1−/− and Ido2−/− C57BL6/J mice as follows. Tissue
lysates were minced in PBS plus protease inhibtors (1 mM
phenylmethylsulphonylfluoride [PMSF], Protease Inhibitor
Cocktail set III [Calbiochem cat. no. 539134], 10 mM E-64
[Sigma cat. no. E3132]), adjusted to 1× RIPA (50 mM Tris–
HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg·ml−1
Aprotinin, 5 µg·ml−1 Leupeptin, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS), vortexed and incubated on ice
for 30 min and then clarified by centrifugation at 12 000
r.p.m. for 15 min at 4°C in a microfuge. Clarified lysates
(100 µg each) were incubated on a rocker shaker for 1 h at
4°C with RIPA-equilibrated Dynabeads Protein A (Invitrogen
cat. no. 100.01D). Beads were removed and anti-IDO2 mAb
(Origen cat. no. TA501378) was added with fresh RIPA-
equilibrated Dynabeads Protein A for a further incubation,
after which the beads were collected by gentle centrifuga-
tion, washed thrice with RIPA buffer and re-suspended in
20 µl SDS sample buffer for SDS–PAGE (10% Novex Tris-Gly
system [Invitrogen]) and western-blot analysis. Briefly, after
transfer to nitrocellulose, the blot was incubated with shaking
at room temperature for 1 h in 5% non-fat dry milk in PBS/0.1%
Tween, then incubated for 1 h with 2 µl affinity-purified rabbit
anti-IDO2 antibody (2) in blocking buffer and finally washed
thrice for 10 min each in PBS/0.1% Tween. Blots were devel-
oped by incubation for 1 h at room temperature with 2 µl anti-
rabbit IgG HRP, light chain (Jackson Immunologicals cat.
no. 211-032-171), washed thrice with PBS/0.1%Tween and
treated briefly with the Pico-ECL system (Pierce).
Steady-state levels of serum kynurenine were determined in
naive mice using methods described previously (22).
Flow cytometric analysis of cytokines and leukocytes
Flow cytometric data were acquired on a BD FACSCanto II or
Cyan ADP flow cytometer and analyzed using FACSDIVA (BD
Biosciences) software. Leukocytes were analyzed as previ-
ously described (23) using the cell surface markers noted in
Supplementary Table I, available at International Immunology
Online. Multiplexed cytokine analysis was conducted using
cytometric bead array (BD Biosciences). Tissue homogen-
ates were centrifuged and supernatant was added to beads
in the array according to the manufacturer’s instructions.
Cytokine concentrations were calculated by comparison
to standard curves using FACSArray analysis software (BD
Mixed leukocyte assays
Assays for regulatory DC activity and Treg activity were con-
ducted in the same manner as described previously (24, 25),
except that cells from Ido2−/− mice instead of Ido1−/− were used.
Mice were sensitized with 3% oxazolone (Sigma) on their
shaved abdomen (50 µl) and hind footpads (5 µl each). Six
days later, mice were elicited with 20 µl of 1% oxazolone on
the left ear (experimental site) or 20 µl 100% ethanol on the
right ear (control site). After 24 h, ear thickness was measured
using a dial gauge (Fowler, A&M Industrial Supply, Rahway,
NJ, USA) with the difference determined in swelling between
the oxazolone-treated ear and the vehicle-treated ear. Trials
performed in multiple mice were replicated at least once.
Oxazolone-treated mice were sensitized as described above.
On day 5, all mice were retro-orbitally injected with Evan’s
blue dye (5 mg·ml−1). After 2 h, the first group of mice was
elicited with 1% oxazolone and the second group of mice
was elicited with 20 µl 85% lactic acid. Ears were measured
and harvested at the maximal swelling (4 h for lactic acid and
24 h for oxazolone). After thickness measurements, ears were
harvested from euthanized animals and dried in a 55°C oven
for 6 h, after which they were weighed, minced and placed in
1 ml formamide overnight. Samples were filtered and OD620
was measured to assess extravasated dye using a standard
curve of Evan’s blue dye. Vascular leakage was measured by
the amount of Evan’s blue dye per milligram of dried tissue
from the oxazolone- or lactic-acid-treated ear minus the value
of leakage in the vehicle-treated ear from the same animal.
