2570? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 9? ? ? September 2007
Plasmacytoid dendritic cells from
mouse tumor-draining lymph nodes
directly activate mature Tregs via
Madhav D. Sharma,1,2 Babak Baban,3 Phillip Chandler,2,4 De-Yan Hou,1,2 Nagendra Singh,2
Hideo Yagita,5 Miyuki Azuma,6 Bruce R. Blazar,7 Andrew L. Mellor,2,4 and David H. Munn1,2
1Department of Pediatrics, School of Medicine, 2Immunotherapy Center and Cancer Center, 3Department of Pathology, and
4Department of Medicine, School of Medicine, Medical College of Georgia, Augusta, Georgia, USA. 5Department of Immunology,
Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan. 6Department of Molecular Immunology, Tokyo Medical and Dental University, Bunkyo-ku,
Tokyo, Japan. 7Department of Pediatrics and Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA.
A subset of DCs in murine tumor-draining lymph nodes (TDLNs)
can express high levels of the tryptophan-degrading enzyme
indoleamine 2,3-dioxygenase (IDO) (1). In other settings, IDO has
been shown to contribute to maternal tolerance toward the allo-
geneic fetus, regulation of autoimmune disorders, and creation of
tolerance to transplanted tissues (2–4). Transfection of IDO into
tumor cells protects them from immune-mediated rejection (5),
while inhibiting IDO in tumor-bearing hosts allows conventional
chemotherapy to disrupt tolerance toward established tumors
and trigger anti-tumor immune responses (6, 7). Thus, IDO is
emerging as a potentially important tolerogenic mechanism in
patients with cancer (8).
In vitro studies of IDO+ DCs from murine TDLNs have shown
that these cells are potently and dominantly suppressive for T cell
activation (1, 7, 9). Even a small minority of IDO+ DCs is capable
of inhibiting all T cell responses in culture, including dominant
inhibition of T cells responding to antigens presented by other
nonsuppressive APCs (1). In vivo, pharmacologic activation of the
IDO pathway systemically can completely inhibit clonal expansion
of large numbers of alloreactive T cells (10). However, the num-
ber of IDO+ DCs in that become activated in spleen or TDLNs is
tiny (less than 1% of total cells, and typically less than 25% of total
DCs), and it is unclear how the effects of IDO could create such
potent and dominant immunosuppression.
Recently it has been shown that IDO can bias naive CD4+ T
cells to differentiate into Foxp3+ Tregs in vitro (11). This impor-
tant finding thus linked IDO to the potent Treg system, which is
known to be a key mechanism of immunosuppression in tumor-
bearing hosts (12). However, de novo differentiation of Tregs
from naive precursor cells is a slow process, requiring many days,
whereas we knew from in vitro studies that IDO created dominant
suppression within hours (prior to the first cell division of the
suppressed T cells) (9, 13). Therefore we hypothesized that there
existed a pathway by which IDO could directly activate the latent
suppressor function of mature, preexisting Tregs; and further, that
this pathway would be active in TDLNs in vivo.
Tregs from TDLNs are highly activated. We first tested the activation
status of Tregs from TDLNs. B16 melanoma tumor cell lines were
implanted in syngeneic C57BL/6 (B6) mice. Cell lines included
B78H1–GM-CSF (a subline of B16 transfected with GM-CSF; ref.
14), the noninfected B16F10 subline of B16, and B16-OVA (the
B16F10 subline transfected with ovalbumin). Mice were studied
on days 7–11 after tumor implantation. All TDLNs contained a
Nonstandard?abbreviations?used: B6, C57BL/6; GCN2, general control nondere-
pressing-2 kinase; IDO, indoleamine 2,3-dioxygenase; 1MT, 1-methyl-d-tryptophan;
OVA, chicken ovalbumin; PD-1, programmed cell death 1; PD-L, PD ligand; pDC,
plasmacytoid DC; TDLN, tumor-draining LN.
Conflict?of?interest: D.H. Munn and A.L. Mellor have intellectual property interests
in the therapeutic use of IDO and IDO inhibitors and receive consulting income from
NewLink Genetics Inc.
Citation?for?this?article: J. Clin. Invest. 117:2570–2582 (2007). doi:10.1172/JCI31911.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
population of cells that constitutively expressed IDO (Figure 1A),
which was not seen in non-TDLNs (contralateral LNs). We have
previously shown (1) that these IDO+ cells are a subset of DCs
expressing plasmacytoid surface markers (CD11c+B220+) and coex-
pressing the marker CD19 (shown in Supplemental Figure 1; sup-
plemental material available online with this article; doi:10.1172/
JCI31911DS1). The IDO+ cells in TDLNs of all 3 tumor lines were
similar; for most cell-sorting experiments B78H1–GM-CSF tumors
were used, as in our previous publications (1, 9), because these gave
the highest yield of pDCs (Supplemental Figure 1). However, pDCs
from tumors without GM-CSF gave similar functional results, and
all key findings were confirmed with both types of tumors.
Figure 1B shows analysis of Foxp3+CD4+ Tregs in TDLNs.
Both the TDLN and contralateral LNs contained a similar per-
centage of Tregs. However, when these Tregs were sorted by flow
cytometry (CD4+CD25+ cells, more than 90% Foxp3+) and tested
for functional suppressor activity, the Tregs from TDLNs were
potently and spontaneously suppressive, whereas the Tregs from
contralateral LNs showed no spontaneous suppressor activity
(Figure 1C). Tregs from TDLNs showed essentially complete sup-
pression at a ratio of Tregs to readout T cells of greater than 1:100,
which was as potent as the most highly activated Tregs achievable
in vitro (15, 16). In these experiments, the goal was to test whether
the Tregs from TDLNs were constitutively activated in vivo (as
opposed to becoming activated during the readout assay). We
therefore selected a readout system that was MHC mismatched to
the B6 Tregs (comprised of TCR-transgenic A1 T cells and splenic
DCs, both on the CBA strain background). The use of an alloge-
neic readout assay minimized any possible activation of the Tregs
by the APCs in the readout assay, and no additional mitogen or
Treg activation by DCs from TDLNs. (A) Contralateral LNs and TDLNs from mice with B16F10 and B78H1–GM-CSF tumors (day 7–11). B16-
OVA tumors were identical to B16F10. Red color identifies IDO by immunohistochemistry. One representative of 3–6 experiments per cell line.
