The Immunoregulatory Enzyme IDO Paradoxically Drives B
Grant N. Scott,* James DuHadaway,* Elizabeth Pigott,* Natalie Ridge,*†
George C. Prendergast,* Alexander J. Muller,* and Laura Mandik-Nayak2*
Rheumatoid arthritis (RA) is a chronic and debilitating inflammatory autoimmune disease of unknown etiology. As with a
variety of autoimmune disorders, evidence of elevated tryptophan catabolism has been detected in RA patients indicative of
activation of the immunomodulatory enzyme IDO. However, the role that IDO plays in the disease process is not well
understood. The conceptualization that IDO acts solely to suppress effector T cell activation has led to the general assumption
that inhibition of IDO activity should exacerbate autoimmune disorders. Recent results in cancer models, however, suggest
a more complex role for IDO as an integral component of the inflammatory microenvironment necessary for supporting
tumor outgrowth. This has led us to investigate the involvement of IDO in the pathological inflammation associated with RA.
Using the K/BxN murine RA model and IDO inhibitor 1-methyl-tryptophan, we found that inhibiting IDO activity had the
unexpected consequence of ameliorating, rather than exacerbating arthritis symptoms. 1-Methyl tryptophan treatment led
to decreased autoantibody titers, reduced levels of inflammatory cytokines, and an attenuated disease course. This alleviation
of arthritis was not due to an altered T cell response, but rather resulted from a diminished autoreactive B cell response, thus
demonstrating a previously unappreciated role for IDO in stimulating B cell responses. Our findings raise the question of
how an immunosuppressive enzyme can paradoxically drive autoimmunity. We suggest that IDO is not simply immunosup-
pressive, but rather plays a more complex role in modulating inflammatory responses, in particular those that are driven by
autoreactive B cells.
The Journal of Immunology, 2009, 182: 7509–7517.
and debilitating destruction of cartilage and bone (1). K/BxN mice
spontaneously develop a joint inflammatory disease that shares
many characteristics with human RA, including cellular infiltrates,
proinflammatory cytokines, autoantibodies, and cartilage and bone
destruction (2, 3). This model uses a TCR transgene, KRN, that
when present in a genetic background expressing the I-Ag7MHC
class II molecule leads to the development of arthritis (2). Arthritis
can be induced either spontaneously by breeding KRN with mice
expressing I-Ag7(K/BxN model) or by transferring serum from
arthritic mice into any naive strain of mice (serum transfer model)
(3). In K/BxN mice, the autoreactive T and B cells both recognize
the glycolytic enzyme glucose-6-phosphate isomerase (GPI) as an
heumatoid arthritis (RA)3is an inflammatory autoim-
mune disease characterized by chronic inflammation of
the synovial joints, eventually leading to a progressive
autoantigen and disease severity correlates with rising titers of anti-
GPI Ig in the serum (3–6). However, as in human RA, the factors
responsible for triggering the initiating autoimmune response in
K/BxN mice are unknown.
IDO is an IFN-?-inducible enzyme that catalyzes the initial and
rate-limiting step in the degradation of tryptophan (7, 8). An im-
munoregulatory role for IDO was suggested by the observation
that administration of the bioactive IDO inhibitor 1-methyl-tryp-
tophan (1MT) (9) elicited MHC-restricted, T cell-mediated rejec-
tion of allogeneic mouse concepti (10, 11). IDO has also been
shown to be a critical driver of immune escape in cancer (12).
This, coupled with data that IDO could suppress activation of ef-
fector T cells in vitro (13), led to the concept of IDO as an im-
munosuppressive actor involved in the establishment of acquired
peripheral immune tolerance.
If IDO were simply immunosuppressive, then it would be ex-
pected to play an inhibitory role in autoimmune responses. Indeed,
this is consistent with some reports using 1MT in the context of
inducible mouse models of autoimmunity, including experimental
autoimmune encephalomyelitis, collagen-induced arthritis, and tri-
nitrobenzene sulfonic acid-induced colitis (14–16). However,
other data, such as that reported in a mouse model of inflammatory
airway disease, show IDO can also play an activating role in driv-
ing Th2-mediated inflammatory responses (17). These data appear
to be more in line with the countervailing hypothesis that increased
IDO activity may, in some instances, contribute positively to in-
flammatory responses. This may be the more relevant model with
regard to autoimmunity in humans as elevated tryptophan degra-
dation has been shown to correlate with disease activity in both RA
and systemic lupus erythematosus patients (18, 19).
The first direct evidence that IDO could contribute to inflam-
matory disease pathology was the recent finding that elevated IDO
is an integral component of the severe cutaneous inflammation
*The Lankenau Institute for Medical Research, Wynnewood, PA 19096; and†St.
Joseph’s University, Philadelphia, PA 19131
Received for publication December 23, 2008. Accepted for publication April 6, 2009.
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.
1This work is supported by the Lankenau Hospital Foundation (to L.M.-N. and
G.C.P.). N.R. is supported by a research assistantship fellowship from the Brook J.
Lenfest Foundation. A.J.M. is the recipient of grants from the Department of Defense
Breast Cancer Research Program (BC044350), the Concern Foundation, and the
Lance Armstrong Foundation and G.C.P. is the recipient of National Institutes of
Health Grants CA109542, CA82222, and CA100123.
