Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells by
Stereoisomers of 1-Methyl-Tryptophan Correlates
with Antitumor Responses
1,2Alexander J. Muller,
4Andrew L. Mellor,
5Madhav D. Sharma,
1,3George C. Prendergast,
5,7and David H. Munn
1Immunotherapy Center and Departments of
5Lankenau Institute for Medical Research, Wynnewood, Pennsylvania;
7Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania
4Biostatistics, Medical College of Georgia, Augusta, Georgia;
6NewLink Genetics Corporation, Ames, Iowa; and
Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive
enzyme that contributes to tolerance in a number of biological
settings. In cancer, IDO activity may help promote acquired
tolerance to tumor antigens. The IDO inhibitor 1-methyl-
tryptophan is being developed for clinical trials. However,
1-methyl-tryptophan exists in two stereoisomers with poten-
tially different biological properties, and it has been unclear
which isomer might be preferable for initial development. In
this study, we provide evidence that the D and L stereoisomers
exhibit important cell type–specific variations in activity. The
L isomer was the more potent inhibitor of IDO activity using
the purified enzyme and in HeLa cell–based assays. However,
the D isomer was significantly more effective in reversing the
suppression of T cells created by IDO-expressing dendritic
cells, using both human monocyte–derived dendritic cells and
murine dendritic cells isolated directly from tumor-draining
lymph nodes. In vivo, the D isomer was more efficacious as
an anticancer agent in chemo-immunotherapy regimens using
cyclophosphamide, paclitaxel, or gemcitabine, when tested
in mouse models of transplantable melanoma and transplant-
able and autochthonous breast cancer. The D isomer of
1-methyl-tryptophan specifically targeted the IDO gene
because the antitumor effect of D-1-methyl-tryptophan was
completely lost in mice with a disruption of the IDO gene
(IDO-knockout mice). Taken together, our findings support
the suitability of D-1-methyl-tryptophan for human trials
aiming to assess the utility of IDO inhibition to block host-
mediated immunosuppression and enhance antitumor immu-
nity in the setting of combined chemo-immunotherapy
regimens. [Cancer Res 2007;67(2):792–801]
The immunoregulatory enzyme indoleamine 2,3-dioxygenase
(IDO) has been implicated as an immunosuppressive and
tolerogenic mechanism contributing to maternal tolerance toward
the allogeneic fetus (1), regulation of autoimmune disorders (2–5),
and suppression of transplant rejection (6, 7). IDO can also be
expressed by cancer cells in a variety of human malignancies (8, 9).
In murine models, transfection of immunogenic tumor cell lines
with recombinant IDO renders them immunosuppressive and
lethally progressive in vivo, even in the face of otherwise protective
T-cell immunity (8). In humans, expression of IDO by ovarian and
colorectal cancer cells has been found to be a significant predictor
of poor prognosis (9, 10).
IDO can also be expressed by host antigen-presenting cells
(APC). APCs with the potential to express IDO include human
monocyte–derived macrophages (11), human monocyte–derived
dendritic cells cultured under specific conditions (12–19), and
certain subsets of murine dendritic cells (20–25). In murine tumor
models, IDO+dendritic cells displaying a plasmacytoid phenotype
(CD11c+B220+) have been found at increased levels in tumor-
draining lymph nodes (22). These have been shown to suppress
T-cell responses in vitro and create antigen-specific T-cell anergy
in vivo (22, 25). In humans, IDO+cells of host origin have been
shown in draining lymph nodes of patients with melanoma, breast
cancer, and other tumors (13, 22, 26, 27). In patients with malignant
melanoma, the presence of these IDO-expressing cells in sentinel
lymph node biopsies was correlated with significantly worse
clinical outcome (22, 28). Thus, expression of IDO, either by host
cells or by tumor cells, seems associated with poor outcome in a
number of clinical settings.
These findings have prompted interest in development of IDO
inhibitor drugs for cancer immunotherapy (29). The most widely
studied of these has been 1-methyl-tryptophan (30–32). Recently, it
was shown that 1-methyl-tryptophan displays marked synergy with
a number of clinically relevant chemotherapeutic agents when used
in combined chemo-immunotherapy regimens (33). In that study,
the combination of 1-methyl-tryptophan with cyclophosphamide,
cisplatin, doxorubicin, or paclitaxel was able to cause regression
of established tumors in a demanding model of autochthonous
HER-2/neu–induced murine breast cancers (33). From a clinical
standpoint, combining an immunomodulatory agent, such as
1-methyl-tryptophan, with conventional chemotherapy drugs
represents an attractive strategy, and a sound mechanistic rationale
supporting such chemo-immunotherapy approaches is now being
However, a key unanswered question regarding 1-methyl-
tryptophan has been which of the two available stereoisomers
(D and L) should be developed initially for clinical trials. The two
isomers differ significantly in their effects on the recombinant IDO
enzyme in vitro (37), and they could potentially have different
biological effects, bioavailability, and off-target toxicities. Most of
the studies in the literature have employed the racemic (DL)
mixture of 1-methyl-tryptophan comprising both isomers, thus
leaving unanswered the question of which stereoisomer would be
Note: Supplementary data for this article are available at Cancer Research Online
Requests for reprints: David H. Munn, Immunotherapy Center, Medical College of
Georgia, CN-4141, Augusta, GA 30912. Phone: 706-721-7141; Fax: 706-721-8732; E-mail:
email@example.com or George C. Prendergast, Lankenau Institute for Medical Research,
Wynnewood, PA 19096. E-mail: firstname.lastname@example.org.
I2007 American Association for Cancer Research.
Cancer Res 2007; 67: (2). January 15, 2007
best suited for use in chemo-immunotherapy regimens. The goal
of the present study was to compare the biological activity of the
D and L isomers of 1-methyl-tryptophan in vitro and in vivo, to ask
whether their pattern of efficacy in vitro correlated with their
observed antitumor effect in vivo.
Materials and Methods
Additional methods available online. Detailed description of mice,
published methods, and statistical analyses are available online at http://
Reagents. 1-Methyl-D-tryptophan (45,248-3), 1-methyl-L-tryptophan
(44,743-9), and 1-methyl-DL-tryptophan (86,064-6) were obtained from
Sigma-Aldrich (St. Louis, MO). For in vitro use, these were prepared as a
20 mmol/L stock in 0.1 N NaOH, adjusted to pH 7.4, and stored at ?20jC
protected from light.
Autochthonous breast cancer model. Multiparous female MMTV-Neu
mice, maintained as described (33), have a high incidence of autochthonous
mammary gland carcinomas. Tumor-bearing mice were enrolled randomly
into experimental groups when tumors reached 0.5 to 1.0 cm in diameter.