Original magnification, ×200. (B) TDLNs and contralateral LNs were stained for CD4 and intracellular Foxp3. Numbers indicate quadrant percent-
ages. Representative of 6 experiments using B16-OVA and B78H1–GM-CSF. (C) Tregs (CD4+CD25+) from TDLNs and contralateral LNs were
sorted and added to readout assays, which were comprised of 1 × 105 A1 T cells plus CBA DCs plus H-Y peptide. Proliferation (incorporation of
[3H]thymidine deoxyribose, [3HTdR]) is shown for a representative experiment. In all similar figures, the ratio of Tregs to A1 cells is shown below
the axis (bars show SD of replicate wells). The lower graph shows data from 8 independent experiments using the tumor types shown (cpm
were normalized to the proliferation in control assays receiving no Tregs, to permit comparison across experiments). (D) CD11c+ DCs were har-
vested from TDLNs, pulsed with OVA peptide, and injected subcutaneously into recipient mice preloaded with OT-I. One group of mice received
implantable sustained-release 1MT pellets at 5 mg/day (IDO blocked), while the other received vehicle control pellets (IDO active). After 4 days,
the LNs draining the site of DC injection were harvested and the Tregs sorted and tested in vitro for spontaneous suppressor activity in readout
assays (A1 T cells + CBA DCs). Representative of 3 experiments; bars show SD of replicate wells.
2572? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
anti-CD3 crosslinking was added. In assays of this type, resting
Tregs do not show suppression (17, 18), whereas activated Tregs
are suppressive (19). Thus, this assay allowed us to measure the
degree of Treg activation in vivo.
IDO+ DCs from TDLNs activate Tregs in vivo. To test the hypothesis
that Treg activation in TDLNs might be related to the presence
of IDO-expressing DCs, we isolated the DC population (CD11c+
cells) from TDLNs and transferred them to new, non–tumor-bear-
ing hosts. (The phenotype of this mixed DC population has been
previously described [ref. 1] and typically contained 30%–50%
IDO-expressing CD19+ pDCs.) To test for the contribution of
IDO, recipient mice were treated with the IDO-inhibitor drug
1-methyl-d-tryptophan (1MT) (7) beginning at the time of DC
adoptive transfer or with vehicle control. Because we have shown
that IDO does not become fully active until IDO+ DCs present
antigen to responding T cells (20), the DCs were pulsed with a
peptide from chicken ovalbumin (OVA) and hosts pre-loaded
with OVA-specific T cells (OT-I). Four days after DC transfer, host
Tregs were sorted from LNs draining the site of DC injection and
tested for suppressor activity. Figure 1D shows that Tregs exposed
to DCs from TDLNs became potently activated and this activation
was blocked when recipient mice were treated with 1MT. Thus, the
DC fraction from TDLNs, by itself, was sufficient to activate rest-
ing Tregs in new hosts, in an IDO-dependent fashion.
Activation of Tregs by IDO in vitro. (A) Resting Tregs were cocultured with TDLN pDCs plus OT-I plus feeder cells (all on B6 background, MHC
b-haplotype) as described in Methods (IDO-activated Tregs). After 2 days the Tregs were re-sorted and added to readout assays (A1 T cells +
CBA DCs, k-haplotype background). Controls Tregs were activated in identical cultures with 1MT added to block IDO activity. Graph shows the
mean of 5–8 pooled experiments, using pDCs from B78H1–GM-CSF and B16-OVA tumors; error bars show SD. (B) Tregs were activated as
described above or in identical cultures containing 1MT (to block IDO) and anti-CD3 mAb (αCD3) plus IL-2 (IDO-activated Tregs). After 2 days,
Tregs were re-sorted and tested in readout assays. Data points show the means for pooled values from 3 independent experiments. (C) Tregs
were activated in cocultures as described above, and APCs were either TDLN pDCs, non-pDC fraction from the same TDLN (CD11c+B220–),
pDCs from mice without tumors, or TDLN pDCs from IDO-KO mice. Graphs show 1 representative of 3–4 similar experiments for each group
(bars show SD of replicate wells). (D) Tregs were activated with TDLN pDCs as described above, with or without 1MT. Tregs were re-sorted and
added to readout assays in the lower chamber of transwell plates; upper chambers received readout assays without Tregs. Thymidine incorpora-
tion was measured separately in each chamber. One of 3 experiments; *P < 0.01 by ANOVA. (E) IDO-activated Tregs were sorted and added to
readout assays containing A1 T cells plus either CBA DCs or CBA B cells. One of 3 experiments; *P < 0.01 by ANOVA.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
IDO+ pDCs from TDLNs activate resting Tregs in vitro. To study the
mechanism of IDO-induced Treg activation, we used the 2-step
model shown in Figure 2. Resting Tregs, from spleens of mice with-
out tumors, were cocultured with IDO+ DCs from TDLNs, then
re-sorted and transferred to readout assays (A1 T cells + CBA DCs)
to measure suppression. The IDO+ DCs were enriched from TDLNs
by sorting for the plasmacytoid DCs (pDCs) fraction, which we
have previously shown to include essentially all of the IDO+ DCs in
TDLNs in our system (Supplemental Figure 1). Similar to human
DCs (20), DCs from TDLNs required triggering signals from T cells
at the time of antigen presentation in order to express functional
IDO enzymatic activity. This was supplied by allowing the pDCs
to present OVA peptide to OT-I. Cocultures also contained a feed-
er layer of T-depleted spleen cells, as described in Methods. After
30–48 hours, cocultures were harvested and the Tregs recovered by
sorting for CD4+ cells (since the Tregs were the only CD4+ cells in
the cocultures, they could be unambiguously recovered).
Figure 2A shows that resting Tregs exposed to IDO+ pDCs medi-
ated potent suppression of T cell proliferation in readout assays.
In contrast, if IDO was blocked by adding 1MT to the activation
cultures, then the re-sorted Tregs showed no suppressor activ-
ity (similar to the resting Tregs from contralateral LNs shown in
Figure 1C). In the remainder of this article, we will refer to Tregs
activated by IDO+ pDCs from TDLNs as “IDO-activated Tregs,”
since IDO was necessary for activation, recognizing that additional
signals besides IDO may also be supplied by these TDLN pDCs.
IDO-activated Tregs were able to suppress CD8+ T cells as well
as CD4+ T cells in the readout assays (Supplemental Figure 2).
Activation occurred within 30 hours and was sufficiently rapid
that IDO-activated Tregs were able to suppress all proliferation
of readout cells, even if the A1 cells and CBA DCs were added
directly to the Treg activation assay at the beginning of cultures
and allowed to activate in parallel (shown in Supplemental
Figure 3). The A1 T cells in the readout assays were suppressed by
activated Tregs, but they were not killed, as shown by the fact that
recovery of CD4+ cells at the end of 3 days was 95% ± 8% of the
expected cell number compared with controls (n = 5 experiments),
and Annexin V staining at the end of the 3-day assay was negative
(Supplemental Figure 4).