Two-stage inflammatory skin papillomagenesis
Mice of 6–8 weeks of age were subjected to a classical
protocol of two-stage carcinogenesis by topical application
of the mutagen DMBA and the inflammatory phorbol ester
12-O-tetradecanoylphorbol-13-acetate (PMA) as described
in detail elsewhere (21).
IDO1 deficiency causes altered splicing and ablation of
IDO2 function in macrophages
Metz et al. (2) suggested that d-1MT did not inhibit tryptophan
catabolism mediated by IDO1 but rather by IDO2. However,
in IDO1-deficient mice, anti-tumor responses to d-1MT treat-
ment were abolished, arguing that IDO1 was essential for
d-1MT bioactivity at some level. One interpretation of these
results was that IDO1 and IDO2 might genetically interact,
such that IDO1 acts upstream to influence IDO2 expres-
sion or activity. In evaluating this hypothesis, we compared
the structure and sequence of IDO2 RNA transcripts in
Ido1−/− and WT mice. Primary peritoneal macrophages were
placed in culture for 48 h and then left untreated or stimu-
lated with IFN-γ, after which total RNA was isolated 24 h
later for RT–PCR amplification and TA cloning of IDO2 tran-
scripts. Sequence analysis of >100 individual cDNA isolates
from each cell population revealed that IDO2 messages in
IFN-γ-stimulated WT cells were largely full-length transcripts
(Fig. 1A, top panel), whereas IDO2 messages prepared from
IFN-γ-stimulated Ido1−/− cells were mainly alternately spliced
in a manner that deleted 36 nt from the 5′ end of exon 4,
generating an in-frame excision of 12 aa from the full-length
IDO2 enzyme (Fig. 1A, bottom panel). Comparing the ratio of
alternately spliced to full-length message in these two pop-
ulations of IFN-γ-stimulated macrophages (as quantitated
by the individual cDNA isolates sequenced), we found that
at Thomas Jefferson University on February 10, 2014
Page 4 of 11 IDO2 supports inflammatory signaling
~60% of the total IDO2 messages expressed in Ido1−/− cells
were alternately spliced, whereas only ~20% of the total IDO2
messages in WT cells were alternately spliced (as depicted
in Fig. 1A).
We further examined IDO2 messages by qRT–PCR analy-
sis in macrophages, B cells and liver where previous analyses
and public in silico data had indicated IDO2 is expressed (2).
First, we confirmed expression of the variant splice isoform
of IDO2 in primary peritoneal macrophages that were stimu-
lated by LPS + IL-4 instead of IFN-γ (Fig. 1B). Under these
conditions, there was no change in the ratio of alternately
spliced to full-length message illustrating specificity in the
IFN-γ response. However, in Ido1−/− macrophages, there was
a strong relative up-regulation of IDO2 expression overall,
suggesting compensation with IDO1 loss. In B lymphocytes,
we found that basal levels of IDO1 and IDO2 expression were
Fig. 1. Ido1 genetic ablation leads to predominant expression in hematopoietic cells of a variant Ido2 RNA splice isoform that is enzymatically
deficient. (A) Altered Ido2 RNA sequence. RT–PCR products generated from RNA isolated from primary peritoneal macrophages stimulated
with IFN-γ were subjected to DNA sequencing. Analysis of individual generating an in-frame 12 aa deletion. Through sequence analysis of
>100 clonal IDO2 cDNA isolates, it was found that ~20% of IDO2 messages were alternately spliced in WT cells compared with ~60% in
Ido1−/− cells. (B) Altered Ido2 RNA expression in macrophages. Primary peritoneal macrophages were isolated from WT or Ido1−/− mice and
stimulated for 24 h with LPS ± IL-4. RNA was isolated for RT–PCR and agarose gel fractionation. (C) Altered Ido2 RNA expression in B lympho-
cytes. Resting B cells isolated from WT or Ido1−/− splenocytes were cultured overnight and left unstimulated (UI) or stimulated 24 h with LPS
or LPS + IL-4. RNA isolated from harvesting was subjected to RT–PCR and agarose gel fractionation. (D) Ectopic expression of the variant Ido2
splice isoform. Full-length and exon 4 splice variant cDNAs were cloned into the tet-regulatable expression vector pcDNATO4 and introduced
by DNA-mediated transfection into T-Rex 293 cells (Invitrogen). Stable clones treated with doxocycline to induce transgene expression were
subjected to western-blot analysis with an IDO2 antibody (2). (E) Reduced kynurenine production by the variant Ido2 splice isoform. Cells were
treated for 48 h with doxocycline and kynurenine levels were determined from culture supernatants. The experiment was performed in triplicate
with data presenting the mean and standard error.
