Glucocorticoid-Induced TNF Receptor Family Related Gene
Activation Overcomes Tolerance/Ignorance to Melanoma
Differentiation Antigens and Enhances Antitumor Immunity1
Teresa Ramirez-Montagut,2* Andrew Chow,* Daniel Hirschhorn-Cymerman,*
Theis H. Terwey,* Adam A. Kochman,* Sydney Lu,* Randy C. Miles,†Shimon Sakaguchi,‡§
Alan N. Houghton,* and Marcel R. M. van den Brink*
Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have
shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression
and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice
challenged with B16 melanoma. Rejection requires CD4?, CD8?, and NK1.1?cells and is dependent on IFN-? and Fas ligand and
independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice
depleted of CD25?cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell co-
stimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16
and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild
hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in
our model that GITR stimulation expanded the absolute number of FoxP3?cells in vivo. Thus, we conclude that overall, GITR
stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity. The
Journal of Immunology, 2006, 176: 6434–6442.
the immune system’s repertoire contains autoreactive T and B cells
that, when activated properly, may reject malignant cells that ex-
press unaltered or altered self-Ags (1). Because tumor Ags are
often self-Ags, high-affinity T cells are either deleted in the thy-
mus, remain ignorant, or become tolerant in the periphery, prob-
ably due to a population of CD4?CD25?immunosuppressor reg-
ulatory T cells (Treg)3(2). In mouse models, Ab depletion of Treg
using anti-CD25 and anti-CD4 Abs has been demonstrated to in-
crease rejection against chemically induced tumor and enhance
vaccination against melanoma differentiation Ags (3–8). Still, de-
pletion of CD4?and/or CD25?T cells has little effect on the
he immune system can recognize and reject primary de-
veloping tumors, a concept known as cancer immunosur-
veillance. In melanoma, cancer immunity exists because
rejection of spontaneous tumors and may have a detrimental effect
on the generation of CD8?antitumor immunity (6). Other groups
have shown a new strategy to inhibit Treg suppression, via the
stimulation of glucocorticoid-induced TNF receptor (TNFR) fam-
ily related protein (GITR), a member of the TNFR superfamily that
is constitutively expressed at high levels on Treg (9–12). GITR
expression is not restricted to Treg, and recent studies have shown
that although GITR stimulation inhibits Treg suppression (9, 10,
13), it also stimulates CD4?and CD8?T cells (12, 14–18). GITR
stimulation has been shown to induce tumor rejection in a con-
comitant immunity model (19), and expression of GITR ligand on
tumor cells can delay tumor growth and increase T cell infiltration
(20). Therefore, we tested whether in vivo stimulation of GITR can
break tolerance to differentiation Ags and enhance antitumor im-
munity to B16 melanoma.
Materials and Methods
Mice and tumor lines
C57BL/6, BALB/c, Rag 1?/?, and IFN-??/?, pfp?/?, and gld mice (fe-
males, 8–10 wk old) were obtained from The Jackson Laboratory. All
experiments were performed in accordance with our institutional guide-
lines. The B16F10 (B16) mouse melanoma cell line was originally ob-
tained from I. Fidler (MD Anderson Cancer Center, Houston, TX) (21, 22)
and passaged intradermally to ensure reproducible and aggressive tumor
growth. TGL tumor lines (B16-TGL and RENCA-TGL) were generated by
transducing B16 and RENCA with a retroviral vector containing a fusion
reporter gene coding for HSV1-TK, enhanced green fluorescent protein,
and firefly luciferase (23). After transduction, individual clones with high
enhanced green fluorescent protein expression were sorted into 96-well
plates using a FACSVantage DiVa (Becton Dickinson) cell sorter. Tissue
culture medium consisted of RPMI 1640 supplemented with 10% heat-
inactivated FCS, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 2 mM
*Department of Medicine and Immunology Program, Memorial Sloan-Kettering Can-
cer Center, New York, NY 10021;†Hampton University, Greensboro, NC;‡Depart-
ment of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto Uni-
versity, Kyoto, Japan; and§Laboratory for Immunopathology, Institute of Physical
and Chemical Research, Research Center for Allergy and Immunology, Yokohama,
Received for publication August 31, 2005. Accepted for publication March 3, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants HL69929, HL72412, and CA107096 from the
National Institutes of Health, Awards from the Emerald Foundation and The Exper-
imental Therapeutics Center of Memorial Sloan-Kettering Cancer Center funded by
William H. Goodwin and Alice Goodwin, the Commonwealth Foundation for Cancer
Research (to M.R.M.v.d.B.), National Cancer Institute Grants CA33049, CA56821,
CA47179, CA59350, and Swim Across America (to A.N.H.).