Figure 2B shows a quantitative comparison of IDO-activat-
ed Tregs versus the same Tregs activated using the widely used
approach of anti-CD3 crosslinking (21). Both activation cultures
contained identical cell populations, but the anti-CD3 cultures
received 1MT to block IDO plus anti-CD3 and recombinant IL-2 to
Suppression by IDO-activated Tregs requires the PD-1/PD-L pathway. (A) Tregs were activated with IDO+ pDCs as described in Figure 2, then
1 × 104 sorted Tregs were added to readout assays (A1 T cells + CBA DCs). After 24 hours, cultures were harvested and stained for PD-L1 and
PD-L2 relative to CD11c. Percentages indicate the proportion of cells that are dual-positive (right-upper quadrant). One of 3 experiments. (B)
IDO-activated Tregs (5,000/well) were added to readout assays (A1 T cells plus either wild-type CBA DCs or IDO-KO DCs on the CBA back-
ground). Readout assays received either no additive, 1MT, or a cocktail of blocking antibodies against PD-1, PD-L1, and PD-L2 (50 μg/ml each).
Control Tregs received 1MT during the activation step. One of 3 experiments; *P < 0.01 by ANOVA. (C) Tregs were activated with IDO+ pDCs
or in identical cultures containing 1MT to block IDO and αCD3 plus IL-2 to activate the Tregs. After sorting, Tregs were added to readout assays
(A1 T cells + CBA DCs) with or without PD-1/PD-L–blocking antibodies as shown. Graphs show the mean ± SD of 10 independent experiments
with IDO-activated Tregs and 3 experiments with αCD3-activated Tregs, using TDLN pDCs from B78H1–GM-CSF and B16-OVA tumors. (D)
IDO-activated Tregs (1 × 104/well) and αCD3/IL-2–activated Tregs (2 × 104/well) were prepared as described in the previous panel and added to
readout assays with or without recombinant IL-2, anti–IL-10 plus anti–TGF-β blocking antibodies (100 μg/ml each), PD-1/PD-L–blocking antibod-
ies, or no additive (-0-). Error bars show SD for replicate wells in 1 of 4 similar experiments. *P < 0.01 by ANOVA.
2574? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
activate the Tregs. After activation and sorting, the IDO-activated
Tregs mediated potent suppression, while the anti-CD3-activated
Tregs were activated but quantitatively less suppressive (50% inhibi-
tion at a Treg/target cell ratio of 1:10, which is consistent with the
findings of others using the anti-CD3 system; refs. 16, 21).
Figure 2C shows similar cocultures, but with the TDLN pDCs
replaced by various DCs that do not express IDO. The left graph
(positive control) shows Tregs cocultured with TDLN pDCs
(IDO+). The middle left graph shows cocultures using the non-
plasmacytoid (CD11c+B220NEG) DCs from the same TDLNs. The
middle right graph shows cocultures using pDCs from LNs of
mice without tumors. The right graph shows cultures contain-
ing pDCs isolated from TDLNs of tumors grown in IDO-knock-
out (IDO-KO) hosts. Only the plasmacytoid DC fraction derived
from TDLNs and with an intact host IDO gene were able to acti-
vate Tregs. These data, combined with the complete abrogation
of activation by 1MT (Figure 2A), support a mechanistic role for
IDO in mediating Treg activation.
We next tested whether IDO-activated Tregs required physical con-
tact with readout T cells in order to cause suppression (Figure 2D).
IDO-activated Tregs were added to the lower well of transwell
chambers, and readout cells (A1 T cells + CBA DCs) were placed
in both the lower chamber (in contact with the Tregs) and the
upper chamber (separated by a microporous membrane). Separate
thymidine incorporation assays were performed on each chamber
and showed that the IDO-activated Tregs suppressed those T cells
with which they were in contact but had no effect on T cells sepa-
rated across the membrane.
Suppression by IDO-activated Tregs requires the PD-1/PD-ligand path-
way. Certain forms of T cell suppression by Tregs can be mediated
indirectly via an effect on the target APCs (22). We therefore asked
whether suppression by IDO-activated Tregs required the partici-
pation of the DCs in the readout assays. Figure 2E shows that IDO-
activated Tregs were unable to suppress proliferation of A1 T cells
when B cells were substituted instead of DCs as APCs used in the
readout assay. Similar loss of suppression was seen when anti-CD3/
CD28-coated beads were substituted for the DCs (data not shown).
This suggested that the suppressive effect of IDO-activated Tregs
might be mediated indirectly via an effect on the target DCs.
One mechanism by which DCs may suppress T cells is the inhibi-
tory programmed cell death 1/programmed cell death 1 ligand
(PD-1/PD-L) pathway (23, 24). While this pathway has not previ-
ously been described as a mediator of Treg suppression, related B7
family members have been linked to Treg-induced suppression (25).
Figure 3A shows that IDO-activated Tregs caused upregulation of
both PD-L1 and PD-L2 on the DCs (CD11c+ cells) in readout assays.
In contrast, PD-L expression by DCs was low in readout assays with-
out Tregs and in readout assays receiving Tregs from activation cul-
tures in which IDO was blocked with 1MT (Figure 3A). Even read-
out assays receiving Tregs that had been activated with anti-CD3
plus IL-2 did not show upregulation of PD-Ls on DCs (Figure 3A).
Thus, the upregulation of PD-Ls on DCs appeared associated spe-
cifically with the form of Treg activation created by IDO.
We therefore asked whether blocking the PD-1/PD-L pathway
in the readout assay would prevent suppression by IDO-activated
Tregs. To ensure that the pathway was fully blocked, we added
IDO-induced activation requires GCN2 in Tregs. (A) Activation cultures were set up with Tregs, TDLN pDCs, OT-I, and feeder cells, with or without
1MT. After 2 days, intracellular staining was performed for CHOP expression in Tregs (CD4+ cells). The percentages show the fraction of Tregs that
were CHOP+. One of 9 similar experiments. (B) As in the preceding panel, Tregs derived from wild-type mice are compared with GCN2-KO mice
(each assay with OVA, without 1MT). One of 3 experiments. (C) Tregs from GCN2-KO mice or wild-type controls were activated with IDO+ pDCs as
described in Figure 2 and re-sorted, and 5,000 Tregs were added to readout assays (A1 T cells + CBA DCs), with and without PD-1/PD-L–blocking
antibodies. One of 3 similar experiments. *P < 0.01 by ANOVA. (D) Tregs from wild-type mice were activated with IDO+ pDCs, re-sorted, and tested
in readout assays with and without added 10× tryptophan (250 μM). Bars show SD for replicate wells. One of 3 similar experiments.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
a cocktail of antibodies against PD-1, PD-L1, and PD-L2 to the
readout assays. Blocking the PD-1/PD-L pathway completely
abrogated the ability of IDO-activated Tregs to suppress T cell
proliferation (Figure 3B). In contrast, adding 1MT to the readout
assay or using DCs from IDO-KO mice had no effect on T cell
suppression. Thus, while IDO was strictly required to activate
the Tregs initially (see Figure 2A), suppression of target cells by
IDO-activated Tregs was independent of IDO and was dependent
on the PD-1/PD-L pathway.