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IDO2 supports inflammatory signaling Page 5 of 11
elevated or unaffected by LPS ± IL-4 treatment, respectively
(Fig. 1C). In B cells lacking IDO1, we found that IDO2 mes-
sages were not only strongly relatively elevated, as in mac-
rophages, but also strongly shifted in structure towards the
alternately spliced isoform (Fig. 1C). These observations fur-
ther suggested compensatory up-regulation of IDO2 in the
absence of IDO1 and offered further evidence of specificity
in alternate splice control of IDO2 message. In contrast to
these results, we observed no relative change in IDO2 mes-
sage level or splicing in livers isolated from Ido1−/− mice (data
not shown). Thus, the variation we documented represented
a tissue-specific event in IDO1-deficient mice occurring only
in hematopoietic cells of the innate and adaptive immune
To assess the functional impact of this alternate splicing
event, we cloned the exon 4 splice variant and the WT mes-
sage into the doxycycline-inducible expression vector pcD-
NATO4 for stable expression in T-Rex 293 human cells. Clonal
cell lines treated with doxycycline exhibited similar levels of
steady-state expression as confirmed by western-blot analy-
sis (Fig. 1D). The splice variant documented (∆4) generated a
truncated IDO2 protein relative to the WT isoform as expected
(Fig. 1D). Notably, under the same conditions, kynurenine
production by the splice variant was greatly diminished com-
pared with the WT isoform (Fig. 1E). This result supported the
hypothesis that IDO1 affects IDO2 function in macrophages
and B lymphocytes at the level of RNA splicing. In providing
evidence of a genetic interaction between these genes, our
findings presented the possibility that a subset of functions
ascribed to IDO1 in Ido1−/− mice might actually be explained
by a mosaic loss of function in IDO2 in those animals.