2Address correspondence and reprint requests to Dr. Laura Mandik-Nayak, The Lan-
kenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA
19096. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: RA, rheumatoid arthritis; ASC, Ab-secreting cell;
LN, lymph node; dLN, draining LN; GPI, glucose-6-phosphate isomerase; 1MT,
1-methyl-tryptophan; Treg, T regulatory cell; b.i.d., twice a day.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
produced by topical application of PMA, necessary for supporting
tumor outgrowth (20). Along these lines, we report here finding
that IDO activity is also elevated in the serum of K/BxN mice at
the earliest stages of joint inflammation. Importantly, the onset of
arthritis was delayed and disease severity alleviated by treatment
of these mice with the IDO inhibitory compound 1MT at this early
stage of disease progression. In contrast, if 1MT was administered
after this time point, it was no longer effective in treating joint
inflammation. The alleviation of joint inflammation with 1MT was
not due to a reduction in T regulatory cells (Tregs) or an altered Th
cell cytokine profile, but resulted from a diminished autoreactive B
cell response. These results provide the first indication that IDO
can contribute to the development of autoimmune disease pathol-
ogy by supporting the activation of autoreactive B cells, adding to
the growing body of evidence that IDO does not simply counteract
inflammation through its ability to suppress T cells, but rather is a
key constituent in the complex milieu of factors that shape the
Materials and Methods
KRN TCR-transgenic (Tg) mice on a C57BL/6 background have been
described previously (2). NOD mice were purchased from The Jackson
Laboratory. To obtain arthritic mice, homozygous KRN Tg C57BL/6 mice
were crossed with NOD mice yielding KRN (C57BL/6 ? NOD)F1mice
designated K/BxN. To obtain arthritic KRN B6.g7 mice, KRN C57BL/6
mice were crossed with C57BL/6 mice expressing the IAg7MHC class II
molecule to yield KRN C57BL/6 mice expressing IAb/g7(termed KRN
B6.g7). All mice were bred and housed under specific pathogen-free con-
ditions in the animal facility at The Lankenau Institute for Medical Re-
search. Studies were performed in accordance with the National Institutes
of Health and the Association for Assessment and Accreditation of Labo-
ratory Animal Care guidelines with approval from the Lankenau Institute
for Medical Research Institutional Animal Care and Use Committee.
Arthritis induction by K/BxN serum transfer
Serum was collected and pooled from 8-wk-old arthritic K/BxN mice. To
induce arthritis, 150 ?l of serum was injected i.p. into naive C57BL/6 mice
on day 0. Arthritis induced by this method is transient, beginning 48 h after
serum transfer and resolving 2–3 wk later (3).
Administration of 1MT
Mice were given a 400-mg/kg dose (100 ?l of total volume) of D/L-1MT
(Sigma-Aldrich) diluted in Methocel/Tween 80 (0.5% Tween 80 and 0.5%
methylcellulose (v/v in water); Sigma-Aldrich) twice daily by oral gavage
(p.o.) using a curved feeding needle (20-gauge 11⁄2 in; Fisher) as previously
described (21). The p.o. dose of 1MT (400 mg/kg) was selected based on
pharmacokinetic studies done previously in the laboratory. A dose titration
of 1MT (50–800 mg/kg) performed at 1 h after administration (Cmaxfor
1MT) showed that the maximum amount of 1MT in the serum (99.1 ? 6.4
?M) is achieved at the 400-mg/kg dose. We have confirmed, using K/BxN
mice, that the maximum level of 1MT in the serum of this strain is also
achieved at 400 mg/kg and that increasing the dosage to 800 mg/kg does
not further increase the serum level of 1MT. Control mice were given an
equal volume of carrier alone (Methocel/Tween 80). 1MT was adminis-
tered on a twice a day (b.i.d.) schedule, once in the morning and once in the
evening Monday to Friday. For the initial experiments, 1MT (2 mg/ml) was
also administered in water bottles on the weekends. No difference was seen
between mice that received 1MT via water bottles on weekends and mice
that were dosed only Monday to Friday. Therefore, in later experiments,
mice were only given 1MT by oral gavage Monday to Friday. For the mice
that spontaneously develop arthritis (K/BxN and KRN B6.g7), 1MT treat-
ment was started at 21 days of age. For mice whose arthritis was induced
by serum transfer, 1MT treatment was started 6 h before the administration
of arthritic serum.
The two rear ankles of K/BxN or KRN B6.g7 mice were measured starting
at weaning (3 wk of age). Measurement of ankle thickness was made above
the footpad axially across the ankle joint using a Fowler Metric Pocket
Thickness Gauge. Ankle thickness was rounded off to the nearest 0.05 mm.
At the termination of the experiment, ankles were fixed in 10% buffered
formalin for 48 h, decalcified in 14% EDTA for 2 wk, embedded in par-
affin, sectioned, and stained with H&E.