Figure 3C compares the role of the PD-1/PD-L pathway in suppres-
sion mediated by IDO-activated Tregs versus the suppression medi-
ated by Tregs activated by anti-CD3 plus IL-2. Suppression by IDO-
activated Tregs was completely prevented by blocking PD-1/PD-L in
the readout assay, whereas suppression by anti-CD3-activated Tregs
was unaffected by PD-1/PD-L blockade. In contrast, Figure 3D shows
that suppression by anti-CD3-activated Tregs was fully reversed by
adding recombinant IL-2 to the readout assay or by blocking IL-10
and TGF-β, although these manipulations had no effect on suppres-
sion by IDO-activated Tregs. Thus, the mechanisms of suppression
by IDO-activated Tregs and anti-CD3-activated Tregs were distinct
and could be unambiguously distinguished based on sensitivity to
PD-1/PD-L blockade, exogenous IL-2, and IL-10/TGF-β blockade.
GCN2 is required for Treg activation. We next asked whether Tregs
responded to IDO via the GCN2 pathway. GCN2 is activated by
reduced levels of amino acids, as might occur when IDO depletes
tryptophan (26). We have previously shown that IDO activates
GCN2 in CD8+ effector T cells, leading to cell-cycle arrest and anergy
in these cells (9). As diagrammed in Figure 4, activation of GCN2
can be detected by measuring the downstream marker gene CHOP/
gadd153 (9). Treg activation cultures were set up as described in the
MHC-dependent and MHC-independent steps in IDO-induced Treg activation. (A) B6 Tregs were activated with IDO+ pDCs as described in Figure 2,
with or without anti–CTLA4-blocking mAb (10 ug/ml) during the activation step. Activated Tregs were re-sorted and tested in readout assays (A1
T cells + CBA DCs). Bars show SD for replicate wells in 1 of 4 similar experiments. (B) CHOP induction in Tregs is MHC restricted. Cultures were
set up as described in Figure 4A and cells stained for CHOP after 2 days. The left plot shows assays using Tregs that were MHC matched to the
IDO+ pDCs (B6 background); the middle plot shows assays with MHC-mismatched (CBA) Tregs. The right plot shows cultures with MHC-matched
B6 Tregs but with 100 μg/ml blocking antibody to IAb. Controls without blocking antibody or with irrelevant antibody were similar to the first plot and
are not shown. One of 4 experiments. (C) Left: Activation cocultures were set up as described in Figure 2 using MHC-mismatched (CBA) Tregs.
After 2 days, CBA Tregs were re-sorted and added to readout assays (A1 T cells + CBA DCs). Right: Identical assays, except that CBA Tregs
were mixed with Thy1.1 congenic B6 Tregs (10,000 each) during the activation cocultures, then each Treg population was re-sorted and tested
separately. Error bars show SD for replicate wells in 1 of 3 similar experiments, using TDLN pDCs from B78H1–GM-CSF and B16-OVA tumors.
2576? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
Figure 2 legend and CHOP expression measured by intracellular
staining after 2 days. Figure 4A shows that CHOP was upregulated
when IDO was active and was expressed in both OT-I (visible as the
CD4– population) and Tregs (CD4+). In these studies, approximately
half of the Tregs upregulated CHOP, which could reflect an intrin-
sic heterogeneity in our CD25+ Treg population. Blocking IDO with
1MT abrogated CHOP expression in OT-I as expected and also pre-
vented CHOP induction in Tregs, showing that both events were IDO
dependent (Figure 4A). Figure 4B shows that Tregs derived from mice
lacking functional GCN2 (GCN2-KO mice) showed no IDO-induced
upregulation of CHOP. Consistent with this, GCN2-KO Tregs
were unable to undergo functional activation by IDO (Figure 4C).
GCN2-KO Tregs were still able to undergo anti-CD3-induced activa-
tion (data not shown), so they were not globally deficient in suppres-
sor activity. Finally, Figure 4D shows that IDO-induced activation
of wild-type Tregs was blocked by adding excess tryptophan to the
activation cultures. Taken together, these data were consistent with
the hypothesis that a tryptophan withdrawal stress, imposed by IDO
and sensed via the GCN2 pathway, was required for Treg activation
by IDO+ pDCs.
Direct activation of mature Tregs is more potent than de novo differentiation of new Tregs. (A) Activation cocultures were set up as described in
Figure 2 using Thy1.1-congenic B6 Tregs. To these were added CD4+CD25– (naive, nonregulatory) T cells from A1 mice plus CBA spleen DCs.
Parallel groups received either no H-Y antigen for the A1 cells, H-Y, or H-Y plus 1MT. (All cultures received OVA peptide for the OT-I). After
2 days, cocultures were stained for CD4, Foxp3, and Thy1.1. The smaller dot plots show similar cultures in which the A1 cells and OT-I were
labeled with CFSE prior to addition, then analyzed for cell division at the end of the assay. CFSE histograms for the A1 cells (CD4+CFSE+) are
superimposed. One of 4 experiments. (B) Assays were set up as described in the previous panel, using Thy1.1 congenic Tregs plus nonregula-
tory CD4+CD25– cells from wild-type B6 mice, activated with αCD3 mAb. Dot plots show upregulation of Foxp3 in this model using CD4+CD25–
cells prelabeled with CFSE. After 2 days the Treg and non-Treg populations were sorted separately based on Thy1.1 expression and tested in
readout assays (A1 T cells + CBA DCs). One of 3 similar experiments; error bars show SD.
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IDO-activated Tregs in TDLNs. (A) Tumors were grown in wild-type or IDO-KO hosts. Tregs from day 7 TDLNs were sorted and added to readout
assays (A1 T cells + CBA DCs) with and without PD-1/PD-L blocking antibodies. Mean ± SD of 4 pooled experiments with B78H1–GM-CSF,
4 experiments with B16-OVA, and 3 experiments with IDO-KO hosts (2 with B78H1–GM-CSF and 1 with B16-OVA). (B) Wild-type mice were
treated throughout tumor growth with vehicle control or sustained-release 1MT. Tregs from day 7 tumors were tested in readout assays as
described above with added isotype, PD-1/PD-L–blocking antibodies, or a combination of anti–PD-1/PD-L plus IL-2 plus anti–IL-10/TGF-β anti-
bodies. One of 3 experiments using B78H1–GM-CSF and B16-OVA. (C) Upper panels: CFSE-labeled OT-I were injected into mice with B16-OVA
tumors (days 7–8) with and without oral 1MT administration after transfer. After 4 days, TDLNs and contralateral LNs (CLN) were stained for
the 1B11 activation marker. Percentages show the CFSE+ OT-I in total LN cells. Histogram shows 1B11 on OT-I in TDLNs. Representative of
4 transfers each. Lower panels: Similar experiments as described above using OT-IGCN2-KO cells transferred into WT or GCN2-KO hosts bearing
B16-OVA tumors. One of 3 similar experiments. (D) B78H1–GM-CSF tumors were treated on day 11 with vehicle (control), cyclophosphamide
(CY; 150 mg/kg), or cyclophosphamide plus 1MT pellets. Seven days later cells from TDLNs were harvested and added to readout assays (allo-
specific BM3 T cells plus B6 splenocytes, as described in ref. 1). One group in each readout assay also received 1MT added during the assay,
as shown on the last bar of each graph. One of 3 experiments.