Tumor volume was measured at the beginning and end of the 2-week
B16F10 and 4T1 tumor models. B16F10 melanoma (American Type
Culture Collection, Manassas, VA) were established in B6 mice by s.c.
injection of 5 ? 104cultured cells. B78H1-GM-CSF (38), gift of H. Levitsky,
(Johns Hopkins University, Baltimore, MD) was implanted by s.c. injection
of 1 ? 106cells. Orthogonal diameters were measured, and the x?y product
(tumor area) was reported. The use of the orthotopically implanted 4T1
breast cancer line (39) has been described in detail (40). Tumors were
implanted by injection of 1 ? 104cells in 50 AL volume into the mammary
fat pad of 6- to 10-week-old BALB/c females. In some experiments,
luciferase-transfected 4T1 cells (4T1-luc) were used for bioluminescence
imaging, as described in the Supplementary Material.
Administration of 1-methyl-tryptophan and chemotherapeutic
agents. Detailed protocols for administration of 1-methyl-tryptophan,
orally and by s.c. pellets, in conjunction with chemotherapy, are given in the
Human and mouse mixed lymphocyte reactions. Human and murine
allogeneic mixed lymphocyte reactions (allo-MLR) were done as detailed in
the Supplementary Material and have been previously described (14, 22).
Western blots. Western blots were done using affinity-purified
polyclonal rabbit antibody against peptides from the NH2-terminal and
COOH-terminal portion of human IDO, as previously described (13) and as
specified in detail in the Supplementary Material.
Cooperativity effect of s.c.
chemotherapy or radiation in B16F10 melanoma. We first
evaluated the racemic DL mixture of 1-methyl-tryptophan as a
component of chemo-immunotherapy using three tumor models:
a stringent established (day 7) B16F10 melanoma, orthotopically
implanted 4T1 breast carcinoma, and autochthonous breast tumors
arising in HER-2/neu–transgenic mice. Figure 1A shows established
B16F10 tumors treated with DL-1-methyl-tryptophan (20 mg/d by
14-day s.c. copolymer pellet; ref. 1), with or without a single injec-
tion of cyclophosphamide (150 mg/kg). DL-1-methyl-tryptophan
alone had no effect on tumor growth, and cyclophosphamide alone
induced only a transient growth delay. However, the combination
of DL-1-methyl-tryptophan + cyclophosphamide resulted in a
sustained growth delay and prolonged survival. In all experiments,
the end of the study period was defined as the time when all of
the mice in the vehicle-only group reached their ethical surrogate
end point (tumor area z300 mm2). At the point when all mice in
the control group had reached this end point, all mice in the
Figure 1. Effect of parenteral DL-1-methyl-tryptophan (DL-1MT) in B16F10
tumors. A, B16F10 tumors were implanted in syngeneic C57BL/6 mice.
Beginning on day 7, mice were treated as shown with timed release s.c. pellets of
DL-1-methyl-tryptophan (20 mg/d) plus cyclophosphamide (CY; 150 mg/kg i.p. ?
1 dose). Three identical experiments were done (a representative example is
shown), and the pooled results were analyzed in a three-experiment ? 2 group
ANOVA. *, P < 0.05. B, identical experimental design showing that the effect of
DL-1-methyl-tryptophan was lost when hosts were immunodeficient Rag1-KO.
Groups were not significantly different by ANOVA. C, similar experimental
design, except that 500 cGy of whole-body cesium-137 irradiation replaced the
cyclophosphamide. One of four similar experiments. *, P < 0.05, ANOVA.
Inhibition of IDO by D-1-Methyl-Tryptophan
Cancer Res 2007; 67: (2). January 15, 2007
DL-1-methyl-tryptophan + cyclophosphamide group were still
surviving. Figure 1B shows that the effect of DL-1-methyl-tryptophan
was lost in immunodeficient Rag1-knockout (Rag1-KO) hosts,
indicating that the antitumor effect of DL-1-methyl-tryptophan
was entirely immune mediated.
Whole-body irradiation has many of the same effects as chemo-
therapy when combined with antitumor immunotherapy (41). We
tested DL-1-methyl-tryptophan in combination with 500 cGy whole-
body irradiation (Fig. 1C). In these experiments, there was consi-
derable variability in the effect of the radiation component alone on
tumor growth, but in all experiments, the effect of DL-1-methyl-
tryptophan plus radiation was superior to radiation alone.
Cooperativity between oral
cyclophosphamide in treating 4T1 breast carcinoma isografts.
We next asked whether DL-1-methyl-tryptophan showed efficacy
via the oral route. For these studies, we tested chemo-immuno-
therapy of the poorly immunogenic 4T1 breast tumor model,
implanted orthotopically in mammary tissue of syngeneic hosts.
Because orthotopic 4T1 tumors are highly invasive and their
margins are difficult to measure conventionally, we followed the
tumor size using luciferase-transfected 4T1 (4T1-luc) tumors
imaged following luciferin challenge. Oral DL-1-methyl-tryptophan
was given by gavage twice daily, five times a week, combined with a
weekly single i.p. dose of cyclophosphamide, beginning at the time
of tumor implantation. As shown in representative scans in Fig. 2A,
cyclophosphamide alone produced a modest reduction in tumor
size, but the combination of cyclophosphamide + DL-1-methyl-
tryptophan produced a marked decrease in tumor size (survival
studies in this model are presented below).
Oral administration of DL-1-methyl-tryptophan in combina-
tion with paclitaxel can elicit regression of autochthonous
breast tumors. We next tested the efficacy of varying durations of
oral DL-1-methyl-tryptophan in combination with paclitaxel for the
treatment of autochthonous tumors arising in MMTV-Neu mice
(33). Mice with tumors were randomly assigned to treatment with
paclitaxel for 2 weeks, with or without addition of 2 to 5 days
of oral DL-1-methyl-tryptophan during the first week, as indicated
in Fig. 2B. Paclitaxel alone caused a minor reduction in the rate of
tumor growth, but tumors continued to increase in size during the
study period despite paclitaxel. The addition of oral DL-1-methyl-
tryptophan produced a progressive reduction in the rate of tumor
growth with increasing duration of 1-methyl-tryptophan, such that
treatment with 4 and 5 days of DL-1-methyl-tryptophan reversed
tumor growth, and caused regression of the established tumors
during the treatment period. Five days of administration via the
oral route was at least as effective as parenteral delivery of the drug
at a comparable daily dose, using implantable s.c. pellet (the last
treatment group and the route reported in our previous study;
In vitro comparison of D versus L isomers of 1-methyl-
tryptophan. We next used in vitro models to compare the different
isomers of 1-methyl-tryptophan for their biological effects, using
two readouts: (a) activity of the IDO enzyme measured as
Figure 2. Oral DL-1-methyl-tryptophan in orthotopic 4T1 and autochthonous
MMTV-Neu tumors. A, orthotopic tumor isografts were established in the
mammary fat pad. Treatment was initiated concurrent with tumor
challenge, using cyclophosphamide i.p. at 100 mg/kg, once a week and
DL-1-methyl-tryptophan oral gavage at 400 mg/kg per dose, twice daily, five times
a week. Bioluminescence imaging of 4T1 tumor cell line transfected with
luciferase, showing the effect of each treatment on tumor burden. Treatment
received by each mouse is indicated. Images were produced at 4 wks following
the initiation of treatment. B, MMTV-Neu mice bearing 0.5 to 1.0 cm
spontaneous tumors were treated for 2 wks with either vehicle alone, paclitaxel
alone (13.3 mg/kg i.v. q. M/W/F), or paclitaxel plus oral DL-1-methyl-tryptophan
(400 mg/kg i.v. twice daily, given for up to 5 d during the first week, as indicated
in the legend). Paclitaxel was given i.v. at over the 2-wk treatment period. The
last group received s.c. pellets of 1-methyl-tryptophan, as in Fig. 1. Fold changes
in individual tumor volumes over the 2-wk period are plotted for each group.