2Address correspondence and reprint requests Dr. Teresa Ramirez-Montagut, Labo-
ratory of Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller Uni-
versity, 1230 York Avenue, Box 226, New York, NY 10021. E-mail address:
3Abbreviations used in this paper: Treg, regulatory T cell; TNFR, TNF receptor;
GITR, glucocorticoid-induced TNFR family related gene; FasL, Fas ligand.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
Tumor challenge and measurement
C57BL/6 mice were challenged by intradermal inoculation of 3 ? 104or
1.2 ? 105(high dose) B16 cells, and tumor diameter was measured every
2 days. Mice rechallenged received an intermediate dose of B16 cells (6 ?
104cells). For tumor-free Kaplan-Meier curves, a mass of 2 ? 2 mm was
considered as (?) for tumor. For bioluminescence experiments, C57BL/6
or BALB/c mice received 1 ? 105cells of B16-TGL or RENCA-TGL i.v.
and were given 150 mg/kg i.p. of D-luciferin (Xenogen). Fifteen minutes
after D-luciferin injection, mice were anesthetized with isoflurane and
placed into the Xenogen IVIS bioluminescence imaging system (Xenogen)
in a supine position and recorded for 5 min. Pseudocolor images showing
the whole body distribution of bioluminescent signal were superimposed
on the conventional grayscale photographs.
C57BL/6 mice were intradermally challenged with 3 ? 104B16 cells and
treated with 1 mg/mouse of rat IgG control Ab (n ? 50 mice) or DTA-1
(n ? 50 mice) after 1, 4, and 9 days from challenge. Twenty-one days after
challenge, spleens and draining inguinal lymph nodes were harvested and
pooled. CD8?cells and CD4?were positively selected using MACS beads
(Miltenyi Biotec). Purified cells (?98% purity) were adoptively transferred
into Rag 1?/?hosts. Groups of 8–10 mice received either CD4?cells
alone (24 ? 106cells/mouse), CD8?alone (12 ? 106), or CD4?and CD8?
at a 1:2 ratio (12 ? 106CD8?and 24 ? 106CD4?cells per mouse). One
day after adoptive transfer, Rag 1?/?mice were challenged intradermally
with 3 ? 104B16 cells, and tumor diameter was measured every 2 days.
DTA-1 treatment and Ab depletions
Mice received 1 mg/mouse of affinity-purified DTA-1 mAb (9) or rat IgG
control Ab (Anogen) injected i.p. at the specified time points. Mice were
depleted of Gr-1?(clone Rb6-8C5), CD4?(GK1.5), CD8?(2.43), and
NK1.1?(PK136), and CD25?cells (PC61) by i.p. injection of 500 ?g of
the mAbs (bioreactor supernatants) at days ?7, ?4, ?4, and ?7 from
tumor challenge. For depletion of Treg before tumor challenge, mice were
depleted with 500 ?g of PC61 by i.p. injection at days ?7 and ?4. Flow
cytometry was used to confirm ?98% depletion of target cells for at least
7 days after the first injection.
Anti-mouse CD16/CD32 FcR block (clone 2.4G2) and all of the following
fluorochrome-labeled and purified Abs against murine Ag were obtained
from BD Biosciences: CD4 (clone RM4-5), CD8 (53-6.7), CD62L (MEL-
14), CD122 (TM-B1), CD44 (IM7), CD45R/B220 (RA3-6B2), Gr-1 (RB6-
8C5), CD25 (PC61), CD69 (H1.2F3), isotype controls: rat IgG2a- (R35-
95), rat IgG2a- (B39-4), rat IgG2b (A95-1), hamster IgG group 1 liter
(Ha4/8), streptavidin-FITC, -PE, and -PCP. Biotinylated anti-murine GITR
(BAF524) was obtained from R&D Systems. T cells were washed in PBS
with 2% FBS and 0.1% sodium azide and incubated for 15 min at 4°C with
anti-CD16/CD32 FcR block. Subsequently, cells were incubated for 30
min at 4°C with Abs and washed twice. Stained cells were analyzed on a
FACSCalibur flow cytometer (BD Biosciences) with CellQuest or Flowjo
software (Tree Star). For detection of FoxP3?cells (eBioscience), spleno-
cytes and draining lymph nodes were harvested, counted, and 10 ? 106
cells were incubated in 2% paraformaldehyde at 37°C for 30 min, washed
three times with PBS, and fixed in 80% methanol at ?20°C overnight.