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CTLA4 blockade prevents Treg activation in cocultures. Tregs them-
selves have been reported to upregulate IDO expression in DCs
(27). This occurs via binding of cell surface CTLA4 on Tregs to
B7.1/B7.2 molecules on DCs, resulting in B7-mediated induction
of IDO (10, 28). Consistent with this, we found that the addition
of Tregs to cocultures of TDLN pDCs plus OT-I significantly
increased IDO enzymatic activity (measured as production of
kynurenine, the first major metabolite of tryptophan produced by
IDO) and that this Treg-induced enhancement was prevented by
blocking CTLA4 in cocultures (Supplemental Figure 5). Likewise,
blocking CTLA4 significantly inhibited IDO-induced functional
activation of Tregs in cocultures (Figure 5A). Thus, IDO caused
activation of Tregs, but a reciprocal interaction with the Tregs
appeared necessary for full induction of IDO.
Distinct MHC-restricted and MHC-unrestricted components of activa-
tion. We next asked whether interaction with MHC molecules on
the pDCs was required for Treg activation. Figure 5B shows that
induction of CHOP expression in Tregs was strictly dependent on
interaction with the MHC molecules expressed on the IDO+ pDCs.
CHOP was not induced if the Tregs and pDCs were mismatched at
MHC class II (Figure 5B) or if interaction with MHC was blocked
by antibody against IAb (the MHC-II allele expressed by B6 mice).
Consistent with this, Figure 5C shows that MHC-mismatched
(CBA) Tregs did not become activated during coculture with
IDO+ B6 pDCs. However, if the CBA Tregs were mixed with MHC-
matched (B6Thy1.1) Tregs, then both populations became activated
and both mediated suppression via the characteristic IDO-induced
PD-1/PD-L–dependent mechanism (Figure 5C). This suggested
that an MHC-restricted interaction between the pDCs and Tregs
was required to trigger the effects of IDO (perhaps as part of the
same CTLA4-dependent activation step described above), but once
IDO was triggered it could then affect other Tregs in the cultures
in an MHC-unrestricted fashion.
One potential mechanism to explain this MHC-unrestricted
effect of IDO might be secretion of soluble metabolites of tryp-
tophan (11). Supplemental Figure 6 presents indirect evidence
consistent with this possibility. However, we have not been able to
directly reproduce the effects of IDO+ pDCs, including the induc-
tion of PD-1/PD-L–mediated suppressor activity, using purified
tryptophan metabolites alone. Thus, while the data shown in
Supplemental Figure 6 suggest that tryptophan metabolites are
important participants in IDO-induced Treg activation, their spe-
cific role remains to be determined.
IDO preferentially activates preexisting Tregs. It has been previously
shown that IDO can promote de novo differentiation of Foxp3+
Tregs from naive CD4+ T cells in vitro (11). We therefore asked
whether IDO in our system would induce naive CD4+ cells to dif-
ferentiate into Foxp3+ cells. Cocultures were set up as shown in the
diagram in Figure 6A and were comprised of IDO+ pDCs, OT-I,
feeder cells, mature Tregs (Thy1.1 congenic), and a population of
CD4+CD25– T cells (naive male-specific A1 T cells isolated from
female mice). CBA splenic DCs were also added to serve as APCs for
the A1 cells. After 2 days, cocultures were harvested and stained for
intracellular Foxp3. Figure 6A shows analysis of the CD4+ popula-
tion from such an experiment. In the absence of their cognate H-Y
peptide, none of the A1 cells expressed Foxp3 at the end of culture.
In the presence of H-Y peptide, there was upregulation of Foxp3 in
up to 95% of A1 cells, depending on the experiment. Upregulation
of Foxp3 was prevented when IDO activity was blocked by 1MT.
The smaller dot plots show data from similar assays in which
the A1 T cells and OT-I were labeled with CFSE, demonstrating
that the A1 T cells remained in a nondivided state when IDO
was active but divided when IDO was blocked by 1MT. Further
studies demonstrated that the IDO-arrested A1 cells upregulated
CD25 and CD44 in response to antigen (thus showing evidence
of attempted activation), even though they could not divide (data
not shown). Thus, it was the combination of antigenic stimula-
tion by H-Y peptide plus forced cell-cycle arrest by IDO that led to
upregulation of Foxp3 in the A1 cells.
To confirm that upregulation of Foxp3 was not a peculiarity
of the TCR-transgenic A1 system, similar experiments were per-
formed using nontransgenic (polyclonal) CD4+CD25– cells from
wild-type B6 mice activated with anti-CD3 crosslinking, as in pre-
vious studies (11). Identical upregulation of Foxp3 was observed in
this system (in which the naive CD4+ cells were identified by CFSE
staining; Figure 6B, left dot plot). To ask whether the de novo
Foxp3-expressing cells acquired functional activity, the cells were
re-sorted and tested for suppressor activity. Figure 6B shows that
the mature Tregs from these cocultures became potently activated
for suppression, whereas the naive CD4+ population acquired only
a small amount of suppressor activity (100-fold less than mature
Tregs on a per-cell basis). Thus, within the length of time that our
activation assays were performed, newly differentiated Foxp3+ cells
acquired little functional activity in response to IDO, whereas the
mature, preexisting Tregs became rapidly and potently activated.
IDO-induced Treg activation in TDLNs. In all the preceding studies,
resting Tregs were activated by IDO in vitro. We now asked whether
Tregs isolated directly from TDLNs showed evidence of constitu-
tive activation by IDO in vivo. Based on data shown in Figure 3D,
findings consistent with IDO-induced Treg activation were defined
Proposed hypothetical model of IDO-induced Treg activation based
on synthesis of results from the in vitro models. The interaction of rest-
ing Tregs with IDO+ pDCs results in activation of the Tregs through a
combination of the GCN2 activation and tryptophan metabolites. Acti-
vated Tregs then suppress target T cells in an IDO-independent fashion,
involving PD-ligand expression on the target DCs, and PD-1 expression
(presumably on the target T cells). In addition, bystander CD4+ T cells
responding to other antigens, if exposed to the conditions created by acti-
vating Tregs and IDO+ pDCs, are biased to differentiate into new Tregs.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
as spontaneous ex vivo suppression that was dependent of the
novel PD-1/PD-L pathway and resistant to IL2 and IL-10/TGF-β
blockade. The “conventional” component of Treg activity was
defined as suppression that was reversed by IL2 and IL-10/TGF-β
blockade and was indifferent to PD-1/PD-L.