Anti-GPI ELISA. Mice were bled once a week between 3 and 11 wk of
age. Sera were stored at ?20°C before analysis. Serum samples were
plated at an initial dilution of 1/100 and diluted serially 1/5 in Immulon II
plates coated with GPI-His (2 ?g/ml). Recombinant GPI-His protein was
generated and purified as described previously. The serum titer was defined
as the reciprocal of the last dilution that gave an OD more than three times
Anti-Ig ELISA. Tissue culture supernatants were plated in triplicate undi-
luted on Immulon II plates coated with unlabeled donkey anti-mouse total
Ig (Jackson ImmunoResearch Laboratories). Purified mouse IgM, IgG1,
IgG2a, IgG2b, and IgG3 (Southern Biotechnology Associates) were used
to generate standard curves. The total amount of Ig in the supernatant
was calculated from the standard curve using Prism 4 software (Graph-
For all ELISAs. Donkey anti-mouse total Ig- HRP (Jackson Immuno-
Research Laboratories), goat anti-mouse IgM-HRP, IgG1-HRP, IgG2a-
HRP, IgG2b-HRP, IgG2c-HRP, or IgG3-HRP (Southern Biotechnology
Associates) were used as secondary Abs. Ab was detected using ABTS
Spleen or lymph node (LN) cells were plated at 4 ? 105cells/well and
diluted serially 1/4 in Multiscreen HA mixed cellulose ester membrane
plates (Millipore) coated with GPI-His (2 ?g/ml). The cells were incubated
on the Ag-coated plates for 4 h at 37°C. The Ig secreted by the plated cells
was detected by alkaline phosphatase-conjugated goat anti-mouse total Ig
secondary Ab (Southern Biotechnology Associates) and visualized using
NBT/5-bromo-4-chloro-3-indolyl phosphate substrate (Sigma-Aldrich).
Cells from the draining LNs (dLNs) of carrier- or control-treated K/BxN
mice were harvested and cultured in either medium alone or PMA (50
ng/ml) plus ionomycin (500 ng/ml) for 16 h. The supernatants were then
harvested and analyzed for the levels of IL-2, IL-4, IL-5, IL-6, IL-12,
TNF-?, IFN-?, and MCP-1 by cytometric bead array (BD Biosciences) and
IL-17 by ELISA.
Cytometric bead array. For the cytometric bead array, samples were
stained according to the manufacturer’s instructions and analyzed on a
FACSCanto II flow cytometer (BD Biosciences) using FACSDIVA soft-
ware (BD Biosciences). Cytokine concentrations were calculated by com-
paring to standard curves using cytometric bead array analysis software
Anti-IL-17 ELISA. Tissue culture supernatants were plated in duplicate on
anti-IL-17-coated wells (clone TC11-18H10). IL-17 was detected with a
biotinylated anti-IL-17 secondary Ab (clone TC11-8H4.1; BD Bio-
sciences) followed by streptavidin-HRP. ABTS was used as a substrate.
The amount of IL-17 in the supernatant was calculated by comparison to a
standard curve generated with rIL-17 (R&D Systems) using Prism software
Joint dLNs (popliteal, axillary, and brachial) were harvested from K/BxN
mice ages 4 wk (prearthritic, early arthritic), 6 wk (acute arthritic), and 8
wk (chronic arthritic). Epididymis from K/BxN- and IDO-deficient mice
were used as positive and negative controls, respectively. Pooled LNs from
two mice each or epididymis were frozen in LN2, mechanically disrupted
with a mortar and pestle, and lysed in radioimmunoprecipitation buffer
containing protease and phosphatase inhibitors. One microgram of total
protein for each LN sample and 50 ?g from each epididymis sample were
immunoprecipitated with 1 ?g of affinity-purified polyclonal rabbit anti-
IDO1 (gift from R. Metz, NewLink Genetics, Ames, IA), separated by
SDS-PAGE, and blotted to an Immobilon-NC membrane (Millipore). The
blots were then incubated with 2.5 ?g of monoclonal rat anti-IDO (clone
mIDO-48; BioLegend), and detected with HRP-conjugated anti-rat Ig
(Southern Biotechnology Associates) using ECL reagent (Pierce) accord-
ing to the manufacturer’s instructions.
Serum was collected from prearthritic, early arthritic, acute arthritic, and
chronic arthritic K/BxN mice. The serum was diluted in water (1/4 v/v),
deproteinated, and analyzed by HPLC coupled to electrospray ionization
7510 IDO DRIVES B CELL-MEDIATED AUTOIMMUNITY
tandem mass spectroscopy (liquid chromatography/mass spectrometry/
mass spectrometry) analysis as previously described (22). Quantitation of
kynurenine was based on analysis of two daughter ions.
Joint dLNs were harvested from carrier or 1MT-treated K/BxN mice.
One ? 106cells were stained with anti-CD4-PE-Cy7 (GK1.5) and anti-
CD25-allophycocyanin (3C7; BioLegend), fixed/permeabilized with
Fixation/Permeabilization solution, and then stained intracellularly with
anti-Foxp3-PE (FJK-16s; eBioscience). Samples were analyzed on a
FACSCanto II flow cytometer (BD Biosciences) using FACSDIVA soft-
ware (BD Biosciences). Data were analyzed using CellQuest software (BD
Biosciences). Gating on live lymphocytes was based on forward and side
scatter, with 100,000 events collected for each sample.
In vitro stimulation
B cells from non-Tg C57BL/6 spleens were purified using a MACS sep-
aration system with paramagnetic anti-CD43 beads (Miltenyi Biotec). Cell
purity was ?95%. In some experiments, the cells were also labeled with 5
?M CFSE (Invitrogen). In brief, 106B cells were cultured for 3 days in a
1-ml total volume in either medium alone (IDMEM, 10% FCS, 5 ? 10?5
M 2-ME), 0.5–25 ?g/ml LPS (Escherichia coli strain 0111:B4; Sigma
Aldrich), 1–50 ?g/ml goat anti-mouse IgM (Fab?)2(Jackson Immuno-
Research Laboratories), or 100-2000 ng/ml anti-CD40 (clone 1C10; R&D
Systems) plus 50 ng/ml IL-4 (eBioscience). The IDO inhibitor DL-1MT (in
DMSO plus 0.1 N HCl) or vehicle alone was added at a final concentration
of 100 ?M.