Cells were washed three times with PBS and incubated with anti-CD16/
CD32 FcR block for 15 min. Subsequently, cells were incubated with Ab
against cell surface markers, incubated for 30 min at 4°C, washed twice in
PBS, and analyzed by flow cytometry.
Peptides and ELISPOT
Peptides analyzed were synthesized by Genemed Synthesis and used at
?80% purity, as confirmed by HPLC. Peptides tested include the follow-
ing: gp100/pmel 17 peptide gp10025–33(24), dopachrome tautomerase/ty-
rosinase-related protein 2 (DCT) DCT181–188(25) and DCT363–370, and
TYRP1/gp75 (gp75), gp75455–462, gp75481–489, and gp75522–529(J. A.
Guevera-Patino, M. E. Engelhorn, M. J. Turk, C. Liu, F. Duan, A. D.
Cohen, T. Merghoub, J. D. Wolchok, A. N. Houghton, submitted for pub-
lication.) For ELISPOT analysis (19), multiscreen-IP plates (Millipore)
were coated with anti-mouse IFN-? Ab in PBS (clone AN18; Mabtech),
incubated overnight at 4°C, washed with PBS, and blocked with RPMI
1640 plus 7.5% FBS for 2 h at 37°C. CD8?T cells were harvested from
spleen and inguinal lymph nodes, purified using anti-CD4 and anti-CD8
MACS magnetic beads (Miltenyi Biotec), and plated at a concentration of
2.5 ? 105cells/well. B16 targets cells were pretreated for 24 h with Con
A supernatant (T-Stim Culture Supplement Rat with CON A; BD Bio-
sciences), detached using a nonenzymatic method (Cell Dissociation So-
lution (1?) Nonenzymatic; Sigma-Aldrich), irradiated, and plated at 1 ?
104cells/well. Target EL-4 leukemia cells were pulsed with 10 ?g/ml
peptide for 1 h and plated at 2 ? 104cells/well. After 20-h incubation for
CD8?T cells and 48 h for CD4?T cells at 37°C, plates were washed with
PBS plus 0.05% Tween and incubated for 2 h at 37°C with biotinylated Ab
against mouse IFN-? (clone R4-6A2; Mabtech). Spots were counted with
an automated ELISPOT reader system with KS 4.3 software (Carl Zeiss
Log-rank analysis was performed using Kaplan-Meier curves. For all other
analysis, nonparametric unpaired Mann-Whitney U test was used.
A single dose of an anti-GITR agonist Ab (DTA-1) induces a
delay in tumor progression
We analyzed two solid tumor cell lines, the spontaneous melanoma
B16 and renal cell carcinoma RENCA, and established that both
tumor lines were negative for GITR expression (Fig. 1A). To as-
sess whether in vivo GITR stimulation could result in tumor delay
or rejection, the agonist Ab against GITR (clone DTA-1) was in-
jected into BALB/c mice challenged with RENCA-TGL (Fig. 1B).
Mice that received a single dose of DTA-1 the day before the
challenge had increased survival when compared with the control
group. This antitumor effect was confirmed in C57BL/6 mice chal-
lenged with B16 (without TGL) intradermally (Fig. 1, C and D) or
by tail vein (B16-TGL) and treated with a single dose of DTA-1
(data not shown).
GITR stimulation induces tumor rejection that requires T cells
and NK/NKT cells
We analyzed GITR expression on different effector populations of
the adaptive and innate immune system (data not shown). GITR
was present at low levels on B cells, at intermediate levels on
CD4?, CD8?, NK, NKT cells, granulocytes, and macrophages,
and at high levels on Treg.
DTA-1 treatment at day ?1 reduced tumor growth more effec-
tively than the other regiments, suggesting that costimulation of
effector T cells by DTA-1 was more important for tumor rejection
than the modulation of Treg. Based on this rationale, we decided
to administer DTA-1 three times, starting 1 day after tumor inoc-
ulation (Fig. 2). Most mice that were challenged with a high dose
(Fig. 2A) or low dose of B16 (Fig. 2B) and treated three times with
DTA-1 rejected the tumor. Next, we examined the contribution of
Gr-1?, CD4?, CD8?, and NK1.1?cells in tumor rejection by
depleting the host with specific Ab. Gr-1 depletion had no impact
on DTA-1-mediated tumor rejection (Fig. 2C), whereas mice de-
pleted of CD4?cells (Fig. 2D), CD8?cells (Fig. 2E), or both (Fig.