Points, mean fold change for each group (also listed in the box below the graph);
bars, SE. *, fully regressed tumors are included in the calculation of the mean
and SE. For the statistical analyses (arrows), the two comparisons of interest
were vehicle alone versus paclitaxel alone and paclitaxel alone versus
paclitaxel + D-1-methyl-tryptophan ? 5 d. Significance was determined at
P < 0.025 using a two-group Wilcoxon exact test.
Cancer Res 2007; 67: (2). January 15, 2007
production of kynurenine from tryptophan and (b) a biological
readout measured as the ability to prevent the suppression of T-cell
proliferation caused by IDO-expressing dendritic cells.
Figure 3A shows enzyme kinetics (kynurenine production) using
recombinant human IDO enzyme in a cell-free assay system. Using
the recombinant enzyme, the L isomer of 1-methyl-tryptophan
functioned as a competitive inhibitor (Ki= 19 Amol/L), whereas
the D isomer was much less effective (no Kifound at 1-methyl-
tryptophan concentrations up to 100 Amol/L). The DL mixture was
intermediate, with a Kiof 35 Amol/L. These values are consistent
with the published literature for studies using cell-free enzyme
assays for IDO (37).
We next tested the different isomers in a biological assay, based
on the intracellular IDO enzyme expressed by living cells (in this
case, HeLa cells activated with IFNg; Fig. 3B). Kynurenine
production by HeLa cells showed a pattern of inhibition similar
to that of the cell-free recombinant enzyme, with L-1-methyl-
tryptophan being more effective than D-1-methyl-tryptophan. In
other studies (data not shown), similar results were obtained using
the murine MC57 tumor cell line transfected with recombinant
mouse IDO and also the simian COS cell line transfected with
human IDO: in each of these transfected cell lines, L-1-methyl-
tryptophan was superior to D-1-methyl-tryptophan at inhibiting
In contrast to the behavior of cell lines, when primary human
monocyte–derived dendritic cells were used as the IDO-expressing
cells (Fig. 3C), the D isomer of 1-methyl-tryptophan was found to be
at least as effective as the L isomer in its ability to inhibit IDO
activity (measured as kynurenine production in culture super-
natants). In these assays, dendritic cells were activated physiolog-
ically by exposure to T cells in allo-MLRs, rather than with
recombinant IFNg, because we have previously shown that IFNg
alone is not sufficient to activate functional IDO in dendritic cells
prepared by this protocol (13, 14).
Figure 3. Effect of different isomers on
in vitro enzyme assays and T-cell
proliferation. A, enzyme kinetics, measured
as kynurenine (KYN) production in cell-free
assay, for purified recombinant human IDO,
showing the effect of the L, DL, and D forms
of 1-methyl-tryptophan in the presence of
varying concentrations of L-tryptophan
substrate. B, intracellular IDO enzyme
activity (measured as kynurenine
production in culture supernatants) by
IFNg-activated HeLa cells, showing
inhibition by different isomers of
1-methyl-tryptophan. % Inhibition of
maximal kynurenine production; lines
show interpolated EC50for each isomer.
C, intracellular IDO activity (kynurenine
production in MLR supernatants) by human
monocyte–derived dendritic cells (DC)
activated in allo-MLRs; lines show EC50.
Combined average of three experiments
using three different donors. D, effect of
1-methyl-tryptophan isomers on T-cell
proliferative responses. Proliferation was
measured by thymidine incorporation in
allo-MLRs using either human T cells
stimulated by IDO-expressing human
monocyte–derived dendritic cells (1 of 10
experiments, using a variety of different
donor combinations), or mouse T cells
stimulated by IDO-expressing plasmacytoid
dendritic cells from tumor-draining lymph
nodes, as described in Materials and
Methods (one of three experiments).
As controls, purified human T cells without
dendritic cells were activated with
immobilized anti-CD3 + anti-CD28
antibodies (one of three experiments).
Inhibition of IDO by D-1-Methyl-Tryptophan
Cancer Res 2007; 67: (2). January 15, 2007
In addition to kynurenine production, we and others have shown
that IDO suppresses proliferation of T cells responding to antigens
presented by IDO+dendritic cells (13, 14, 22). Figure 3D shows a
comparison of the different 1-methyl-tryptophan isomers on
human T-cell proliferation in allo-MLRs stimulated by IDO+
monocyte–derived dendritic cells (similar to the MLRs shown in
Fig. 3C, but using T-cell proliferation as the readout). Using this
readout, the D isomer was found to be reproducibly superior to
either the L isomer or the DL mixture, typically eliciting a 2- to
3-fold greater maximum level of T-cell proliferation. A similar
pattern was seen using murine T cells (Fig. 3D). For mice, allo-
MLRs were done using IDO+dendritic cells isolated directly from
murine tumor-draining lymph nodes, as previously described (22).
These tumor-activated dendritic cells were used to present a
constitutive allo-antigen to BM3 TCR-transgenic T cells (specific
for the H2Kbantigen expressed by the C57BL/6 dendritic cells). In
this model, just as in the human system, the D isomer of 1-methyl-
tryptophan was superior in supporting activation and proliferation
of T cells, compared with either the L or DL forms.
To test for nonspecific (off-target) effects of 1-methyl-tryptophan
on the T cells themselves, control experiments were done using
purified human T cells stimulated by immobilized anti-CD3 + anti-
CD28 antibodies (i.e., without any dendritic cells present to express
IDO). Under these conditions, none of the 1-methyl-tryptophan
preparations had any detectable effect on T-cell proliferation
(Fig. 3D). Additional studies (shown in Supplementary Fig. S1) were
done further evaluating the D isomer, using MLRs stimulated by
dendritic cells derived from mice with a targeted disruption of the
IDO gene (IDO-KO mice). MLRs using IDO-KO dendritic cells
showed that the effects of the D isomer were completely lost when
the stimulating dendritic cells lacked IDO. Thus, the D isomer of
1-methyl-tryptophan exerted its effects in MLR specifically by tar-
geting the IDO gene expressed by the dendritic cells, not through
an off-target effect.