IDO2 is non-essential for development, basal serum
kynurenine levels or hematopoiesis
To explore in vivo functions of IDO2, we generated trans-
genic mice genetically deficient in this gene. Exons 9 and
10 encoding the catalytic region were targeted based on
confirmation of their requirement in recombinant IDO2 for
tryptophan catabolic activity (data not shown). Starting from
a BAC genomic clone containing the murine Ido1 and Ido2
genes, we designed a conditional deletion plasmid vector
that would allow the construction of complete, tissue-spe-
cific or mosaic knockout (KO) mice by standard methods
(Fig. 2A). Briefly, homologous genomic recombination of the
plasmid in independent murine embryonal stem cell clones
was confirmed by restriction digestion and end-sequencing
analyses (data not shown). Mice bred from positive chimeric
animals generated by clonal microinjection were crossed with
EIIa-cre transgenic mice to achieve germline recombination
of the conditional Ido2-null allele, which was homozygosed
in subsequent generations as confirmed by PCR analysis of
tail DNA (Fig. 2B). We confirmed the loss of expression of
Ido2 message in the liver of Ido2−/− mice, where this gene
is normally most highly expressed (2), and we also exam-
ined whether Ido1 expression might be affected as a com-
pensatory event in Ido2−/− mice. RT–PCR analysis of liver
RNA confirmed Ido2 expression in WT but not Ido2−/− mice,
as expected (data not shown). This result was validated fur-
ther by IP/western-blot analysis of liver and kidney extracts
prepared from WT, Ido1−/− and Ido2−/− mice (Fig. 2C), showing
loss of IDO2 protein expression in Ido2−/− mice. We observed
similar Ido1 expression levels in livers from WT or Ido2−/− mice,
consistent with non-compensation and a distinct function for
Ido2 (Supplementary Figure S1 is available at International
Immunology Online). Basal kynurenine levels in serum were
comparable between Ido2−/− and WT mice (Fig. 2D). This
result was expected, given the narrow range of IDO2 expres-
sion relative to IDO1 and TDO, the latter of which would be
expected to contribute more significantly to systemic kynure-
nine production because of their much broader and higher
levels of expression relative to IDO2.
A comparison of immune cell profiles in naive Ido2−/− and
WT mice showed no significant differences. Briefly, cells
were harvested and analyzed from the thymus, bone mar-
row, spleen, lymph nodes and peritoneal cavity (equal num-
bers of males and females) and analyzed by flow cytometry
for expression of specific developmental and lineage mark-
ers. No differences were found in the relative numbers
of any of the following leukocyte cell populations exam-
ined (Supplementary Figure S2 is available at International
Immunology Online). We saw little discernable change in
pro-B, pre-B, immature or mature B cells in the bone mar-
row, and no change in double negative, double positive or
single positive CD4 or CD8 T cells in the thymus. There was
no change in the relative percentages of B-1 or B-2 B cells
in the peritoneal cavity of Ido2−/− mice. Similarly, there was
no change in the relative number of leukocytes in the spleen
and lymph nodes, including follicular, marginal zone or total B
cells; CD4+ or CD8+ T cells; NKT cells; or macrophages and
neutrophils. Lastly, no differences were noted in lymphocyte
activation markers CD25, CD44, CD62L and CD69 on these
cell types. We conclude that IDO2 does not grossly affect
hematopoietic differentiation in naive mice, consistent with
previous characterizations of IDO1-deficient mice and with
the expectation that IDO2 was similarly likely to act as a modi-
fier rather than regulator of immunity.
IDO2 is essential for IDO1-dependent T-cell suppression
IDO1 acts to control the activation and differentiation of Treg
in a variety of settings, including in cancer (25–29). Given
evidence suggesting genetic epistasis between IDO1 and
IDO2, we wished to determine whether IDO2 deletion might
affect IDO1-mediated Treg generation in settings where an
essential function for IDO1 has been established. Thus, we
compared the activity of Treg isolated from WT and Ido2−/−
mice treated with CpG oligonucleotides in an ex vivo T-cell
suppression assay containing responder A1 T cells, APC
and H-Y peptide that has been described previously (25). In
this assay, ex vivo proliferation of A1 T cells was restricted
by WT Treg activated in vivo, and suppression mediated by
the Treg was reversed by mAbs against programmed death
1(PD-1)/PD-1 ligand 1 (PD-L1)/PD-L2 that block PD-1/PD-L1/
PD-L2 interactions, a validated hallmark of IDO1-activated
Tregs (25). Notably, Ido2−/− Treg isolated 24 h after CpG oli-
gonucleotide treatment did not suppress ex vivo proliferation
of A1 T cells, and addition of PD-1/PD-L1 blocking mAbs
did not further enhance A1 proliferation (Fig. 3). This effect
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Page 6 of 11 IDO2 supports inflammatory signaling
phenocopied the effect of Ido1−/− Treg generated under the
same conditions (25), supporting a functional role for IDO2 in
Treg control and genetic interaction with IDO1.