2F) were unable to reject B16, even in the presence of DTA-1,
indicating that T cells are necessary for DTA-1-induced tumor
rejection. Mice depleted of NK1.1?cells (Fig. 2G) developed tu-
mors rapidly, and addition of DTA-1 was associated with only low
levels of rejection comparable to the nondepleted rat IgG control
group, indicating that NK/NKT were also required for B16 rejec-
tion, which is consistent with other published experiments (26, 27).
Prior studies have indicated that anti-CD25 Ab depletion of Treg
can enhance antitumor immunity against highly immunogenic tu-
mors but has little or no effect against nonimmunogenic tumors
(3–7). Experiments treating B16 with anti-CD25 administration
range from a delay in B16 progression without rejection (7) to low
rate rejection (3). Our results show a delay in B16 progression in
CD25-depleted mice compared with the control group, although
this result did not reach statistical significance. Addition of DTA-1
to anti-CD25 did not result in improved tumor protection (Fig.
2H). Because the presence of the Ab can be detected up to 15–21
6435The Journal of Immunology
days after the first injection (3), these results are consistent with
abrogation of immunity due to a block in IL-2 signaling by the
anti-CD25 Ab (28–30) or, alternatively, depletion of recently ac-
tivated effector cells (6). Rag 1?/?mice that lack T and B cells, but
have increased NK activity, were challenged with B16 and treated
with DTA-1 or control Ab (Fig. 2I). No difference in tumor take
was observed between these two groups. Overall, these results
confirm that both T and NK cells are required for tumor rejection.
To discriminate between the effects of DTA-1 on Treg from that
on effector T cells CD25 depletion was done twice, at 7 and 4 days
before tumor inoculation (Fig. 3A). Mice depleted of CD25?cells
and treated with control rat IgG developed tumors at the same rate
as nondepleted mice treated with the control Ab. All mice depleted
of CD25?and treated with DTA-1 rejected tumors, whereas 80%
of nondepleted mice treated with DTA-1 rejected tumors. Al-
though CD25 depletion and DTA-1 treatment seemed to be en-
hancing tumor rejection, the comparison between both groups did
not reach statistical significance. These results indicate that DTA-1
rejection of B16 is dependent on effector T cells rather than abro-
gation of Treg suppression.
To demonstrate whether T cells were sufficient for tumor rejec-
tion, CD4?and CD8?cells purified from spleens and draining
lymph nodes of mice challenged with B16 and treated with DTA-1
or control Ab were adoptively transferred into Rag 1?/?recipients
(Fig. 3B). One day after transfer, the recipients were challenged
with B16 cells. Transfer of CD4?, CD8?T cells, or both derived
from mice treated with control Ab and challenged with B16 did not
affect tumor growth. Conversely, transfer of T cells derived from
mice treated with DTA-1 and challenged with B16 (CD4?, CD8?,
or both) resulted in significant rejection of B16.
To test the therapeutic effect of anti-GITR stimulation, DTA-1
treatment was delayed until mice were bearing tumors of 3-mm
mean diameter. Mice were treated with DTA-1 (1 mg/mouse) 10
and 14 days after challenge. None of the mice under these con-
ditions were able to reject the B16 challenge (data not shown).
Because B16 is a highly aggressive tumor, it remains to be estab-
lished whether in slower progressive tumors, anti-GITR demon-
strates therapeutic effects.
Tumor rejection is dependent on IFN-? and Fas ligand (FasL)
and independent of perforin
A number of studies have demonstrated a role for IFN-? in tumor
immunity (31, 32). To test whether IFN-? was relevant for GITR-
mediated tumor rejection, IFN-??/?and wild-type mice were
treated with DTA-1 and challenged with B16 (Fig. 4). IFN-??/?
mice treated with DTA-1 were not protected from B16 challenge,
demonstrating that this cytokine is required for DTA-1-mediated
tumor rejection. We next tested the requirements for perforin and
FasL in this system. All pfp?/?mice challenged with B16 and
treated with control Ab developed tumors (10 of 10 tumor-bearing
mice), whereas most pfp?/?mice treated with DTA-1 showed re-
jection (2 of 10 tumor-bearing mice). Mice deficient in FasL (gld)
challenged with B16 develop tumors (10 of 10 tumor-bearing
mice), whereas DTA-1 treatment did not enhance tumor rejection
(8 of 10 tumor-bearing mice). We conclude that perforin is not
required for GITR-mediated tumor rejection, whereas IFN-? and
FasL are required for B16 rejection.