Western blots suggest the possible existence of more than
one isoform of IDO. The cell type–specific effects of the different
isomers of 1-methyl-tryptophan prompted us to ask whether there
might be more than one form of IDO expressed in different cells.
Published databases suggested potential alternate splicing isoforms
of human IDO differing primarily in the COOH-terminal portion of
the molecule.8Therefore, we generated polyclonal antibodies
against peptide sequences in the NH2-terminal and COOH-terminal
portions of the IDO molecule for use in Western blots, as described
in the Supplementary Material.
Figure 4A shows Western blots using the two different
antibodies. Samples were prepared from human monocyte–derived
macrophages, as a known source of IFNg-inducible IDO (11). As
shown in Fig. 4A, the NH2-terminal antibody detected a band of
f44 kDa, which was present both before and after IFNg
stimulation, and which showed little apparent change with IFNg.
In contrast, the COOH-terminal antibody detected an antigen of
f42 kDa, which was only visible after IFNg treatment. A similar
pattern of two different constitutive and inducible bands has been
described for IDO expression by in other cell types (42). We and
others have also shown that IDO can be expressed constitutively at
the protein level (e.g., as with the higher molecular weight band)
without necessarily showing enzymatic activity until activated
(13, 43). In other experiments (data not shown), HeLa cells showed
the same pattern of bands and the same response to IFNg, as did
the monocyte-derived macrophages in Fig. 4A.
Figure 4. Evidence for two possible isoforms of human IDO. A, human
monocyte–derived macrophages were prepared as described (11), with or
without IFNg treatment for the final 24 h. Lysates were analyzed by Western blot
using antibodies against the NH2-terminal portion of IDO, the COOH-terminal
portion, or a mixture of the two antibodies. All blots were stripped and reprobed
for h-actin (data not shown) to confirm even loading. B, macrophages, as above,
were treated with or without IFNg, in the presence or absence of cycloheximide
(10 Ag/mL). h-Actin blots (data not shown) confirmed even loading. C, lysates
of macrophages with and without IFNg pretreatment were analyzed by
two-dimensional electrophoresis, followed by Western blotting with the
NH2-terminal–specific anti-IDO antibody. D, human monocyte–derived dendritic
cells were cultured for 7 d as described in Materials and Methods, with or
without addition of a maturation cocktail during the final 48 h. IFNg was added
during the last 24 h. Western blots were done as in (B), with the same blot
stripped and reprobed for each anti-IDO antibody and the h-actin loading control.
8J. Thierry-Mieg et al. AceView: identification and functional annotation of cDNA-
supported genes in higher organisms—Homo sapiens gene INDO, encoding indole-
amine-pyrrole 2,3 dioxygenase. Available from http://www.ncbi.nlm.nih.gov/IEB/
Cancer Res 2007; 67: (2). January 15, 2007
Figure 4B shows that expression of the IFNg-inducible (lower
molecular weight, COOH-terminal) band was blocked by cyclohex-
imide, suggesting that it represented a newly synthesized protein,
rather than a posttranslational modification of the larger isoform.
Although conventional Western blot analysis did not reveal any
obvious change in the larger molecular weight (NH2-terminal)
isoform in response to IFNg, two-dimensional Western blots
(Fig. 4C) revealed that there was a significant IFNg-induced shift in
isoelectric point (up to 2 pH units). Thus, these data revealed that
both forms of IDO were in fact IFNg responsive, with the larger
form appearing to undergo some IFN-induced posttranslational
modification, whereas the smaller form seemed to be synthesized
Regulation of IDO activity in dendritic cells is more complex
than in macrophages, with multiple factors reported to influence
both protein expression and enzymatic activity (17, 19). When we
analyzed human monocyte–derived dendritic cells by Western
blot (Fig. 4D), there was significant up-regulation of the larger
(NH2-terminal) isoform with dendritic cell maturation, whereas
IFNg treatment had no discernible effect on this band in dendritic
cells. The smaller (COOH-terminal) isoform showed no expression
in immature dendritic cells and was not inducible in dendritic cells
by IFNg. However, the COOH-terminal isoform underwent marked
up-regulation with dendritic cell maturation (again independent of
IFNg). Thus, the regulation of the two IDO isoforms in dendritic
cells was complex and differed from their regulation in macro-
phages. However, the essential point was similar for dendritic cells:
that more than one species of IDO was present, and that the
pattern of expression was regulated by biologically relevant
Efficacy of the D isomer of 1-methyl-tryptophan in chemo-
immunotherapy. Based on the superiority of the D isomer in
supporting T-cell activation in vitro, we tested the D isomer of
1-methyl-tryptophan in vivo using the B16F10 model. Established
(day 7) B16F10 tumors were treated with cyclophosphamide plus
D-1-methyl-tryptophan in a design similar to Fig. 1A. However, in
these studies, the dose of the D isomer was reduced 4-fold
compared with the dose of the DL mixture used in Fig. 1A, based on
its superior efficacy in vitro. Even at the lower dose, D-1-methyl-
tryptophan + cyclophosphamide showed significant growth delay
compared with cyclophosphamide alone (Fig. 5A). Similar results
were seen with a second chemotherapeutic agent gemcitabine
(Fig. 5B). Neither gemcitabine alone nor D-1-methyl-tryptophan
alone had a significant effect on B16F10 tumor growth, but
together, the combination produced a significant growth delay.
D-1-methyl-tryptophan had no effect on B16F10 tumors when
used as a single agent, but B16F10 is not a highly immunogenic
tumor; we therefore asked whether D-1-methyl-tryptophan alone
might show an effect if a more immunogenic tumor was used.
B78H1-GM-CSF is a subline of B16 that has been transfected with
granulocyte macrophage colony-stimulating factor (GM-CSF) to
increase recruitment of APCs to the tumor and draining lymph
nodes (44). The tumor is modestly immunogenic, although if
implanted without irradiation, the tumors invariably grow and
kill the host (45). In this somewhat more immunogenic model,
D-1-methyl-tryptophan, as a single agent, was found to have a
modest but reproducible and statistically significant effect on the
growth (Fig. 5C, left). This modest antitumor effect was lost when
the hosts were immunodeficient Rag1-KO mice (Fig. 5C, middle),
showing that the effect of D-1-methyl-tryptophan was immune
mediated. Likewise, the effect of D-1-methyl-tryptophan was lost
when the less immunogenic parental tumor (without GM-CSF) was
used in place of B78H1-GM-CSF (Fig. 5C, right). Thus, D-1-methyl-
tryptophan did show some modest effect as a single agent when
used with an artificially immunogenic tumor. However, this was
substantially less potent than the effect of 1-methyl-tryptophan in
combination with chemotherapy.
Comparison of D versus L isomers in chemo-immunother-
apy. We next did side-by-side comparisons of the different iso-
mers of 1-methyl-tryptophan in chemo-immunotherapy regimens.
Figure 6A shows a comparison of D versus L versus DL forms of
1-methyl-tryptophan in orthotopic 4T1-luc tumors. Each 1-methyl-
tryptophan preparation was given in combination with low-dose
cyclophosphamide (25 mg/kg/dose by oral gavage once per week).