Tregs were sorted from TDLNs and added directly to readout
assays (A1 T cells + CBA DCs). Figure 7A (left and middle panels)
shows that the majority of suppression by TDLN Tregs was pre-
vented by PD-1/PD-L blockade. A small amount of residual PD-1/
PD-L–independent activity remained at the higher Treg/effector T
cell ratios, consistent with a mixture of both conventional and IDO-
induced forms of suppression. Based on the shift in IC50, 75%–90%
of suppression by TDLN Tregs appeared mediated via the PD-1/
PD-L pathway. In contrast, when tumors were grown in IDO-KO
mice, the Tregs in TDLNs completely lacked the PD-1/PD-L–medi-
ated component of suppression (Figure 7A, right panel).
Similar results were obtained when the IDO pathway was
pharmacologically inhibited by administering 1MT during the
period of tumor growth (Figure 7B). To test the hypothesis that
the residual, non–PD-1/PD-L–mediated component of suppres-
sion represented conventional Treg activity, we asked whether
exogenous IL-2 plus anti–IL-10/TGF-β blockade would reverse
this residual component of Treg suppression. Figure 7B shows
that this manipulation completely reversed all of the remaining
components of suppression. In the case of tumors grown in the
absence of IDO (mice administered 1MT; Figure 7B), the conven-
tional (IL-2/anti–IL-10/TGF-β–reversible) form of suppression
accounted for all of the Treg activity in TDLNs, and none of the
Treg activity was PD-1/PD-L dependent.
Inhibition of T cell responses in TDLNs in vivo. We next asked wheth-
er in vivo T cell responses were suppressed in TDLNs. OT-1 were
labeled with CFSE tracking dye and injected intravenously into
mice bearing established B16-OVA tumors (which express the
cognate antigen for OT-I). Figure 7C (upper panels) shows that
OT-I preferentially accumulated in the TDLN 4 days after injec-
tion (6% of cells in TDLN versus 1% in contralateral LN), but they
showed no cell division and no evidence of activation (assessed
as upregulation of the 1B11 T cell activation marker; ref. 29). To
ask whether this lack of response was related to IDO expression,
recipient mice were treated with 1MT. When IDO was blocked
with 1MT, OT-I in TDLNs became able to uniformly upregulate
the activation marker 1B11 (Figure 7C, histograms), thus showing
evidence of attempted activation, although they were still not able
to undergo extensive cell division.
The use of 1MT could not distinguish between direct suppres-
sion of OT-I by IDO itself and an IDO-induced activation of host
suppressor cells (e.g., IDO-activated Tregs). To eliminate any
direct effect of IDO on OT-I, we used OT-I mice bred onto the
GCN2-KO background (OT-IGCN2-KO mice). This rendered the
OT-IGCN2-KO T cells refractory to the direct suppressive effects of
IDO, as we have previously shown (9), but they remained fully
susceptible to suppression by Tregs, since Treg-mediated suppres-
sion was independent of IDO (cf. Figure 3B). Since IDO could not
directly suppress the OT-IGCN2-KO cells, any effect of IDO would
have to be exerted via IDO-responsive host suppressor cells. This
host response to IDO could be controlled by making the recipient
mice either GCN2 sufficient or GCN2-KO, as shown in Figure 7C
(lower panels). When recipient mice were GCN2 sufficient, trans-
ferred OT-IGCN2-KO cells remained fully suppressed in TDLNs;
however, the same OT-IGCN2-KO cells became able to activate if the
recipient mice were GCN2-KO (and hence unable to respond to
IDO). These data thus supported the preceding data using 1MT
and, taken together, were consistent with a population of IDO-
responsive suppressor cells in TDLNs, derived from the host and
activated by IDO in a GCN2-dependent fashion.
Chemotherapy plus 1MT in vivo depletes suppressor activity in TDLNs.
From a clinical standpoint, constitutive activation of Tregs in
TDLNs could represent a formidable barrier to immunotherapy.
Certain chemotherapeutic drugs such as cyclophosphamide have
been reported to partially reduce the number and/or function
of Tregs (30, 31). We and others have shown that 1MT displays
immune-mediated synergistic antitumor effects when combined
with chemotherapy (6, 7). Therefore, we asked whether cyclophos-
phamide combined with 1MT could reduce the suppressor activ-
ity found in TDLNs. For these experiments, we measured the sup-
pressor activity in total, unfractionated TDLN cells, as previously
described (1), because the cell number in TDLNs after chemother-
apy was too small to permit sorting of individual cell populations.
Figure 7D shows that in untreated control mice, the TDLN cells
were intensely suppressive in the readout assays (proliferation of
CD8+ T cells). This suppression was not affected by the addition
of 1MT to the readout assays (final bar in each graph), consistent
with a significant component of suppression by activated Tregs.
Treatment with cyclophosphamide alone reduced the suppressive
activity only slightly. However, administration of cyclophospha-
mide followed by 1MT significantly reduced the suppressor activ-
ity by TDLN cells (Figure 7D). This suggested that the potently
suppressive milieu in TDLNs could be partially alleviated by the
combination of chemotherapy plus 1MT.
The current study demonstrates for the first time a mechanistic
link between IDO, functional activation of Tregs, and the PD-1/
PD-L pathway. Each of these mechanisms is known to be indepen-
dently important in tumor immunology, and strategies targeting
each mechanism are currently in clinical trials or in active preclini-
cal development. We now show that these 3 powerful regulatory
mechanisms are tightly linked at the level of Treg activation in the
TDLN. This linked pathway constitutes a major contributor to
the intensely immunosuppressive milieu present in TDLNs. Since
this suppressive milieu drives T cell anergy and unresponsiveness
to tumor antigens presented in the TDLNs (32), identification of
molecular mechanisms contributing to this suppression repre-
sents an important goal in cancer immunotherapy.
Our findings suggest a hypothetical model (Figure 8) in which
IDO-induced Treg activation proceeds via a self-amplifying loop.