GITR stimulation overcomes tolerance/ignorance to melanoma
differentiation Ags and induces responses against B16
To examine Ag specificity of T cells during tumor rejection, mice
were challenged with B16, treated with DTA-1 or control Ab, and
analyzed by IFN-? ELISPOT (Fig. 5, A and B). Mice that received
B16 and DTA-1 demonstrated T cell responses against three mel-
anoma differentiation Ags (gp100, TYRP1/gp75, and DCT for
CD8?responses) and against syngeneic B16 cells (for CD4?and
CD8?responses). Mice that received B16 and control Ab showed
no detectable IFN-?-producing T cells. Interestingly, even though
these peptide-specific responses were only detected in the DTA-1
group, low-level responses also existed against negative controls
agonist (DTA-1) induces a delay in tumor
progression. A, GITR surface expression of
B16 and RENCA tumor cell lines (gray
histogram, unstained cells; black line, iso-
type control; red line, DTA-1). B, Kaplan-
Meier survival curve of BALB/c mice
challenged i.v. with 1 ? 105RENCA-TGL
cells and treated with a single dose of rat
IgG control Ab or DTA-1 (1 mg/mouse) 1
day before the challenge. C, C57BL/6 mice
challenged intradermally with 1 ? 105B16
and treated with a single dose of rat IgG
control Ab or DTA-1 (1 mg/mouse) at dif-
ferent time points of tumor challenge. D,
The data represent the mean size of intra-
dermal tumors per group at different time
points after challenge. All data are repre-
sentative of two independent experiments
(n ? 10–15 mice per group).
A single dose of anti-GITR
6436 GITR ACTIVATION ENHANCES ANTITUMOR IMMUNITY
(syngeneic EL4 pulsed with irrelevant peptide or syngeneic
splenocytes) (Fig. 5A), suggesting some recognition of self-Ags
during early tumor progression. Furthermore, at day 34 after chal-
lenge, control animals bearing tumors still remained tolerant/igno-
rant to these Ags, whereas Ag-specific responses were detected in
the DTA-1-treated group that rejected tumor (Fig. 5B).
GITR stimulation generates memory against B16 and minimal
To test whether memory against B16 was established in mice that
received DTA-1 and rejected tumor, mice were re-challenged 60–
140 days after the primary challenge. Although all naive control
mice rapidly developed tumors, only 23% of mice rechallenged
with B16 developed tumors (Table I). These results are consistent
with the generation of a memory response against B16.
Careful examination of coat color demonstrated that a small
proportion of mice (5 of 43 mice) that were treated with DTA-1
and rejected B16, developed signs of mild depigmentation (Table
I). Two mice presented mild salt and pepper hair hypopigmentation,
whereas the remaining three developed one or two 2- to 3-mm2
patches of hypopigmented hair at the site of tumor challenge. No
change in coat color was detected in mice treated with control Ab and
challenged with B16. These results indicate that mild autoimmunity
was induced in a small subset of DTA-1-treated mice.
GITR stimulation increases CD4?and CD8?T cell activation
To test whether DTA-1 had an effect on augmenting T cell acti-
vation, mice were challenged with B16, treated with DTA-1, and
sacrificed 7 days after tumor challenge. The analysis of T cell
activation markers showed a significant increase in the absolute
treated with control Ab or DTA-1 after intradermal B16 challenge. All mice received 1 mg/mouse of DTA-1 Ab (DTA-1 treatment) or rat IgG control Ab
(Rat IgG treatment) on days ?1, ?4, and ?9 after challenge. A, Mice were challenged with B16 at a high dose (1.2 ? 105B16 cells). B, Mice were
challenged with B16 at a low dose (3 ? 104B16 cells). C–H, Control groups included mice challenged with B16 (3 ? 104cells) and treated with DTA-1
or control Ab (DTA-1 treatment and Rat IgG treatment). Depletion groups included mice challenged with B16, treated with DTA-1 or rat IgG Ab, and also
depleted of the following: Gr-1?(C), CD4?(D), CD8?(E), CD4?and CD8?(F), NK1.1?(G), and CD25?(H). Depletions were done by i.p. injection
of 500 ?g Ab/mouse at days ?7, ?4, ?4, and ?7 of tumor challenge. These groups are designated as Depletion ? rat IgG and Depletion ? DTA-1. I,
Rag 1?/?mice were challenged with B16 (3 ? 104cells) and treated with DTA-1 (DTA-1 treatment) or rat IgG control Ab (Rat IgG treatment). All data
are representative of at least two independent experiments (n ? 10–20 mice per group).