Although minor effects were observed with the other combina-
tions, only D-1-methyl-tryptophan with cyclophosphamide showed
a statistically significant prolongation of survival relative to cyclo-
phosphamide alone (for clarity, these two groups are re-graphed
together in the second plot). A second, similar experiment showed
the same results, reproducing the survival advantage of D-1-methyl-
tryptophan over L-1-methyl-tryptophan in combination with cyclo-
Figure 6B compares the
D versus L isomers of 1-methyl-
tryptophan in the autochthonous MMTV-Neu breast tumor model.
Both isomers were delivered orally for 5 days, as in Fig. 2C, in
combination with paclitaxel. In this model also,
tryptophan was found to be superior to L-1-methyl-tryptophan
(in these studies, the L isomer showed no effect compared with
Specificity of the D isomer for host IDO in vivo. Finally,
one critical outstanding question was the target specificity of the
D isomer in vivo. We had shown in Supplementary Fig. S1
(Supplementary Material) that the D isomer of 1-methyl-trypto-
phan specifically targeted the IDO gene in vitro. However, it was
possible that in vivo,
D-1-methyl-tryptophan might exert an
antitumor effect via some other off-target mechanism. Figure 6C
addresses this question by comparing tumors grown in wild-type
(IDO sufficient) mice versus tumors grown in IDO-KO mice, each
treated with cyclophosphamide +
tumors that grew in the IDO-KO hosts would, by definition, have
been selected for their lack of dependence on IDO (i.e., they must
necessarily be escape variants that could grow in the absence of
IDO). Thus, if D-1-methyl-tryptophan truly targeted IDO, then treat-
ing tumors grown in IDO-KO mice with D-1-methyl-tryptophan
should have no effect on tumor growth; conversely, if D-1-methyl-
tryptophan was not specific for IDO, then any off-target effects
should be retained in the IDO-KO hosts. Figure 6C shows that
tumors grown in IDO-KO mice became completely refractory to the
effects of D-1-methyl-tryptophan, thus confirming that IDO was the
target of D-1-methyl-tryptophan in vivo, as hypothesized. More
specifically, these studies suggested that in this model, the relevant
target for D-1-methyl-tryptophan was IDO expressed by host cells,
rather than by tumor cells, because the tumor cells were the same
in both cases.
In the current study, we show significant differences in biological
activity between the D and L stereoisomers of 1-methyl-tryptophan.
The L isomer was superior at inhibiting activity of purified
recombinant IDO enzyme in a cell-free assay and also at inhibiting
IDO enzymatic activity in HeLa cells and other cell lines. In
Inhibition of IDO by D-1-Methyl-Tryptophan
Cancer Res 2007; 67: (2). January 15, 2007
contrast, the D isomer was at least as effective as the L isomer at
inhibiting IDO enzymatic activity expressed by human or mouse
dendritic cells. Unexpectedly, the D isomer was found to be
significantly superior to both the L form and the DL mixture when
tested by the biologically important readout of T-cell activation in
MLRs. In vivo, a head-to-head comparison of the antitumor effect
of the two isomers showed that the D isomer was more effective
than the L isomer, using two different tumors and different chemo-
immunotherapy regimens. Thus, the in vitro superiority of the D
isomer for enhancing T-cell activation in MLRs seemed to correctly
predict the superior in vivo antitumor efficacy in the models tested,
whereas the results of the cell-free enzyme assays did not.
The superiority of the L isomer in the cell-free enzyme assay
was expected from the literature (37). However, to our knowledge,
no comparison of the two isomers of 1-methyl-tryptophan has
been previously reported using assays based on intact cells. Such
cell-based systems are important because different cell types may
respond differently to the two isomers, as we have now shown. The
molecular basis for these cell type–specific differences is not yet
known. Possibilities include differential transport into or out of the
cells, different subcellular compartmentalization of the inhibitors,
or altered metabolism by cellular enzymes. It is also possible that
there may be different isoforms of IDO (as could be suggested by
our Western blot data), and these might have different sensitivities
to the two isomers, although this is currently speculative. Finally, it
may be that 1-methyl-tryptophan exerts some of its inhibitory
effects on IDO not by competing directly for the catalytic site but
by altering enzyme activity in another way that does not register in
the cell-free enzyme assay.
Others have also reported efficacy of the D isomer of 1-methyl-
tryptophan for enhancing T-cell responses in vitro and in vivo
(46, 47). Importantly, our data unambiguously showed that the
T cell–enhancing effect of D-1-methyl-tryptophan in vitro was
completely lost when APCs were derived from IDO-KO mice; and,
likewise, the antitumor efficacy of D-1-methyl-tryptophan in vivo
was lost when the tumor-bearing hosts were IDO-KO. Thus, the
molecular target of D-1-methyl-tryptophan was indeed IDO, and
the efficacy of D-1-methyl-tryptophan was not due to some off-
target effect. This would also be consistent with recent studies
using RNA–knock-down techniques, which concluded that the
major molecular target of the DL-mixture of 1-methyl-tryptophan
was IDO, rather than an off-target effect (48).
One critical reason underlying the superior activity of the D
isomer in vivo may be our observation that the L isomer seemed
actively inhibitory for T-cell activation in MLRs. Both isomers were
equally effective at blocking the enzymatic activity of IDO in MLRs
(measured as kynurenine production in the supernatant); yet,
the L isomer could not produce the same high levels of T-cell
proliferation achieved by the D isomer. Revealingly, the DL mixture
also proved less effective than the D isomer alone, suggesting that
the presence of the L isomer actively inhibited T-cell proliferation.
The nature of this inhibition is currently unknown. However, it did
not seem to be due to a direct toxic effect of L-1-methyl-tryptophan
on the T cells themselves because T cells stimulated by mitogen
(i.e., in the absence of IDO-expressing dendritic cells) wereno longer
affected by L-1-methyl-tryptophan. This suggests that the off-target
inhibitory effect of the L isomer might be due to a toxic effect of
L-1-methyl-tryptophan on the IDO-expressing dendritic cell itself
Figure 5. Effect of parenteral D-1-methyl-
tryptophan in the B16F10 model. A, mice
with B16F10 tumors were treated in a
design similar to Fig. 1A, except using the D
isomer of 1-methyl-tryptophan at a 4-fold
lower dose (5 mg/d by timed release
pellets). Cyclophosphamide was given at
150 mg/kg i.p. Three identical experiments
were pooled and analyzed by ANOVA.
*, P V 0.05. B, experimental design similar
to (A), using gemcitabine 120 mg/kg i.p. on
days 8, 11, 14, and 19 following B16F10
tumor implantation. Three experiments
were pooled and analyzed by ANOVA.
*, P < 0.05. C, B78H1-GM-CSF tumors, or
parental tumors without the GM-CSF
transgene, were implanted as indicated.