Statistical significance was determined using an unpaired Student’s t test or
the Mann-Whitney U nonparametric test and Instat Software (GraphPad
1MT delays the rate of arthritis development and alleviates its
To begin to define the role that IDO activation plays in shaping the
inflammatory autoimmune response in K/BxN mice, we inhibited
its activity pharmacologically using the racemic mix of 1MT (DL-
1MT). K/BxN mice were given 1MT, or carrier alone starting at 21
days of age, before the initiation of arthritis. Mice were monitored
for arthritis development by measuring joint inflammation by the
change in ankle thickness and synovial proliferation and inflam-
matory cell infiltrates by histology (Fig. 1). Consistent with
carrier alone (F) by oral gavage starting at the age of 21 days. A, Rear ankles were measured as an indication of arthritis and represented as the mean ankle
thickness ? SEM (top panel) and percentage of mice exhibiting joint inflammation (bottom panel). Representative experiment of four total, showing n ?
5 mice for each treatment. B, Metatarsal joint from carrier- or 1MT-treated K/BxN mouse at 42 days of age stained with H&E (top panel). Scale bar, 500
?m, (bottom panel) scale bar ? 50 ?m. Representative sections from a total of n ? 20 mice for each treatment group. C, Cells from the joint dLNs
(popliteal, axillary, and brachial LNs) were cultured overnight in medium alone or PMA (50 ng/ml) plus ionomycin (500 ng/ml). Cytokines were measured
in culture supernatants by cytometric bead array. Graphs show the mean concentration ? SEM from a representative experiment with n ? 9 carrier-treated
(?) and 12 1MT-treated (u) mice. This experiment was repeated three times. ??, p ? 0.01 and ?, p ? 0.03.
1MT inhibits arthritis development and inflammatory cytokine production. K/BxN mice were treated with 400 mg/kg 1MT b.i.d. (E) or
7511The Journal of Immunology
previous observations in untreated animals (5), K/BxN mice that
received carrier alone developed severe inflammation in their front
and rear paws that began between 28 and 35 days of age. In con-
trast, the rate of inflammation was slower and severity reduced in
mice that had IDO activity blocked with 1MT (Fig. 1A). The in-
cidence of arthritis was also reduced in 1MT-treated mice (Fig.
1A). At the termination of the experiment (7wk of age), rear ankles
were harvested and examined for histological evidence of arthritis
by staining with H&E (Fig. 1B). Carrier-treated mice showed clas-
sic signs of arthritis, with a greatly expanded synovium, pannus
formation, and inflammatory cell infiltrates. Similar to what was
observed by measuring ankle thickness, joints from 1MT-treated
mice showed a reduction in the severity of arthritis with minimal
synovial expansion and fewer infiltrating inflammatory cells. Con-
sistent with the reduction in arthritis, 1MT treatment also reduced
the levels of the inflammatory cytokines MCP-1 and IL-6 pro-
duced in the dLNs. Levels of IL-10 were also reduced (Fig. 1C).
The K/BxN model is an F1between C57BL/6 and NOD. Several
immune abnormalities have been described in NOD mice, includ-
ing decreased numbers of Tregs and reduced APC function (23,
24). Additionally, young female NOD mice have been reported to
have a defect in IDO activation attributable to a peroxynitrite-
induced blockade of IFN-? signaling in dendritic cells (25). These
abnormalities could complicate the interpretation of the 1MT stud-
ies in K/BxN mice. Therefore, we repeated the 1MT experiments
using KRN B6.g7 mice. KRN B6.g7 mice are C57BL/6 mice that
express both the KRN TCR Tg and the IAg7MHC class II mol-
ecule necessary for KRN T cell activation, but lack the rest of the
NOD-associated genes (2). KRN B6.g7 mice were given 1MT or
carrier alone, starting at 3 wk of age and monitored for arthritis
development (Fig. 2A). Similar to what was seen in K/BxN mice,
1MT delayed the rate of onset and attenuated the severity of ar-
thritis in KRN B6.g7 mice. Therefore, the antiarthritic effect of
1MT is independent of the NOD genetic background.
1MT exerts its inhibitory effect early in the response
Arthritis in K/BxN mice occurs in two phases, the initiation phase
that is dependent on GPI-specific T and B cells (3), and the effector
phase that occurs once anti-GPI Ab is produced and is dependent
upon neutrophils, macrophages, and mast cells (26–28). 1MT in-
hibition of IDO could affect one or both of these stages to block
arthritis development. In the K/BxN model, these two phases can
be separated experimentally by transferring serum from arthritic
mice to naive non-Tg recipients, bypassing the T and B cell-de-
pendent initiation phase (29). To test whether 1MT could inhibit
the effector stage, arthritis induced by serum transfer was com-
pared in carrier and 1MT-treated C57BL/6 mice (Fig. 2B). Carrier-
treated mice began to develop inflammation in their front and rear
paws 2 days after serum transfer, with a peak of inflammation 8
days after transfer. 1MT-treated mice developed arthritis with
identical kinetics, demonstrating that 1MT was unable to inhibit
the effector phase of arthritis.