IDO2 loss does not phenocopy IDO1 loss in suppressing
To assess the functional significance of IDO2 in the context
of IDO1-associated pathophysiology, we compared the sus-
ceptibility of Ido2−/− and WT mice to DMBA + PMA-induced
skin papillomagenesis, a well-established model of inflam-
matory cancer where IDO1 has been shown to have a criti-
cal role (21). Briefly, mice were exposed to a single topical
application of the Hras-activating mutagen DMBA followed
by weekly topical applications of PMA, which triggers a local
inflammation that leads to papilloma development. Previous
work has shown that Ido1−/− mice are resistant to this carci-
nogenic protocol (21). In contrast to the resistance of Ido1−/−
mice, we found that Ido2−/− mice displayed the same rate of
papilloma formation as WT mice (Fig. 4). Thus, IDO1 and
IDO2 did not phenocopy each other in inflammatory skin car-
cinogenesis, establishing that in vivo IDO2 and IDO1 have
distinct biological functions.
IDO2 is critical for contact hypersensitivity
To assess the impact of IDO2 loss on a classical adaptive
inflammatory response, we compared WT and Ido2−/− mice
for their susceptibility to hapten-induced contact hypersen-
sitivity (CHS). Topical application of a hapten antigen on the
Fig. 2. Generation of an Ido2-deficient mouse. (A) Gene deletion strategy. The targeting vector included positive (Neo) and negative (DTA) selec-
tion strategies. The DTA expression cassette distal to the specific IDO2 targeting sequences negatively selected for non-homologous recombina-
tion and the neomycin cassette flanked by sites for FLP recombinase positively selected for homologous recombination. LoxP sites flanking Cre
recombinase and the Ido2 murine exons 9 and 10 provided the means for Cre-dependent excision of this region encoding the catalytic domain of
IDO2 in the desired ES cell recombinant. (B) Genetically targeted mice. Tail DNA was screened by PCR for the expected Ido2 deletion. (C) IDO2
and IDO1 expression in liver from WT and Ido2−/− mice. Total RNA isolated from tissues harvested from euthanized animals were analyzed by
qRT–PCR. (D) Serum kynurenine levels. Steady-state levels of kynurenine were quantitated in serum by an LC/MS-based method as described
before (22). The experiment was repeated once with data obtained from samples processed in triplicate (mean ± standard error).
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IDO2 supports inflammatory signaling Page 7 of 11
abdomen and footpads triggers migration of Langerhans cells
(LC) to regional lymph nodes where MHC class II-restricted
presentation occurs. Following a second application on the
ear, a strong recall response occurs accompanied by ear
swelling. In this effector phase, CD8+ T cells are recruited and
secrete pro-inflammatory cytokines including IFN-γ, IL-1, IL-6
and IL-8 (keratinocyte-derived chemokine [KC]), with CD4+
T cells also recruited as a negative feedback mechanism to
ameliorate the response via production of IL-4 and IL-10 (30).
Biologically, the CHS response is quantitated by the degree
of swelling of the treated ear relative to the untreated ear
(31). We compared the degree of ear swelling in WT mice to
Ido2−/− and Ido1−/− mice after challenge with the well-estab-
lished contact sensitizer oxazolone. Relative to WT mice, ear
swelling after oxazolone challenge was reduced 37.9% in
Ido2−/− mice and 18.4% in Ido1−/− mice (Fig. 5A and B). In
contrast to the significant impact on ear swelling observed
in the CHS assay, Ido2−/− mice did not demonstrate any dif-
ference in swelling relative to WT mice when challenged
with lactic acid, a non-specific irritant that inflames the skin
through innate-only mechanisms (Fig. 5C). Similar outcomes
were observed when the degree of inflammation-induced
vascular leakiness was assessed using a modified Miles
assay (32) (Fig. 5D). Taken together, these results establish
a biological role for IDO2 in regulating the adaptive immune
response in the context of CHS.