Beginning at the time of implantation,
mice received 14-day pellets of
D-1-methyl-tryptophan (5 mg/d) or vehicle
control. Left, three experiments were
pooled and analyzed by ANOVA.
*, P = 0.011. Middle, all hosts were
Rag1-KO. Right, tumors lacked the
GM-CSF transgene (neither of these
groups showed significant
Cancer Res 2007; 67: (2). January 15, 2007
(e.g., rendering it less able to present antigen to the Tcells). Perhaps
consistent with such an off-target effect on dendritic cells, it has
recently been reported that exposure of dendritic cells in vitro to the
DL-mixture of 1-methyl-tryptophan at 1,000 Amol/L (much higher
than the maximum concentration used in the current study) caused
alteration in dendritic cell function, which did not seem related to
the effect of DL-1-methyl-tryptophan on IDOitself (49). Alternatively,
the T cells might be sensitive to some metabolite of the L isomer
generated by the dendritic cells. In either case, it seems that the
D isomer of 1-methyl-tryptophan escaped this off-target inhibitory
effect on T-cell activation, perhaps precisely because it was not the
Although the D isomer showed superior efficacy in our chemo-
immunotherapy models, the L isomer proved better at inhibiting
IDO in HeLa cells and in mouse tumor cell lines transfected with
IDO. Thus, it may be that in certain biological contexts the L isomer
Figure 6. D-1-methyl-tryptophan provides greater survival benefit in combination therapy, in an IDO-dependent fashion. A, 4T1-luc orthotopic isografts were
established in the mammary fat pad. Cyclophosphamide was given at 25 mg/kg orally once a week, and 1-methyl-tryptophan (D, L, or DL) given at 400 mg/kg by
oral gavage twice daily, five times a week by gavage, beginning at the time of tumor implantation. Top, time to endpoint for all groups; bottom, only the
cyclophosphamide versus cyclophosphamide + D-1-methyl-tryptophan groups, for clarity. The comparisons of interest were between D-1-methyl-tryptophan +
cyclophosphamide versus cyclophosphamide and L-1-methyl-tryptophan + cyclophosphamide versus cyclophosphamide. Because survival data were not censored,
groups were analyzed using a two-group Wilcoxon exact test; statistical significance was determined at P < 0.025. The combination of D-1-methyl-tryptophan +
cyclophosphamide showed a significant survival benefit over cyclophosphamide alone (P = 0.024), whereas L-1-methyl-tryptophan + cyclophosphamide was
not different from cyclophosphamide alone (P = 0.14). B, MMTV-Neu mice with tumors were treated for 2 wks as in Fig. 2B, receiving either vehicle alone,
paclitaxel alone, or paclitaxel (13.3 mg/kg q. MWF) plus oral D-1-methyl-tryptophan or L-1-methyl-tryptophan for 5 d, as indicated. For statistical analysis, the
comparisons of interest were D-1-methyl-tryptophan + paclitaxel versus paclitaxel alone and L-1-methyl-tryptophan + paclitaxel versus paclitaxel alone. Significance
was determined at P < 0.025 using a two-group Wilcoxon exact test. The fold change of the D-1-methyl-tryptophan + paclitaxel group was significantly smaller than
that of paclitaxel alone (P = 0.012), whereas paclitaxel + L-1-methyl-tryptophan was not different from paclitaxel alone (P = 0.85). C, effects of the D isomer of
1-methyl-tryptophan require an intact host IDO gene. B16F10 tumors were grown in either wild-type B6 hosts or IDO-KO hosts on the B6 background. All groups
received cyclophosphamide, with or without oral D-1-methyl-tryptophan (2 mg/mL in drinking water). Analysis by ANOVA showed that cyclophosphamide +
D-1-methyl-tryptophan was significantly different (*, P < 0.05) than cyclophosphamide alone for the wild-type hosts, but there was no effect of D-1-methyl-tryptophan
when tumors were grown in IDO-KO hosts.
Inhibition of IDO by D-1-Methyl-Tryptophan
Cancer Res 2007; 67: (2). January 15, 2007
might be preferable, whereas in other contexts, the D isomer is
superior. This might become relevant where the target of 1-methyl-
tryptophan is IDO expressed by the tumor cells themselves,
rather than by host dendritic cells. However, the data from our
in vitro T-cell activation models and from our in vivo chemo-
immunotherapy models suggest that in these systems, the bene-
ficial effect of the D isomer on T-cell activation is the key advantage,
rendering the D isomer superior in these settings. Furthermore,
based on the fact that efficacy of D-1-methyl-tryptophan was lost
when the host mice were genetically deficient in IDO (Fig. 6C), our
data suggest that the molecular target of D-1-methyl-tryptophan in
our system was the IDO activity expressed specifically by host APCs,
not by the tumor cells themselves.
In the murine models used in this study, relatively high doses
of 1-methyl-tryptophan were required to see an antitumor effect.
However, this seems to represent a peculiarity of 1-methyl-
tryptophan pharmacokinetics in mice. Preclinical pharmacology
studies in both rats and canines (to be published elsewhere) show
that these animals require significantly lower doses per kilogram
to achieve plasma levels in the same range. These lower doses
should be readily achievable clinically.
The combination of 1-methyl-tryptophan with chemotherapy
(cyclophosphamide, paclitaxel or gemcitabine) was more potent
against established tumors than either 1-methyl-tryptophan or
chemotherapy alone. Regimens featuring chemotherapy plus
immunotherapy are receiving increasing attention (34, 35). In part,
this is because they are readily applicable in the clinic because
patients do not have to be denied standard chemotherapeutic
agents to receive immunotherapy. In addition, there is a sound
mechanistic rationale underlying combined chemo-immunother-
apy. Chemotherapy causes death of tumor cells, thus releasing
tumor antigens into the host antigen-presentation pathway (34).
In addition, certain chemotherapy drugs seem to decrease the
number and activity of regulatory T cells (50, 51), which may assist
the immunotherapy regimens in breaking tolerance to tumor
antigens. Finally, the recovery phase from chemotherapy-induced
lymphopenia seems to constitute a favorable window for reacti-
vating previously tolerized T cells (41). However, despite these
effects, chemotherapy alone does not elicit an effective antitumor
immune response. We hypothesize that one reason for this failure
is because the antigens released by chemotherapy are presented
first in the tumor-draining lymph nodes. We and others have
previously shown that tumor-draining lymph nodes are a highly
tolerogenic microenvironment (52), due at least in part to the
presence of IDO-expressing APCs (22, 25). Thus, IDO+host APCs
may play an important pathogenic role in helping the tumor
re-establish immunologic tolerance toward itself after it is
disrupted by chemotherapy. Based on our current data, we
hypothesize that the addition of an IDO inhibitor drug during this
post-chemotherapy period may allow the tumor-bearing host to
mount an effective immune response to tumor antigens during this
post-chemotherapy window of opportunity.
Received 8/7/2006; revised 10/11/2006; accepted 10/25/2006.