Three doses of DTA-1 induce B16 rejection that requires T cells and NK/NKT cells. Kaplan-Meier tumor-free curves of C57BL/6 mice
6437The Journal of Immunology
numbers of CD4?CD44highT cells (Fig. 6A). We gated on CD44
and CD62L (Fig. 6B) to determine the percentage of central mem-
ory (CD44highand CD62L?) vs effector T cells (CD44highand
CD62L?). There was a significant increase in the percentage of
effector T cells in the DTA-1-treated mice compared with control
mice (Fig. 6C). We also noted an increased percentage of
CD44highCD4?and CD8?T cells in draining lymph nodes of the
DTA-1-treated group compared with control mice (Fig. 6D).
GITR stimulation increases the expansion of FoxP3?cells
Previous studies have demonstrated that GITR-stimulated Treg
proliferate in the presence of IL-2 (11–13, 16, 33). We repeated
these experiments and found that, indeed, Treg in the presence of
CD3 stimulation and IL-2 readily expand, and proliferation is in-
creased in the presence of CD28 or DTA-1 (data not shown), con-
firming its costimulatory role on Treg. We next tested whether
systemic administration of DTA-1 to naive hosts could expand the
absolute number of CD4?CD25?T cells. In both C57BL/6 and
BALB/c mice, weekly treatments with 1 mg/mouse of DTA-1 in-
creased the absolute number of CD4?CD25?T cells compared
with mice treated with control Ab (data not shown).
Because in vivo GITR stimulation expands Treg, we tested
whether once Treg had been stimulated through GITR, the ex-
panded cells could still suppress effector T cells. High precursor
frequencies and strong proliferation is observed during allogeneic
responses, thus we used this model to test suppression (Fig. 7).
Treg were purified and cultured with anti-CD3 and anti-GITR for
a week and subsequently tested in a MLR (Fig. 7A). GITR-stim-
ulated cultured Treg were able to suppress alloactivation of
CD4?CD25?and CD8?CD25?to a better extent than fresh Treg.
Cultured Treg are better suppressors than freshly isolated Treg,
presumably due to the fact that all GITR-cultured Treg added to
the MLR had been polyclonally stimulated with anti-CD3. Next,
we tested whether Treg isolated from mice treated with DTA-1
maintained a suppressive activity ex vivo (Fig. 7B). Naive mice
were treated with 1 mg/mouse of a control Ab (rat IgG) or with
DTA-1. Twenty-four hours after treatment, Treg were isolated and
tested in a MLR. Treg derived from DTA-1-treated mice were
equally efficient at suppressing alloactivation than Treg derived
from the control group. These results suggest that once GITR stim-
ulation has occurred, Treg regain their suppressive activity.
To test the role of DTA-1 administration on Treg expansion in
tumor-bearing mice, T cells from spleens and lymph nodes of mice
challenged with B16, treated with DTA-1, and sacrificed 12 days
after tumor challenge were analyzed by flow cytometry (Fig. 8). It
has been demonstrated that FoxP3 is a required and restricted
marker for natural Treg (34, 35). Mice treated with DTA-1 have
increased percentage and absolute number of splenic FoxP3?
cells. The percentage of FoxP3?in lymph node from these mice
compared with the control group was similar, but the absolute
ated by costimulation of effector cells rather than modulation of Treg.
Kaplan-Meier tumor-free curves of C57BL/6 mice treated with control Ab
or DTA-1 after intradermal B16 challenge. All mice received 1 mg/mouse
of DTA-1 Ab or rat IgG control Ab on days ?1, ?4, and ?9 after chal-
lenge. A, Control nondepleted mice were treated with DTA-1 or rat IgG Ab
(Rat IgG treatment or DTA-1 treatment). Two groups of mice were de-
pleted of CD25?cells by injection of 500 ?g of Ab per mouse 7 and 4 days
before B16 challenge. One group received DTA-1 (CD25 depletion ?
DTA-1), and the other group received the control Ab (CD25 depletion ?
rat IgG). All mice were challenged intradermally with 3 ? 104B16 cells.
B, Adoptive transfer of T cells from tumor-bearing DTA-1-treated mice
protect Rag 1?/?mice from B16 challenge. C57BL/6 mice were chal-
lenged intradermally with 3 ? 104B16 cells and treated with 1 mg/mouse
of DTA-1 Ab or control rat IgG Ab 1, 4, and 9 days after challenge. On day
21, CD4?and CD8?T cells purified from splenocytes and lymph nodes of
tumor-bearing mice were adoptively transferred at a 2:1 ratio into Rag 1?/?
mice. One day after transfer, Rag1?/?recipients were challenged intrad-
ermally with 3 ? 104B16 cells. A control group of mice did not receive
T cells (No T cells). Mice that received T cells from donors treated (Tx)
with control rat IgG Ab are labeled as CD4, CD8, and CD4?CD8 from rat
IgG Tx, and mice that received T cells form DTA-1-treated donors are
labeled as CD4, CD8, and CD4?CD8 from DTA-1 Tx. Comparisons be-
tween all groups that received cells from rat IgG-treated mice (blue lines)
vs all groups that received T cell from DTA-1-treated mice (red lines) are
statistically significant (p ? 0.04).