Role of IDO2 in CHS is mechanistically distinct from IDO1
We next compared the mechanistic contributions of IDO1 and
IDO2 to inflammatory responses in the CHS model. In CHS,
cytokines are key drivers of inflammation along with immune
cell chemotaxis and maturation/differentiation. Therefore, we
investigated cytokine signaling 24 h after oxazolone re-expo-
sure. Both treated and untreated ears on each mouse were
harvested for evaluation. Supernatants from tissue homogen-
ates were analyzed using a cytokine array that included IL-1β,
IL-2, IL-4, IL-5, IL-6, IL-10, IL-12/IL-23-p40, IL-13, IL-17A,
IFN-γ, TNF-α, MCP-1, MIP-1α, RANTES, G-CSF and GM-CSF.
In untreated ears, baseline levels for every cytokine were sim-
ilar in WT, Ido1−/− and Ido2−/− mice, as expected. In treated
ears, Ido2−/− mice displayed significantly lower induction of
the inflammatory cytokines IL-6, IFN-γ and TNF-α (Fig. 6A).
Additionally, induction of the chemotactic cytokine MCP-1/
CCL2 was attenuated significantly along with the two key
hematopoietic cytokines GM-CSF and G-CSF (Fig. 6A). The
pattern in these responses was completely distinct from that
observed in Ido1−/− mice where the responses trended in the
opposite direction of those produced by Ido2 loss (Fig. 6A).
Fig. 3. IDO2 ablation attenuates IDO1-dependent Treg generation. Treg suppression assays were performed essentially as described before
(25). Briefly, MACS-enriched CD4+CD25+ cells obtained from WT or Ido2−/− mice treated i.v. with 100 µg CpG 1826 oligonucleotide were mixed
with MACS-enriched CD4+ T cells from A1 TCR-transgenic mice, MACS-enriched CD11c+ APC from female CBA spleen and H-Y-Ek cognate
peptide. IDO1 dependence in this assay was discriminated as established previously by adding a mixture of antibodies against PD-1, PD-L1
and PD-L2 (25). The experiment was repeated once with data obtained from samples processed in triplicate (mean ± standard error).
Fig. 4. IDO2 is dispensable for inflammatory skin papillomagenesis
unlike IDO1. C57BL6/J WT mice (n = 8) and Ido2−/− mice (n = 10)
were administered a single topical application of 400 nM DMBA at
week 0 followed by twice weekly applications of 10 µg of PMA during
weeks 1–20. Papillomas arising during this protocol were counted twice
weekly. The data present the number of papillomas per mouse plotted
as mean values from each group ± SE. Significance was measured with
a non-parametric two-tailed Mann–Whitney test. The experiment was
repeated once with data presented as mean ± standard error.
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Page 8 of 11 IDO2 supports inflammatory signaling
Ido2 loss produced no observable effect on the remaining
cytokines in the panel (data not shown).
To assess whether the decrease in affected cytokines
reflected a more proximal defect in their production by
immune cells, we purified lymphocytes from auricular lymph
nodes isolated from animals treated to elicit CHS as before.
These primary cells were cultured ex vivo with PMA and iono-
mycin to investigate whether they were inherently defective
in cytokine production. Interestingly, analysis of cell super-
natants revealed a similar production of cytokines regardless
of genotype (Fig. 6B). Thus, the defect observed in vivo was
not recapitulated ex vivo, suggesting that Ido2 loss acted
indirectly to block cytokine production by cells that were oth-
erwise competent. Taken together, the results suggest that
IDO1 and IDO2 both contribute to CHS but that they diverge
in the mechanism of action. Accordingly, these results offer
further mechanistic evidence that the functions of IDO2 and
IDO1 are different, since while both genes contribute to CHS
they do not act similarly in how they regulate key contributing
This study provides the first direct physiological evidence
that IDO2 functions in immune control. Initial characteriza-
tion of the genetically deficient mouse strain reported here
argues that IDO2 is genetically linked but non-redundant with
IDO1, its closest relative among the four tryptophan catabolic
enzymes expressed in mammals. Unlike ablation of murine
IDO1 or TDO, which are more widely and strongly expressed,
ablation of murine IDO2 does not affect systemic blood levels
of kynurenine, leaving open some existing questions about
the enzymology and substrate specificity of IDO2 but offering
a useful tool to address them in a physiological context that
has been lacking to date. Our findings extend the concept
that IDO2 functions differently from IDO1, as introduced ini-
tially by cell-based studies suggesting differences in effector
As has been shown to be the case with deficiency in IDO1,
deficiency in IDO2 did not appear to impair development or
hematopoietic differentiation in the naive immune system (33).