We hypothesize that when IDO+ pDCs present antigen to effec-
tor T cells in the presence of mature, resting Tregs, this initiates
a GCN2-dependent activation of the Tregs by IDO. In other cells,
GCN2 is known to activate a downstream stress-response path-
way, resulting in a coordinated program of changes in gene expres-
sion (26, 33). In the case of CD8+ effector T cells, we have shown
that activation of the GCN2 pathway leads to cell cycle arrest and
anergy (9). In the case of Tregs, we now show that GCN2 signaling
is critical for allowing IDO-induced functional activation. Based
on our data (Figure 5A and Supplemental Figure 5), we speculate
that the activating Tregs reciprocally induce high levels of IDO in
pDCs, via CTLA4-B7 interaction (27), leading to increased produc-
tion of tryptophan metabolites. These metabolites then complete
the full activation of the Tregs (as suggested by data shown in
2580? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
Figure 5C and Supplemental Figure 6), resulting in emergence of
the novel, highly potent PD-1/PD-L–dependent form of suppres-
sion that we describe. This model is speculative, but it is consistent
with our data, and the 2 key features of the model are clear and
well supported: direct IDO-induced activation of mature Tregs
and PD-1/PD-L–dependent suppression by IDO-activated Tregs.
A role for PD-1/PD-L as a downstream suppressor mechanism
for Tregs has not been previously described. However, induc-
tion of a another suppressive B7 family member, B7-H4, on
APCs by Tregs has been recently shown (25). In our system, the
PD-1/PD-L mechanism of suppression was only found with the
IDO-induced form of Treg activation and was not seen with the
widely studied anti-CD3-induced form of Treg activation. The
PD-1/PD-L pathway has been the focus of considerable interest
because it has been found to mediate clonal exhaustion and T
cell anergy in HIV and other chronic viral infections (34) as well
as tolerance to self antigens and immune suppression in cancer
(35). Our findings provide a novel mechanistic link between the
PD-1/PD-L system, Tregs, and IDO.
Tregs isolated from TDLNs in vivo were constitutively activated,
displaying spontaneous suppressor activity that was as potent as
the highest levels reported for Tregs extensively activated in vitro
(15, 16). The majority of this constitutive Treg activity in TDLNs
was mediated via the novel IDO-induced, PD-1/PD-L–dependent
mechanism. We demonstrate the existence of 2 distinct, clearly
distinguishable forms of Treg activity — the conventional form
elicited by anti-CD3 crosslinking, in which suppression was depen-
dent on IL-10/TGF-β, was reversed by IL-2, and was unaffected by
PD-1/PD-L blockade; and the novel IDO-induced form, which was
not dependent on IL-10/TGF-β, was not reversed by IL-2, and was
strictly dependent on the PD-1/PD-L pathway. Under IDO-suf-
ficient conditions, 75%–90% of the constitutive Treg activity in
TDLNs was due to the IDO-induced form of Treg activity. This
IDO-induced component was completely lost when tumors were
grown in IDO-KO mice or in mice treated with an IDO-inhibitor
drug during tumor growth. Under these chronically IDO-deficient
conditions, tumors showed a compensatory increase in the form
of Treg activity that was not dependent on IDO, consistent with
emergence of tumor escape variants (36). However, while tumors
were thus able to compensate for artificial genetic or pharmaco-
logic ablation of IDO, from a clinical standpoint human patients
would normally be IDO sufficient. Thus, the key observation in our
system was that 75%–90% of the naturally occurring Treg activity in
TDLNs was of the IDO-induced, PD-1/PD-L–dependent form.
In vitro, IDO activity also promoted de novo upregulation of
Foxp3 expression in naive CD4+ T cells. This finding is not novel,
since the pathway has already been described by Fallarino and col-
leagues (11). In our system, the mature, preexisting Tregs activated
by IDO were 100-fold more potent on a per-cell basis than the newly
differentiated Foxp3+ cells. In human T cells, it is known that Foxp3
upregulation does not necessarily connote stable commitment to
Treg differentiation (37, 38), so it is possible that not all of the newly
derived Foxp3+ cells would go on to become Tregs. Nevertheless, it
is relevant to note that IDO is potentially linked to the Treg lineage
at 2 points, the rapid and potent activation of mature Tregs that we
describe and the potential for de novo differentiation of new Tregs.
IDO-induced Treg activation was almost entirely prevented
by blockade of CTLA4. CTLA4 has multiple regulatory roles in
the immune system, most of which are intrinsic to the CTLA4+ T
cells themselves; however, it is also known that CTLA4 can induce
IDO expression in DCs, via back-signaling through B7 molecules
(27). We hypothesize that CTLA4 on Tregs delivers a signal to
IDO+ pDCs that enhances their normal level of IDO enzymatic
activity and thus increases the production of immunoregulatory
metabolites. Interpretation of such studies is complex because it
is difficult to separate cell-autonomous effects of the antibody on
Treg function from the effect of the antibody on IDO, so further
studies are required. However, from a therapeutic standpoint,
anti-CTLA4 antibodies are in late-stage clinical trials (39), so it is
of interest to note that CTLA4 blockade also interrupts the novel
The human counterpart of the IDO+ pDCs in mouse TDLNs is not
yet established, and human and mouse DC subsets do not always
correspond. However, a prominent population of IDO-expressing
cells is observed in many human TDLNs (40), displaying a charac-
teristic plasmacytoid morphology (41). Recently, human plasmacy-
toid DCs (CD123+BDCA2+) have been shown to upregulate IDO in
response to HIV infection (42); thus, authentic human pDCs can
be induced to express IDO. Further investigation will be required
to specifically identify the IDO-expressing cells found in human
TDLNs. Future studies will also be needed to address the possible
developmental roles of IDO and GCN2 in the differentiation of the
Treg lineage. Preliminary studies demonstrate selective but signifi-
cant functional defects in Tregs derived from IDO-KO, GCN2-KO,
and CHOP-KO mice, suggesting that the IDO pathway may have
broader importance for aspects of normal Treg differentiation.
The current study suggests that patients with cancer may have
abnormally increased Treg activity in TDLNs, due in part to the
effects of IDO. Once tumors are established, simply blocking IDO
was not sufficient to fully reverse the suppressive milieu in the
TDLN (Figure 7C). But even in established tumors, blocking IDO
allowed initial activation of tumor-specific effector T cells in TDLNs,
with attempted cell division. Combining IDO-inhibitor drugs with
chemotherapy may further help to reverse the established suppres-
sive milieu in TDLNs. Therapeutic strategies to block IDO, tumor-
induced Tregs, and the PD-1/PD-L pathway are all currently in clini-
cal or preclinical development. Our demonstration of a molecular
link uniting all 3 of these potent immunosuppressive mechanisms
may have significant implications for cancer immunotherapy.
Additional details of Methods are given in Supplemental Methods online.