DTA-1-induced rejection of B16 is predominantly medi-
FasL and independent of perforin. A, Wild-type and IFN-??/?C57BL/6
mice were treated with control Ab or DTA-1 and challenged intradermally
with 3 ? 104B16 cells. All mice received Ab at 1 mg/mouse on days ?1,
?4, and ?9 after challenge. Data are representative of two independent
experiments (n ? 6–10). B, Wild-type, perforin?/?(pfp?/?), and FasL-
deficient (gld) C57BL/6 mice were treated with control Ab or DTA-1 and
challenged intradermally with 3 ? 104B16 cells. All mice received Ab at
1 mg/mouse on days ?1, ?4, and ?9 after challenge. Data are represen-
tative of two independent experiments (n ? 10).
DTA-1-induced tumor rejection is dependent on IFN-? and
6438 GITR ACTIVATION ENHANCES ANTITUMOR IMMUNITY
number of these cells was increased in the DTA-1-treated group
(data not shown). These results demonstrate an expansion of
FoxP3?cells after DTA-1 treatment and are consistent with pre-
vious studies demonstrating that GITR stimulation induces Treg
proliferation in vitro (11–13, 16, 33).
Our results indicate that GITR stimulation overcomes tolerance/
ignorance to self-Ags (melanoma differentiation Ags), induces tu-
mor rejection that requires T and NK/NKT cells, and is dependent
on IFN-? and FasL, but independent of perforin. In vivo GITR
stimulation induces potent rejection and memory against B16,
whereas it induces mild hypopigmentation and expansion of
We sought to explore the mechanism of B16 tumor rejection.
Adoptive transfer of T cells derived from DTA-1-treated donor
demonstrates that CD4?and CD8?T cells are sufficient for GITR-
mediated rejection of B16. Although most studies implicate CD8?
Table 1. Generation of memory without severe autoimmunity in DTA-1-treated mice that rejected B16
Tumor-bearing mice84%16%23%Depigmented 12%
aDose of B16 cells injected intradermally.
bNumber of tumor-bearing mice per total number of mice.
cNumber of depigmented mice per total number of mice.
104B16 cells treated with rat IgG control Ab (rat) or DTA-1 (DTA) were purified at day 7 (A) and day 37 (B) and tested in IFN-? ELISPOT assays against
EL4 cells pulsed with peptides or against syngeneic B16 tumor pretreated with Con A supernatant. A, CD4?T cell (left panel) and CD8?T cell (right panel)
IFN-? spots at day 7 postchallenge from unchallenged mice (naive; n ? 2 mice), mice challenged with B16 and treated with rat IgG control Ab (rat; n ?
3), and mice challenged with B16 and treated with DTA-1 (DTA-1; n ? 3). B, CD8?T cell IFN-? spots at day 37 postchallenge, from unchallenged mice
(naive; n ? 1), challenged mice treated with rat IgG control Ab (rat; n ? 2), and challenged mice treated with DTA-1 (DTA-1; n ? 4).
DTA-1-treated mice that reject B16 recognize melanoma differentiation Ags. T cells from naive mice (naive) or mice challenged with 3 ?
6439The Journal of Immunology
T cells at the effector arm of tumor rejection, as our results also
demonstrate, it was unexpected to identify B16 rejection mediated
by CD4?T cells derived from DTA-1-treated mice. Studies have
shown that CD4?T cells that infiltrate tumors contain perforin
granules and have the potential to kill by Fas/FasL system (36, 37).