To begin to define the mechanism by which 1MT inhibited the
initiation phase of arthritis development in K/BxN mice, we de-
termined when 1MT administration was required during the course
of arthritis to produce an efficacious response. First, we tested
whether 1MT was able to inhibit an ongoing arthritic response.
K/BxN mice were allowed to develop arthritis and then given 1MT
or carrier alone (Fig. 2C). When administered at this stage, 1MT
had no significant effect on arthritis development, corroborating
the serum transfer experiment, and thus indicating that 1MT
needed to be present earlier in the response to have its antiarthritic
effect. Next, we tested whether treatment with 1MT during the
establishment of disease would be sufficient to produce an antiar-
thritic effect. To accomplish this, K/BxN mice were given 1MT or
carrier alone for 10 days, at which time treatment was stopped and
mice were monitored for arthritis development (Fig. 2D). When
administered for just this short time period, 1MT was able to sig-
nificantly attenuate arthritis in the K/BxN mice, demonstrating that
blocking IDO activity with 1MT during the initiation phase of the
response was sufficient to affect the course of arthritis.
IDO is most active early in the arthritic response
1MT was only effective if administered early in the arthritic re-
sponse, and this short-term exposure was sufficient to attenuate
arthritis development even if the treatment was stopped. This sug-
gested that IDO expression or activity was essential during the
initiation of arthritis and either was inhibited or was unnecessary
once arthritis had been established. To evaluate IDO expression
during the course of arthritis, joint dLNs were harvested from
K/BxN mice at the very onset of arthritis development (pre/early),
response. A, KRN B6.g7 mice were treated with
carrier alone (E) or 1MT (F) starting at 21days
of age and followed for arthritis development. B,
Arthritis was induced in C57BL/6 mice by trans-
fer of arthritic serum on day 0. At the same time,
mice began treatment with either 1MT or carrier
alone. C, K/BxN mice were allowed to develop
arthritis and then were treated with carrier alone
or 1MT b.i.d. starting at the onset of arthritis (33
days of age). Treatment was continued through-
out the remainder of the experiment. D, K/BxN
mice were treated with carrier alone or 1MT
b.i.d. starting at the age of 21 days. Treatment
was stopped 10 days later and the mice followed
for arthritis development. Arthritis development
was assessed by measurements of rear ankle
thickness ? SEM. Representative experiments
of three total showing n ? 5 mice for each treat-
ment regimen. ??, p ? 0.01.
1MT exerts its effect early in the
7512IDO DRIVES B CELL-MEDIATED AUTOIMMUNITY
during the aggressive inflammatory stage (acute) or during estab-
lished arthritis (chronic). IDO protein was immunoprecipitated
from whole LN lysates and detected by Western blotting (Fig. 3A).
IDO protein was detectable at the earliest stage of arthritis and
remained present throughout arthritis development, ruling out dif-
ferential protein expression as an explanation for 1MT’s effects.
To measure IDO activity, serum samples from K/BxN mice were
analyzed for kynurenine levels (Fig. 3B), a breakdown product
indicative of IDO-mediated tryptophan catabolism. A spike in se-
rum kynurenine levels was detected in mice just after the onset of
arthritis. Kynurenine levels decreased once arthritis became estab-
lished in the acute and chronic stages. These data indicate that,
although IDO was expressed throughout the course of arthritis de-
velopment, it was most active at the initiation of the arthritic re-
sponse. Therefore, 1MT was effective at inhibiting arthritis devel-
opment only during the time when IDO was most active, at the
initiation of the autoimmune response.
1MT does not affect Th1/Th2/Th17 cytokine responses
One potential mechanism by which 1MT could inhibit arthritis
development early in the autoimmune response is by skewing the
cytokine profile of the T cells responsible for its initiation. IDO has
been shown to be a modifier of the cytokine profile of T cell re-
sponses in vitro (30). The precise mechanism by which IDO exerts
this effect is unknown, but is thought to involve tryptophan deple-
tion and/or tryptophan catabolites, which have been shown to pref-
erentially affect the survival of Th1, but not Th2 cells (31). Their
effect on Th17 cells has not been assessed.
TNF-? and, more recently, IL-17 have been proposed to be key
cytokines involved in both human and other mouse models of RA
(32–35). The cytokine profile of the anti-GPI T cells driving the
arthritogenic response in K/BxN mice is not well characterized,
although IL-4 is thought to be important to the disease process.
This is because serum anti-GPI Abs are predominantly IgG1, an
IL-4-related isotype, and K/BxN mice deficient in IL-4 show re-
duced disease (36). To determine which cytokines were expressed
during arthritis development in K/BxN mice and whether 1MT
altered this cytokine profile, we measured the cytokine profile se-
creted by T cells in the LNs draining the arthritic joints by ELISA
and/or the flow cytometry-based cytometric bead array (Fig. 4). In
cultures from control-treated K/BxN mice, the Th1 cytokine IFN-?
arthritis. A, IDO protein levels. Joint dLNs were harvested from untreated
K/BxN mice at the pre/early stage of arthritis (4 wk), acute stage of arthritis (6
wk), or chronic stage of arthritis (8 wk). Total LN protein lysates were im-
munoprecipitated with affinity-purified rabbit anti-mouse IDO1, separated by
SDS-PAGE, and then immunoblotted with monoclonal rat anti-mouse IDO Ig.