In contrast, an immunomodulatory role was revealed under
conditions of immune stimulation, where IDO2 was found to
be critical for IDO1-dependent Treg function. A requirement
for IDO2 in Treg induction originally defined for IDO1 (34) is
consistent with evidence reported here for the genetic epista-
sis of IDO2 because there is misregulation of IDO2 expres-
sion in macrophages from Ido1−/− mice. How IDO1 influences
RNA splicing of IDO2 is not yet known but seems likely to be
indirect. In any case, the evident mosaic disruption of IDO2
function in Ido1−/− mice has implications for interpreting immu-
nological deficiencies in Ido1−/− mice, given the possibility that
Fig. 5. IDO2 ablation attenuates oxazolone-induced CHS. (A) Time course. Mice were compared for hapten-specific ear swelling at 24-h inter-
vals for 4 days after elicitation (n = 5 per group). The data present the relative increase in the thickness of treated ears compared with untreated
ears. The data shown are the means summed from four independent replicates. (B) Statistical analysis of 24 h data from (A) by a Mann–Whitney
t-test. **, P < 0.05; ***, P < 0.01. (C) IDO2 ablation affects the adaptive arm of the CHS response. Ears were challenged with either oxazolone
or lactic acid and measured 24 h later (n = 5 per group). (D) Effect of IDO2 ablation on ear vascular permeability. Measurement is based on the
amount of Evan’s blue dye extravasated 24 h following CHS elicitation with oxazolone or lactic acid. The latter experiments were repeated once
and the data shown are means summed with statistical analysis as in (B).
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IDO2 supports inflammatory signaling Page 9 of 11
some deficiencies might be ascribed to loss of IDO2 function.
In human immune physiology, the identification of this func-
tional link between IDO1 and IDO2 is particularly intriguing
to consider given the high frequency of occurrence of two
functionally attenuating Ido2 polymorphisms in the human
population (2) that may result in a spectrum of different levels
of IDO2 functionality. Since APC are a primary site of IDO2
expression, further investigation is needed to understand
how IDO2 may act to initiate, maintain, fix or reverse antigen
tolerance. Along these lines, one recent study in human DC
suggests that IDO2 may help fix basal levels of tolerance,
acting differently than IDO1 which unlike IDO2 in these cells
is induced strongly by pro-inflammatory signals (S. Trabanelli,
Given the observed differences between Ido1−/− and
Ido2−/− mice in CHS mechanism of action and requirements
in inflammatory skin carcinogenesis, where IDO1 is essen-
tial to confer cancer-associated inflammatory determinants to
the skin microenvironment (21, 35), the contributions made
by IDO2 to inflammation are clearly differentiable from IDO1
for reasons that remain mechanistically unresolved. Using
CHS as an initial platform to evaluate contributions to immune
modulation, we found that IDO2 was required for induction of
a number of pivotal cytokines unaffected by IDO1, despite
the outwardly similar reduction in inflammation associated
with loss of either gene in this setting. Specifically, IDO2
was crucial for induction of GM-CSF, G-CSF, IFN-γ, TNF-α,
IL-6 and MCP-1/CCL2, most of which have been causally
implicated in CHS themselves by genetic ablation (36–40).