Because class II Ags are expressed in different solid malignancies
including melanoma (38, 39), CD4?T cells may target these tu-
mors directly and secrete IFN-? to activate macrophages that reject
the tumor. Alternatively, in antitumor responses against class II-
negative tumors, the melanoma tumors may be cross-presented by
host APC that activate CD4?T cells. Once activated in the lymph
node, CD4?T cells migrate back to the tumor, where they become
activated by macrophages that present class II-restricted tumor
Ags. Once locally reactivated, CD4?T cells generate IFN-?-ac-
tivating macrophages and NK for tumor lysis (40). A similar IFN-
?-dependent mechanism may operate in GITR-mediated CD4?re-
jection of B16. Although IFN-? up-regulates the expression of
class I and induces the expression of class II on B16 cells cultured
in vitro, expression of class I and class II in B16 cells harvested
from tumor-bearing mice is heterogeneous for both molecules
(data not shown). Because the recipients of the immunized T cells
were Rag 1?/?mice, it is possible that tumor-specific CD4?T
cells migrate to the tumor, become reactivated, secrete IFN-?, tar-
get the tumor directly, and/or activate macrophages and NK cells
that eradicate B16. Importantly, because the purified cells used in
the adoptive transfer were not depleted of CD25?cells before
transfer into Rag 1?/?recipients, the CD4?immune cells con-
tained normal numbers of Treg. These mice rejected B16, indicat-
ing that CD4?effector cells can induce tumor rejection even in the
presence of Treg. These results are consistent with a previous
study in which three mice that were adoptively transferred with
CD4?T cells from donors immunized with B16 and treated with
cells and treated with a 1 mg/mouse of rat IgG control Ab or DTA-1 at days ?1 and ?4 were harvested at day 7 and analyzed by flow cytometry. A, Splenic
T cells gated on CD3?, CD4?, or CD8?, and CD44high. B, Analysis of T cells gated on CD3?, CD4?, or CD8?, comparing CD44 vs CD62L expression.
C, Percentage of T cells gated on CD44?CD62L?(black), CD44?CD62L?(light gray), CD44highCD62L?(dark gray), and CD44highCD62L?(white) in
the right panel. D, Inguinal draining lymph nodes (DLN) and nondraining lymph nodes (NDLN) from animals treated with rat IgG control Ab or DTA-1
were harvested 7 days after tumor challenge, pooled, and analyzed by flow cytometry. Data are representative of two independent experiments (n ? 4 mice
per group). ?, p ? 0.05.
GITR stimulation increases CD4?and CD8?T cell activation. Spleens from C57BL/6 mice intradermally challenged with 3 ? 104B16
6440 GITR ACTIVATION ENHANCES ANTITUMOR IMMUNITY
anti-CD25, rejected tumors (3). Because our results indicate that
IFN-? and FasL are required for DTA-1-mediated B16 rejection,
these cytolytic mechanisms, especially FasL, may be involved in
CD4?rejection of B16. Future adoptive transfer experiments us-
ing CD4?T cells deficient in FasL or other effector molecules will
elucidate the pathways involved in CD4?DTA-1-mediated tumor
Our data indicate an important role for IFN-? in B16 rejection,
consistent with its role in immunosurveillance and tumor rejection
(31). Although IFN-? produced by CD8?T cells responding to
CTL epitopes inhibit the generation/activation of CD4?CD25?
Tregs (41), recent studies have indicated that IFN-? production by
Treg is required for suppression (42–44). Our results demonstrate
that the predominant role for IFN-? in GITR-dependent tumor re-
jection is on effector T cells, because GITR stimulation does not
protect IFN-??/?hosts from B16 challenge.
Finally, we hypothesize that priming against tumors occurs and
is strongly enhanced by GITR stimulation. We believe that GITR
stimulation may enhance antitumor immunity through a very po-
tent costimulatory signal on effector cells, which allows for its
remarkable activity against nonimmunogenic aggressive tumors
such as B16. This strong costimulation of effector T cells allows
for long-term memory to be generated. Although other groups
have determined that GITR stimulation inhibits Treg suppression
(9–12), our experiments do not demonstrate that GITR stimulation
abrogates Treg suppression, but clearly demonstrate that GITR co-
stimulation of effector T cells allows for rejection of the aggressive
B16 melanoma line. Future experiments with T cells deficient in
GITR (GITR?/?) will demonstrate whether tolerance/ignorance is
broken through a strong costimulatory signal provided by GITR
stimulation to the effector T cells or whether, as other groups have
determined, it is due to GITR stimulation on Treg that abrogates
Overall, our results in preclinical mouse models for cancer re-
jection indicate that systemic GITR stimulation breaks self-toler-
ance/ignorance, allows for recognition of tumor Ags, and induces
tumor rejection. GITR stimulation is a potent and promising strat-
egy to enhance tumor immunotherapy without the detrimental ef-
fect of autoimmunity.
T. Ramirez-Montagut thanks Humilidad Gallardo and Nathalie E. Blachere
for helpful comments and Robert B. Darnell for his support in the elabo-
ration of this manuscript.
The authors have no financial conflict of interest.
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