Epididymis from K/BxN mice and IDO1-deficient mice were used as a pos-
itive and negative control, respectively. This is a representative blot of two
pooled from two mice each. B, Serum kynurenine levels. Kynurenine levels
were measured in the serum of individual K/BxN mice at various stages of
arthritis. Each symbol represents an individual mouse with a bar depicting the
the prearthritic (p ? 0.001), acute arthritic (p ? 0.0007), and chronic arthritic
(p ? 0.0001) stages.
IDO is expressed throughout, but has highest activity early in
7–8 wk of age. Cells from the joint dLNs (popliteal, axillary, and brachial LNs) were cultured overnight in medium alone or PMA (P; 50 ng/ml) ?
Ionomycin (I; 500 ng/ml). Cytokines were measured in culture supernatants by cytometric bead array and/or ELISA. Graphs show the mean concentration ?
SEM from a representative experiment with n ? 9 carrier-treated and 12 1MT-treated mice from a total of three separate experiments.
1MT treatment does not alter Th1/Th2/Th17 cytokine levels. K/BxN mice were treated with 1MT (u) or carrier alone (?) and analyzed at
7513The Journal of Immunology
was detected at high levels and Th2 cytokine IL-5 was present at
a low level. IL-17 and TNF-? were both present at high levels, a
profile associated with Th17 cells. This is consistent with the in-
creasing evidence implicating both Th1 and Th17 cells as being
promoters of autoimmunity (37). In contrast to what was predicted
by the serum anti-GPI IgG1 profile, levels of IL-4 were barely
detectable above background. Surprisingly, 1MT did not affect this
cytokine profile. High levels of IFN-?, IL-17, and TNF-?, a low
level of IL-5, and almost no IL-4 were secreted by LN cells from
1MT-treated K/BxN mice (Fig. 4). Together, these data indicate
that 1MT does not inhibit arthritis development by skewing the Th
cytokine profile of the autoimmune response.
1MT-treated K/BxN mice have a normal frequency of Tregs
Tregs have been shown to be important in controlling the aggres-
siveness of autoimmunity in the K/BxN model (38, 39). Therefore,
treatment with the IDO inhibitor 1MT, which we have shown elic-
ited a reduction in the level of IL-6 in the joint dLNs of K/BxN
mice (Fig. 1C), might be inhibiting arthritis development through
suppression of Tregs, given that Treg development is promoted by
IDO and inhibited by IL-6 (40, 41). To test this, the percentage of
Tregs was quantitated by flow cytometry and compared in 1MT-
vs carrier-treated K/BxN mice (Table I). Tregs were elevated in the
dLNs compared with the non-dLN in carrier-treated K/BxN mice
(p ? 0.03) as has been reported for nontreated K/BxN mice (38,
42). Treg frequencies were also increased in the dLNs of 1MT-
treated K/BxN mice (p ? 0.0001). However, there was no differ-
ence in the percentage of Tregs in either dLNs (p ? 0.8) or non-
dLNs (p ? 0.3) in 1MT compared with carrier-treated mice.
Therefore, decreased disease activity in 1MT-treated mice could
not be attributed to a reduction in the percentage of Tregs.
1MT inhibits the anti-GPI B cell response
Abs that recognize the glycolytic enzyme GPI are the key effector
molecules in the disease process in K/BxN mice (4). Previously,
we demonstrated that this pathogenic anti-GPI B cell response was
focused to the LNs draining the arthritic joints (5). To address
whether the reduced arthritis in 1MT-treated mice was the result of
a diminished GPI-specific B cell response, we measured the titers
of anti-GPI Ig in the serum of 1MT- and carrier-treated mice (Fig.
5). Titers of anti-GPI Ig (Fig. 5A) were significantly lower in 1MT
compared with carrier-treated mice, particularly those of the IgG1
isotype required for disease initiation. The effect of 1MT appeared
to be specific to GPI-reactive B cells, as total serum Ig levels were
not different between carrier- and 1MT-treated animals (Fig. 5B).
To determine whether the reduced serum anti-GPI levels were
due to 1MT treatment affecting the location or magnitude of the
anti-GPI B cell response, the number of Ab-secreting cells (ASCs)
was quantitated in the spleen, dLNs, and non-dLNs of carrier- vs
1MT-treated K/BxN mice (Fig. 5C). GPI-reactive ASCs were
present in all three locations in carrier-treated K/BxN mice, with
the largest number in the dLNs. Numbers of anti-GPI ASCs were
significantly reduced in both the spleen and dLN of 1MT-treated
mice. This reduction in serum anti-GPI titers and numbers of anti-
GPI ASCs was found whether the 1MT was administered contin-
uously (long term) or was stopped at the onset of arthritis (short
term). Together, these data demonstrate that inhibition of IDO ac-
tivity with 1MT at the earliest stage of the autoimmune response is
sufficient to diminish the subsequent pathogenic anti-GPI B cell
response and results in reduced arthritis.
B cells, like most APCs, express IDO. Therefore, 1MT could be
acting directly on the B cells or inhibiting their activation through
an indirect mechanism. To distinguish between these two possi-
bilities, the ability of 1MT to affect B cell activation and differ-
entiation was measured in vitro (Fig. 6). Purified B cells were
labeled with CFSE and cultured in vitro with medium alone, LPS,
and anti-IgM (Fab?)2or the T cell mimics anti-CD40 plus IL-4 in
were analyzed at 6–8 wk of age following treatment with either carrier
alone, 1MT throughout the course of the experiment (long term), or 1MT
up to the onset of arthritis (short term). A, The isotype of anti-GPI Ig was
measured by ELISA. IgM, IgG2c, and IgG3 anti-GPI titers were undetect-
able (data not shown). The data are represented as the mean titer of Ig ?