While IFN-γ and TNF-α, associated with Th1 responses, were
decreased in Ido1−/− mice, we saw no changes in IL-2, IL-4,
IL-10 or IL-17A consistent with a lack of skewing in Th2/Th17
populations due to Ido2 deficiency in mice with either naive
or activated immune systems. Changes in IL-6 and MCP-1/
CCL2 were interesting given that their induction in inflamma-
tion-driven models of cancer relies upon IDO1 (17), which is
not the case in CHS where IDO2 is involved instead, suggest-
ing parallels in inflammatory control in CHS versus cancer.
However, there are clearly other distinctions, since this paral-
lel does not address the different requirements of IDO1 and
IDO2 in driving inflammatory skin cancer.
GM-CSF is interesting to consider among the cytokines
affected by loss of IDO2 but not IDO1 during the CHS
response. GM-CSF promotes maturation of LC (41), the
primary APC in the skin. In the CHS response, LC residing
in the skin take up the hapten antigen, undergo maturation
and migrate to draining lymph nodes where they initiate an
adaptive immune response (42). Notably, mice deficient in
the kynurenine receptor AhR (3) display reduced GM-CSF
secretion and impaired LC maturation (41). Thus, one model
for understanding how IDO2 could support CHS invokes
IDO2-mediated kynurenine as an upstream activator of AhR-
dependent LC maturation and GM-CSF secretion, the lack
of which would lead to an attenuated CHS response as
observed. This model is consistent with evidence of crucial
roles for kynurenine and AhR in maturation of bone mar-
row-derived DC and differentiation of Treg (43, 44). Since
the IDO2 promoter is itself a target for AhR activation (45),
it is plausible that IDO2 may act in a feed-forward loop to
reinforce expression of GM-CSF and other cytokines that
Fig. 6. IDO2 ablation attenuates expression of cytokines associated with LC maturation and inflammation in CHS. (A) Cytokine levels in ear tis-
sue. Treated and untreated ears were harvested from euthanized mice 24 h after oxazolone elicitation (n = 5 each group) and cytokines were
quantitated in homogenized tissue extracts by a multiplexed cytokine bead immunoassay. The data are plotted as the mean ± SEM with sig-
nificance relative to baseline evaluated by the Mann–Whitney t-test. Data present the mean from three independent trials. (B) Cytokine levels in
auricular lymph nodes. Treated ears were harvested as above and 1 × 106 cells were plated and stimulated with PMA and ionomycin. Cell culture
supernatants were collected 24 h later and cytokine levels were quantitated as before. The data are plotted as the mean ± SEM with one replicate.
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Page 10 of 11 IDO2 supports inflammatory signaling
promote DC/LC maturation. In any case, such a model may
offer a logical starting point with explanative and predictive
elements to interpret how IDO2 may act in APCs to sup-
port IDO1-dependent Treg induction, an area which clearly
requires further study. In closing, we note that IDO2-deficient
mice will be useful not only to advance studies of how immu-
nometabolism mediates tolerance in normal physiology and
disease but also to gain insight into mechanisms of action of
IDO inhibitors being developed to treat cancer, chronic infec-
tion and other disorders, where early clinical trials suggest
Supplementary data are available at International Immunology
Conflict of interest: R.M., J.B.D., A.L.M., A.J.M. and G.C.P. declare
a conflict of interest due to their various relationships with New Link
Genetics Corporation, which has licensed IDO- and IDO2-related
intellectual property from the Lankenau Institute for Medical Research
and Georgia Regents University. R.M. (formerly of the Lankenau
Institute), J.B.D. and A.J.M. are inventors and shareholders in the
company. G.C.P. and A.L.M. are inventors and shareholders who
have received grant support and compensation from the company in
their role as scientific advisors. C.S., P.C., B.B., L.M.F.M., E.P., M.P.K.,
S.R. and L.M.N. declare no conflict of interest related to the work in
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