Grant support: NIH grants CA103320 (D.H. Munn), CA096651 (D.H. Munn),
CA112431 (D.H. Munn), and CA109542 (G.C. Prendergast); Department of Defense
Breast Cancer Research Program grants BC021133 (G.C. Prendergast) and BC044350
(A.J. Muller); and State of Pennsylvania Department of Health CURE/Tobacco
Settlement Award (A.J. Muller).
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.
We thank Anita Wylds, Jingping Sun, Judy Gregory, Anita Sharma, Jie Huang, Erika
Sutanto-Ward, and P. Scott Donover for expert technical assistance.
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Inhibition of IDO by D-1-Methyl-Tryptophan
Cancer Res 2007; 67: (2). January 15, 2007
- 1 -
Supplemental online material
Figure S1. The D-isomer of 1MT is specific for IDO.
Tumors were grown in either IDO-deficient (IDO-KO) hosts or WT hosts, both on the B6
background. The pDC fraction (CD11c+B220+) containing the IDO+ DCs was sorted from
TDLNs. The balance of TDLN cells, which included all the other APCs, was also collected in
each sort. MLRs were performed using the two populations (pDCs and all other cells) as
stimulators for BM3 T cells, separately and mixed together. The upper panel demonstrates
suppression by the pDC fraction, which was reversed by D-1MT. There was no suppression by
the other-APC fraction, and no effect of D-1MT. Mixing of the two stimulator populations
demonstrated that suppression by IDO was dominant, as previously described (1). However,
when the pDC fraction was derived from IDO-KO mice, there was no suppression by pDCs, and
no effect of D-1MT on T cell proliferation. Thus, IDO-KO DCs lacked the target for D-1MT,
confirming that the D isomer acted on IDO, not via some off-target effect.
Additional detailed methods
Animal studies were approved by the institutional animal use committee of the Medical College
of Georgia or the Lankenau Institute for Medical Research. C57BL/6 (B6) mice and rag1-KO
mice (B6 background) were from Jackson Laboratory (Bar Harbor, ME). FVB/N-
Tg(MMTVneu)202Mul/J mice, homozygous for a rat cNeu transgene under the mouse mammary
tumor virus promoter (MMTV-Neu mice) (2) were from Jackson Laboratory. BALB/cAnNCr
- 2 -
mice were from Charles River Laboratories (Frederick, MD). IDO-KO mice have been
previously described (3).
Sources of 1MT isomers
Studies of the various 1MT isomers in human DCs were performed on at least two different sets
of each compound, supplied in blinded fashion by the Developmental Therapeutics Program,
National Cancer Institute (Bethesda, MD), and results confirmed using multiple different
commercial lots of each isomer obtained from Sigma-Aldrich. All experiments gave similar
results. For in vivo studies, 1-methyl-D-tryptophan was supplied by the Developmental
Therapeutics Program, National Cancer Institute and confirmed using commercially prepared D
and DL isomers (Sigma-Aldrich). 1MT was prepared as a 20 mM stock solution in 0.1 N NaOH
and adjusted to pH 7.4; solutions were stored at 4o C and protected from light.
Administration of 1MT and chemotherapy agents
Administration of 1MT by implantable subcutaneous pellets was performed as described (4, 5).
Pellets tended to release higher amounts during the early period of the infusion, so the treatment
periods stated are the nominal release times.
To prepare 1MT for oral gavage, 1 g of 1MT (Sigma) was added to a 15 ml conical tube
with 7.8 ml Methocel/Tween [0.5% Tween 80/0.5% Methylcellulose (v/v in water; both from
Sigma)]. The mixture was bead milled overnight by adding 1-2 ml by volume of 3 mm glass
beads (Fisher) and mixing by inversion. The next day, the 1MT concentration was adjusted to 85
mg/ml by adding an additional 4 ml Methocel/Tween and mixing again briefly. The 1MT slurry
was administered by oral gavage at 400 mg/kg/dose (0.1 cc/20 g mouse) using a curved feeding
needle (20 G x 1 1/2 in; Fisher). For bid (twice a day) dosing, 1MT was administered once in the
morning and once in the evening.
For administration in drinking water, D-1MT was prepared at 2 mg/ml in water as
described above, supplemented with a small amount of aspartame (2 envelopes per liter) to
improve acceptance by the mice, and filter sterilized. The solution was delivered in standard
autoclaved drinking-water bottles. Mice drank 4.5-5.0 ml/day (similar to consumption of water
without drug). Plasma levels of D-1MT at the end of 6 days were 33-40 uM.
Paclitaxel (Hanna Pharmaceuticals, Wilmington, DE), 6 mg/ml in 50% Cremphor EL /
50% ethanol, was diluted in saline delivered i.v. Cyclophosphamide was from Bristol-Myers
Squibb (Princeton, NJ) or Hanna Pharmaceuticals, and gemcitabine from Eli Lilly (Indianapolis,
In vivo bioluminescence imaging of 4T1-luc tumors
For bioluminescence imaging experiments, luciferase-expressing derivative of the 4T1 cell line
(4T1-luc) was prepared by stable transfection of pCAG-luc (generously provided by Dr. J
Sawicki), which expresses the firefly luciferase gene under the control of the β-actin promoter
and the CMV IE enhancer. Transfection was followed by three rounds of single cell cloning to
establish a cell line with stably expressed luciferase activity. Prior to imaging, tumor-bearing
mice were anesthetized by intramuscular injection of a mixture of 25 mg/kg ketamine/5 mg/kg
xylazine hydrochloride (Hanna Pharmaceuticals, Wilmington, DE). Anesthetized mice were
injected intraperitoneally with 150 mg/kg firefly luciferin (Xenogen, Alameda, CA). At 5 min
after administration of the substrate, in vivo images were acquired with an IVIS charge-coupled-
device camera system (Xenogen). Data analysis was performed with the LivingImage 2.5
- 3 -
software package (Xenogen ).
IDO enzyme assays
Purification of recombinant human His6-tagged IDO produced by E. coli strain BL21DE3pLys,
and the 96-well plate-based spectrophotometric assay to monitor enzymatic activity, were
performed essentially as described (6). Briefly, 1MT enantiomers were solubilized in
dimethylsulfoxide (DMSO) containing 0.1N HCl and added at concentrations of 100, 50, and 0
µM (but maintaining constant DMSO and HCl dilutions of 1:1000) to wells containing the
reaction mixture (6) in which the tryptophan concentration was varied from 0-200 µM, followed
by addition of IDO enzyme. Plates were sealed with plastic wrap and incubated 1 hr in a
humidified 37˚C incubator, after which the reactions were terminated by addition of 12.5 µl 30%
TCA per well. Plates were then resealed in plastic wrap, incubated 30 min at 50˚C to hydrolyze
the reaction product N-formylkynurenine to kynurenine, and centrifuged 10 min at 2400 rpm in a
Sorvall tabletop centrifuge. Supernatants were transferred to a flat-bottom 96-well plate, mixed
with 100 µl Ehrlich reagent (2% p-dimethylamino benzaldehyde w/v in glacial acetic acid), and
incubated 10 min at room temperature. Absorbance at 490 nm was read on a Bio-Tek Synergy
NT plate reader to quantitate the reaction product.} Global nonlinear regression analysis and
computation of best fit Ki values was performed using the Prism4 software package (GraphPad).