SEM from a total of 6 carrier-treated, 6 long-term 1MT-treated, and 10
short-term 1MT-treated mice. B, The total amount of serum Ig was mea-
sured by ELISA. The data are represented as the mean titer of Ig ? SEM
from a total of six mice for each treatment group. C, ASCs from the spleen,
joint dLN, and non-joint dLN were measured using an ELISPOT assay.
The graph shows the mean number of ASCs ? SEM in each organ from an
experiment representative of three in total. n ? 9 carrier-treated, 12 long-
term, and 6 short-term 1MT-treated mice. ?, p ? 0.05; ??, p ? 0.02; and
???, p ? 0.01.
1MT inhibits the anti-GPI B cell response. K/BxN mice
Table I. 1MT treatment does not affect the percentage of Tregsa
24.8 ? 4.5
24.6 ? 3.1
17.3 ? 3.3
15.0 ? 2.4
aCells from the joint dLNs and non-dLNs of carrier- and 1MT-treated K/BxN
mice were stained with Abs to the surface markers CD4 and CD25, fixed, perme-
abilized, and then stained intracellularly for Foxp3. Numbers represent the mean
percentage ? SD of CD4?CD25?Foxp3?cells from n ? 5 carrier- and n ? 10
7514IDO DRIVES B CELL-MEDIATED AUTOIMMUNITY
the presence or absence of 1MT. B cells proliferated robustly to
LPS, anti-IgM, and anti-CD40 plus IL-4. 1MT had no effect on
either the number of cells dividing or the number of cell divisions
(Fig. 6A). The effect of 1MT on Ab secretion was determined by
measuring the amount of Ig secreted into the culture supernatants
(Fig. 6B). Ig was detected at equal levels in cultures with and
without 1MT. Therefore, at least in vitro, 1MT does not directly
affect B cell activation. These data suggest that 1MT affects the
pathogenic B cell response in K/BxN mice through an indirect
The role that IDO plays in regulating immune responses has been
the subject of intense investigation. The bulk of the literature has
focused on investigating the suppressive effects of IDO activity,
predominantly on the activation of T cells (43). The prevailing
theory is that IDO expressed by dendritic cells inhibits T cell ac-
tivation, either directly or indirectly by driving the development
of Tregs (30, 44, 45). In contrast to IDO’s effect on T cell re-
sponses, the role that IDO may play in the B cell responses has not
been evaluated. In this study, we show that administration of 1MT
to K/BxN mice reduced inflammatory cytokines and autoantibod-
ies, resulting in an attenuated course of arthritis. Surprisingly, no
difference was detected in the percentage of Tregs nor in the levels
of Th1/Th2/Th17 cytokines. Instead, the main effect of 1MT ap-
peared to be to suppress the autoreactive B cell response. Our
findings suggest that IDO is not simply an immunosuppressive
enzyme, but rather plays a more complex role that includes sup-
porting the establishment of B cell-mediated inflammatory
RA patients show evidence of elevated IDO activity that corre-
lates with disease activity (18, 46), but it has been unclear what
relevance this has, if any, to the autoimmune response. In K/BxN
mice, IDO activity was highest at the initiation of arthritis and
treatment with the pharmacological inhibitor of IDO 1MT at this
early stage delayed the development of arthritis and reduced dis-
ease severity. Importantly, 1MT exposure was required only dur-
ing the initiation of arthritis to exert its protective effect. In fact,
starting 1MT treatment after disease initiation was no longer ef-
fective. There is precedence for short-term exposure to 1MT hav-
ing a lasting effect on immune cell function (20, 47). Therefore, in
K/BxN mice, we suggest that IDO plays an activating role in es-
tablishing the autoreactive B cell profile at the onset of the auto-
immune response. If IDO activity is inhibited at this critical stage,
the autoreactive B cell profile is not established and subsequent
joint inflammation and damage is reduced.
Although 1MT was effective at alleviating arthritis, 1MT treat-
ment did not completely prevent arthritis development, as most
mice developed an attenuated course of disease. This study, like
most in the literature, used a pharmacological agent to inhibit IDO
activity. A potential caveat of pharmacological inhibitors is that
they may not be fully effective at inhibition or may have off-target
effects. Indeed, 1MT can also inhibit the IDO-related enzyme
IDO2 (48). Additionally, there may be an underlying biological
difference between constitutive loss of IDO due to genetic ablation
vs acute loss through pharmacological inhibition. This is consistent
with studies in pregnancy and tumor models in which compensatory
mechanisms for maintaining tolerance that apparently come into play
in IDO-deficient mice are not as effectively engaged following IDO
inhibitor treatment (49, 50). To address these possibilities, it will be
important to evaluate the impact of genetic loss of IDO and/or IDO2
on the development of arthritis in the K/BxN model.