Mice and tumors. Animal studies were approved by the Institutional Ani-
mal Care and Use Committee of the Medical College of Georgia. TCR-trans-
genic OT-I mice (CD8+, B6 background, recognizing the SIINFEKL peptide
of ovalbumin on H2Kb; ref. 43) and B6.PL-Thy1a/CyJ mice (congenic for
the B6 background but bearing the Thy1.1 allele) were purchased from
The Jackson Laboratory. GCN2-KO mice (B6 background) were a gener-
ous gift from the laboratory of David Ron (New York University School of
Medicine, New York, New York, USA) and have been previously described
(9). A1 mice (CBA background, recognizing an H-Y peptide presented on
IEk) (44), BM3 mice (CBA background, recognizing H2Kb as an alloanti-
gen; ref. 45), and IDO-KO mice (B6 and CBA backgrounds; refs. 10, 46)
have been previously described.
Tumor implantation is described in detail in Supplemental Methods.
Cell lines used were B78H1–GM-CSF (a subline of B16 transfected with
GM-CSF [ref. 14], as used in our previous studies of IDO+ pDCs [refs. 1, 9]);
the B16F10 subline of B16 (ATCC) and B16-OVA subline (parental B16F10
transfected with full-length OVA, clone MO4 [ref. 47], a gift from Alan
Houghton (Memorial Sloan-Kettering Cancer Center, New York, New York,
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 9 September 2007
USA). The use of OVA as a model tumor antigen was informative in our
system, since the goal was to detect the suppression of immune response
to tumor antigens; thus, a strong nominal antigen was an advantage. IDO+
pDCs and activated Tregs found in TDLNs of all 3 tumor lines were similar.
As in our previous publications (1, 9), most experiments requiring sorted
pDCs used the B78H1–GM-CSF tumors because these gave the highest
yield of pDCs (see Supplemental Figure 1). However, pDCs from tumors
without GM-CSF gave similar functional results, and all key findings were
confirmed with tumors with and without GM-CSF.
Reagents and 1MT. 1MT (catalog no. 45,248-3; Sigma-Aldrich) was pre-
pared as described (9) and used at a final concentration of 200 μM. Delivery
of 1MT by sustained-release subcutaneous pellets (5 mg/day) was as previ-
ously described (7). For oral delivery, 1MT was added to drinking water at
2 mg/ml. Recombinant mouse IL-2 (R&D Systems) was used at 10 ng/ml.
Blocking antibodies against PD-L1/B7-DC (clone MIH7) (48), PD-L2
(TY25) (49), and PD-1 (J43) (50) were used as a cocktail at 50 μg/ml each
(or rat IgG1 isotype control). Anti-CTLA4 antibody (clone 9H10, used at
10 μg/ml) and rat anti–IL-10 receptor antibody (used at 100 μg/ml, clone
1B1.3a) were from BD Biosciences; anti-mouse I-Ab (used at 100 μg/ml)
and IgM isotype control were from Southern Biotech; chicken anti–TGF-β/
β2/β3 (MAB1835, used at 100 μg/ml) was from R&D Systems.
Ex vivo Treg assays. Tregs (CD4+CD25+) were sorted from 2–4 pooled
TDLNs and added directly to readout assays containing 1 × 105 CD4+
A1 cells, 2 × 103 CD11c+ DCs from CBA spleen, and 100 nM H-Y peptide
(REEALHQFRSGRKPI). All cultures were performed in V-bottom wells.
For both Tregs and pDCs, it was important to perform sorts rapidly, collect
cells in complete medium on ice, and transfer them promptly into culture,
in order to preserve viability and function.
Treg activation cultures and readout assays. Sorting of pDCs from TDLNs was
performed as described (1, 9). The pDC fraction (CD11c+B220+) was sorted
from 2–6 pooled TDLNs (days 7–11 of tumor growth) and collected in
medium on ice. Activation cultures contained 2 × 103 pDCs, 1 × 105 sorted
CD8+ OT-I, 100 nM SIINFEKL peptide, and 5 × 103 sorted CD4+CD25+
Tregs from spleens of B6 mice without tumors. All cultures received a feeder
layer of 1 × 105 T cell–depleted spleen cells (CD4–CD8–) as described in Sup-
plemental Methods. For anti-CD3-induced activation, the same cultures
received 200 μM 1MT to block IDO plus 0.1 μg/ml anti-CD3 mAb (clone
145-2C11; BD Biosciences — Pharmingen) and 10 ng/ml IL-2. We routinely
added IL-2 to the anti-CD3 activation cultures, although this did not have
any further enhancing effect on suppressor activity over anti-CD3 alone,
presumably because adequate IL-2 was contributed by the activating OT-I
(51). After 2 days, cultures were harvested and stained for CD4 and Tregs
were isolated by sorting for CD4+ cells. Preliminary studies showed that
sorting on either total CD4+ Tregs or the CD4+CD62Lhi subset of Tregs gave
equivalent results, so the total CD4+ Treg population was routinely used.
Re-sorted Tregs were added to readout assays containing 1 × 105 A1 cells,
2 × 103 CD11c+ DCs from CBA spleen (or 5 × 104 B cells, as CD11c–B220+
spleen cells), and H-Y peptide. For assays performed in transwells, Multiwell
96-well insert plates (1 μM pore size; Falcon; BD) were used and the number
of cells in all groups was doubled.
DC and T cell adoptive transfer. The DC adoptive transfer model and adop-
tive transfer of CFSE-labeled T cells have been previously described (1, 9),
and detailed methods are given in Supplemental Methods.
Statistics. Individual thymidine incorporation assays were performed in
triplicate or quadruplicate wells for each data point, and for these analyses,
error bars in the figures indicate SD of replicate wells. Multiple treatment
groups in each experiment were compared using ANOVA. In cases where 1
representative experiment of several is shown, each independent replicate
experiment showed comparable statistical significance between the same
groups by ANOVA. Where multiple experiments were combined for analy-
sis, raw thymidine incorporation counts were normalized to the control
(wells without Tregs) in each experiment to permit comparison across mul-
tiple experiments. For these analyses, the error bars in the figures indicate
the SD of the pooled data.
The authors thank Makio Iwashima and Pandelakis Koni for
insightful discussion; Jingping Sun, Joyce Wilson, Judy Gregory,
and Doris McCool for expert technical assistance; and Jeanene
Pihkala for expert cell sorting. D.H. Munn and A.L. Mellor have
intellectual property interests in the therapeutic use of IDO and
IDO inhibitors, and receive consulting income from NewLink
Genetics Inc. This work was supported by NIH grants CA103320,
CA096651, and CA112431 (to D.H. Munn) and HD41187 and
AI063402 (to A.L. Mellor).
Received for publication February 21, 2007, and accepted in revised
form May 29, 2007.
Address correspondence to: David H. Munn, Room CN-4141, Med-
ical College of Georgia, Augusta, Georgia 30912, USA. Phone: (706)
721-7141; Fax: (706) 721-8732; E-mail: firstname.lastname@example.org.
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Ligation of B7-1/B7-2 by human CD4+ T cells trig-
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