For HeLa cell assays, HeLa human tumor cells (ATCC) were seeded at 4.0 x 104 cells per
well in DMEM/phenol red free media supplemented with 10% FBS (Hyclone) and penicillin-
streptomycin (Gibco). The following day, 1-MT enantiomers or the racemic mixture were
solubilized in DMSO/0.1 N HCl and serially diluted in assay wells while maintaining the
DMSO/HCl dilution constant at 1:1000. 100 ng/ml of human recombinant IFN-γ (R&D
Systems, Minneapolis, MN) was then added per well to stimulate IDO expression. Following
IFN-γ addition, plates were incubated 20 hr at 37˚C in a humidified CO2 incubator. Supernatants
(200 µl media/well) were harvested and analyzed for kynurenine as described (5).
For measurement of kynurenine production by human DCs in allo-MLRs, culture
supernatants were harvested on day 5 of MLR and analyzed by high-performance liquid
chromatography as previously described (1). Similar patterns were also obtained at days 2 and 3
Human monocyte-derived APCs and allo-MLRs.
Work with human materials was performed under protocols approved by the Institutional Review
Board of the Medical College of Georgia. Our systems for culturing IDO+ human monocyte-
derived DCs and macrophages have been previously described (1, 7). For DCs, the features of
the culture system relevant to maximizing IDO expression included the use of leukocytapheresis
followed by counterflow elutriation for preparation of monocytes; culture for 7 days in serum-
free X-vivo15 medium (BioWhitaker, Walkersville, MD) supplemented with GMCSF + IL4;
maturation during the final 48 hrs of culture with TNFα, IL1β, IL6 and PGE2 (but without IFNγ,
CD40-ligand or TLR-ligands); and harvesting of only the non-adherent cell fraction. The PGE2
reagent was found to be labile, and so was frozen in aliquots and mixed fresh for each
experiment. Monocyte-derived macrophages were cultured as previously described (7) using
recombinant human macrophage colony-stimulating factor (R&D Systems), with IFNγ (100
U/ml) added for the last 24 hrs.
For human allo-MLRs, 2.5 × 104 nonadherent DCs were mixed with 5 x 105 allogeneic
lymphocytes in 250 ul of medium (10% fetal calf serum in RPMI-1640) in “V”-bottom culture
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wells (Nalge-Nunc, Rochester, NY). V-bottom wells gave superior IDO activity, as previously
described (1). For mitogen-activated T cell proliferation, purified lymphocytes were activated
with immobilized anti-CD3 antibody plus soluble anti-CD28 (1). After 5 days, proliferation was
measured by 4 hr [3H]thymidine-incorporation assay.
Mouse tumor-draining lymph node pDCs and MLRs
Mouse IDO-expressing DCs from tumor-draining lymph nodes, as used in our previous
publications (8, 9). Tumors were implanted using 1 x 106 B78H1·GM-CSF cells injected
subcutaneously in the anteriomedial thigh of syngeneic wild-type B6 mice, or mice with a
targeted disruption of the IDO gene (IDO-KO mice) (3). After 11 days, inguinal LNs were
removed for cell sorting to enrich for IDO+ DCs, which were contained in the plasmacytoid DC
fraction (CD11c+B220+ cells). These sorted B220+CD11c+ DCs contained all of the IDO-
mediated suppressor activity, and were used as stimulators in allo MLRs as described (8).
Responder cells were 1 × 105 nylon-wool enriched BM3 TCR-transgenic BM3 T cells,
recognizing the -H2Kb allo-antigen expressed on the B6-background DCs, as described (8).
After 3 days, proliferation was measured by 4-hr thymidine incorporation assay. All MLRs were
performed in V-bottom culture wells (Nalge-Nunc, Rochester, NY), and the IDO+ DCs were not
Affinity-purified polyclonal rabbit antibody was raised against the peptide sequence
DLIESGQLRERVEKLNMLC, from the N-terminal portion of the published human IDO
sequence (NM002164) (10), conjugated to KLH, and has been previously described (11).
Affinity-purified polyclonal rabbit antibody against the C-terminal peptide sequence
LKTVRSTTEKSLLKEG conjugated to ovalbumin was prepared similarly. 2D-Western blots
were performed using a Protean IEF cell and Mini-Protean blotting system (BioRad, Hercules,
CA) as described (11). Both antibodies detected single bands consistent with the predicted
molecular weight for one or more splice-variants of IDO (12), and the bands were fully
neutralized in Western blots by their respective immunizing peptides.
To rule out the possibility that the N-terminal epitope (which was constitutively
expressed) might be a spurious cross-reacting band, we conducted a BLAST search of the
Genbank protein database using the immunizing peptide. This peptide sequence matched only
IDO. The N-terminal band was constitutive in macrophages, but validation studies showed that
the band was not found in other cell types (e.g., B cells), and the levels of the N-terminal band
was regulated in DCs by maturation status. Reactivity of the N-terminal antibody was fully
neutralized by the immunizing peptide sequence.
For analysis of variance, the comparison was performed on the two arms that of interest in order
to test for an effect of 1MT when combined with chemotherapy: thus, in all analyses, the
chemotherapy+vehicle arm was compared to chemotherapy+1MT arm in each experiment.
Because the group sizes were small in each individual experiments, 3 identical experiments were
performed and analyzed together where possible. If needed, a log transformation was used on
tumor size to render tumor growth to experiment end a linear trend with homogeneous variance
across time. A regression of tumor size on day was performed for each mouse. The resulting
slopes were interpreted as the rate of growth of tumor size to end-point (euthanasia). Where
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indicated, identical experiments were pooled and analyzed in a 3 Experiment × 2 Group analysis
of variance (ANOVA) where an interaction between experiment and group was investigated.
When comparing the effect of chemotherapy+vehicle and chemotherapy+1MT on the rate of
tumor growth for two types of mice (WT vs. IDO-KO) a 2 Group x 2 Type ANOVA with
interaction was used.
In Fig. 2 and Fig. 6 comparisons of interest were fold-change in tumor size, and age at
death, respectively. Since all mice were alive at the end of the experiments the survival data
were not censored and the mean was an unbiased representation of µ. However, for experiments
using both survival and fold-change measurements, the SDs between groups varied considerably,
and the fold-change measurements were likely not normally distributed; therefore, data were
analyzed using a two group Wilcoxon exact test, due to the distributional issues and the small
sample size. Significance was determined at p < [0.05/(number of comparisons)] for each
experiment. SAS version 9.1.3 was used for all analyses
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