1MT has been used in several other inflammatory disease mod-
els with conflicting results. 1MT exacerbated disease in experi-
mental autoimmune encephalomyelitis and trinitrobenzene sulfonic
acid-induced colitis (14, 16). In collagen- induced arthritis, one
tivation in vitro. Purified B cells were labeled with
CFSE and cultured for 3 days in either medium alone,
20 ?g/ml anti-IgM (Fab?)2, 2 ?g/ml anti-CD40 ? 50
ng/ml IL-4, or 25 ?g/ml LPS with or without 100 ?M
1MT. A, Proliferation was measured by flow cytometry
as a decrease in CFSE intensity. Histograms show plots
representative of three separate experiments, with his-
tograms from carrier-treated cultures (filled) overlayed
with histograms from the corresponding 1MT-treated
cultures (open). B, Total Ig levels were measured in the
culture supernatants. Plots show the mean ? SD of trip-
licate wells from a representative experiment of three
1MT does not directly affect B cell ac-
7515The Journal of Immunology
study showed accelerated disease upon administration of 1MT
(15). However, another study showed 1MT had no effect on its
own, but did reverse the protective effect of immunotherapy with
an Ab to the B7 family molecule 4-1BB (51). In contrast to the
disease-exacerbating effect of 1MT in these models, 1MT admin-
istration was protective in a mouse model of allergic airway in-
flammation (17). In this case, the disease-initiating Th2 response
was inhibited in 1MT-treated mice, suggesting that IDO normally
promotes Th2-mediated inflammatory responses. A similar re-
sponse was reported in vitro where IDO was shown to inhibit Th1
responses and promote Th2 responses (31). We have likewise
shown in this study that 1MT treatment was also protective against
joint inflammation in K/BxN mice. However, in this case, the pro-
tective effect of 1MT was not due to a skewing of the Th cytokine
profile. The autoimmune response in K/BxN mice exhibited char-
acteristics of Th1, Th2, and Th17 responses (Fig. 4 and Ref. 36)
and 1MT treatment did not affect this cytokine profile.
In contrast to the Th1/Th2/Th17 cytokines, cytokines associated
with inflammation, MCP-1, IL-6, and IL-10, were reduced in
1MT-treated mice. MCP-1, a cytokine that plays a key role in
recruiting monocytes into sites of inflammation, has been shown to
be elevated in RA patients (52). Likewise, IL-6, a cytokine thought
to induce inflammatory joint destruction through the recruitment
and induction of inflammatory Th17 cells, is also elevated in RA
patients (53, 54). MCP-1 and IL-6 were also both elevated in con-
trol-treated and reduced in 1MT-treated K/BxN mice. Therefore,
the alleviation of inflammation in 1MT-treated K/BxN mice was
reflected in a reduction of contributory cytokines. At this point,
it is not known whether 1MT treatment caused a reduction in
IL-6 and MCP-1 levels directly or whether the reduced levels
were simply a consequence of the overall reduced inflammatory
response. IL-10 levels have also been shown to increase in re-
sponse to inflammation; however, unlike MCP-1 and IL-6,
IL-10 serves to dampen the response (55). Although the signif-
icance of the reduced IL-10 levels is not clear, it may also be the
result of the overall reduction in the inflammatory response in
the 1MT-treated mice.
The most dramatic effect of 1MT treatment was the reduction
observed in the autoreactive B cell response. Autoantibody-secret-
ing B cell numbers were significantly decreased and titers of anti-
GPI Ab in the serum were greatly reduced in 1MT-treated K/BxN
mice. A role for IDO in driving B cell responses has not been
previously appreciated. B cells, like most APCs, express IDO and
levels increase upon activation (our unpublished observations).
Our experiments do not distinguish between 1MT having a direct
effect on autoreactive B cells or inhibiting their activation by an
indirect mechanism. However, our in vitro experiments demon-
strate that 1MT does not directly inhibit the activation of nonau-
toreactive B cells. IDO expression in another cell type could affect
the environment required for efficient B cell activation and Ab
secretion. Macrophages and dendritic cells, in particular plasma-
cytoid dendritic cells, have been implicated in the IDO-mediated
suppression of T cells (13, 56, 57). However, we were unable to
detect any difference in the percentage or activation status of these
cells in carrier- vs 1MT-treated K/BxN mice (our unpublished ob-
servations). Future experiments will be directed at identifying the
cell type(s) responsible for the 1MT-mediated suppression of ar-
thritis in the K/BxN model.
Recent evidence has shown that topical application of the proin-
flammatory agent PMA drives IDO activity in the regional LNs
and that this was a key component of the inflammatory microen-
vironment required for supporting tumor outgrowth following car-
cinogen exposure (20). The elevation of IDO in response to PMA
in these studies was interpreted as paradoxical because IDO was
considered to be immunosuppressive and yet no indication that
IDO was having a negative impact on the development or severity
of PMA-driven inflammation was observed. In light of the current
study, it is clear that categorizing IDO strictly as an immunosup-
pressive enzyme is an oversimplification and that its involvement
in disease processes such as cancer and autoimmune disorders will
be much more complex. In particular, its role in driving the acti-
vation of autoreactive B cells may have broad clinical implications
for the future utility of IDO inhibitors as potential therapeutic
We thank Dr. Paul Allen (Washington University) for the KRN C57BL/6
and B6.g7 mice, Dr. Richard Metz (NewLink Genetics) for the anti-IDO1
polyclonal Ab and helpful discussions, and Dr. Lisa Laury-Kleintop (Lan-
kenau Institute for Medical Research) for critical reading of this manuscript
and thoughtful input.
J.M. and G.C.P. have intellectual property interests (patents and/or license
fees through the authors’ institutions) in the therapeutic use of IDO and
IDO inhibitors in cancer. Additionally, these same authors are members of
the Scientific Advisory Board for NewLink Genetics Inc. and receive con-
sulting income and/or have financial holdings from this source.
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