icos promotes the function of cd4+ effector t cells during anti … · 2016. 5. 4. · further, it...
TRANSCRIPT
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ICOS promotes the function of CD4+ effector T cells during anti-OX40 mediated tumor rejection
Todd C. Metzger1*, Hua Long1, Shobha Potluri1, Thomas Pertel1, Samantha L. Bailey-
Bucktrout1, John C. Lin1, Tihui Fu2, Padmanee Sharma2,3, James P. Allison2, Reid M.R.
Feldman1*
1Rinat Laboratories, Pfizer Inc., South San Francisco, CA 2Department of Immunology, 3Department of Genitourinary Medical Oncology, University of
Texas MD Anderson Cancer Center, Houston, TX
*Corresponding Authors: Todd Metzger, [email protected], Reid Feldman,
[email protected], Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San
Francisco, CA 94080, (650)615-7429
Disclosure of Potential Conflicts of Interest
J. Lin has ownership interest (including patents) in Pfizer Inc. P. Sharma serves on the scientific advisory boards of and has ownership interest in Jounce Therapeutics and Kite Pharmaceuticals, and consults for Bristol-Myers Squibb, GlaxoSmithKline, Amgen, and AstraZeneca. J.P. Allison serves on the scientific advisory boards of and has ownership interest in Jounce Therapeutics, Neon Therapeutics, and Kite Pharmaceuticals, is a licensor of intellectual property to Bristol-Myers Squibb, Jounce Therapeutics, and Merck, and receives royalties from Bristol-Myers Squibb and Merck. Pfizer provides research support to the M.D. Anderson Immunotherapy Platform as part of an alliance partnership agreement. No potential conflicts of interest were disclosed by other authors.
Running Title: ICOS promotes anti-OX40-mediated tumor immunity
Keywords: ICOS, costimulation, immunotherapy, OX40
Word Count: 2,999
Figures: 3
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Abstract
ICOS is a T cell co-regulatory receptor that provides a costimulatory signal to T cells
during antigen-mediated activation. Antitumor immunity can be improved by ICOS-targeting
therapies, but their mechanism-of-action remains unclear. Here we define the role of ICOS
signaling in antitumor immunity using a blocking, non-depleting antibody against ICOS ligand
(ICOS-L). ICOS signaling provided critical support for the effector function of CD4+ Foxp3- T
cells during anti-OX40-driven tumor immune responses. By itself, ICOS-L blockade reduced
accumulation of intratumoral T regulatory cells (Treg), but it was insufficient to substantially
inhibit tumor growth. Further, it did not impede antitumor responses mediated by anti-4-1BB-
driven CD8+ T cells. We found that anti-OX40 efficacy, which is based in Treg depletion and to a
large degree on CD4+ effector T cell (Teff) responses, was impaired with ICOS-L blockade. In
contrast, the provision of additional ICOS signaling through direct ICOS-L expression by tumor
cells enhanced tumor rejection and survival when administered along with anti-OX40 therapy.
Taken together, our results showed that ICOS signaling during antitumor responses acts on both
Teff and Treg cells which have opposing roles in promoting immune activation. Thus, effective
therapies targeting the ICOS pathway should seek to promote ICOS signaling specifically in
effector CD4+ T cells by combining ICOS agonism and Treg depletion.
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Introduction
ICOS was originally identified as a marker of T cell activation (1), and has since been
found to have important roles in T cell proliferation and cytokine secretion (2). The role of
ICOS in supporting follicular T cell-dependent germinal center responses has been well
documented (3-5), but its contribution to the anti-tumor immune response remains unclear.
Initial studies found that ICOS-L transfection of tumor cells led to increased tumor rejection (6,
7) and resulted in subsequent immunity upon re-exposure to the same tumor (7). More recent
work has found that ICOS-L transfection of B16 tumor cells can promote enhanced anti-tumor
immunity when such cells are irradiated and used for vaccination alongside anti-CTLA-4
treatment (8), and experiments with wild-type or ICOS-/- tumor-bearing mice have shown a
requirement for ICOS signaling to support effective anti-CTLA-4 immunotherapy (9).
Additional studies have found that recombinant ICOS-L can also promote anti-tumor immune
responses (10, 11), although the strong FcR-binding affinity of some of these molecules makes it
difficult to distinguish whether they act through manipulation of ICOS signaling or direct
antibody-dependent cellular cytotoxicity (ADCC)-based depletion of ICOS+ populations.
However, ICOS can support both effector and regulatory populations (12), and in contrast to the
above studies, clinical observations suggest that ICOS promotion of immunosuppressive Tregs
may impair tumor immunity (13), while human melanoma cells have been shown to support the
induction of Tregs in vitro through ICOS-L expression (14). Here, we investigate the role of ICOS
in the context of CD4+ T cell-dependent anti-OX40 immunotherapy, and find a critical role for
ICOS in supporting CD4+ effector T cell function during anti-tumor immune responses.
Combination therapies with ICOS agonism and anti-OX40-based Treg depletion demonstrate the
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potential to enhance anti-OX40 efficacy through provision of additional ICOS signaling and
support the pairing of ICOS agonist therapies with Treg-depleting modalities for immunotherapy.
Materials and Methods
Mice and cell lines
7-8 week old female Balb/c mice were purchased (JAX #000651or Harlan 047) for CT26
tumor growth and flow cytometry experiments. Six week old C57BL/6 mice (Charles River)
were used for MB49 implantation. Balb/c.Foxp3-GFP mice (JAX #006769) were used for RNA
seq analysis. All Balb/c mice were maintained at an AAALAC-approved facility at Rinat, and
studies were conducted according to protocols approved by the Institutional Animal Care and
Use Committee of Rinat, Pfizer Inc.
CT26.wt cells were purchased from ATCC in 2014 and used with minimal passaging.
IMPACT testing for pathogens was performed at the Research Animal Diagnostic Laboratory.
No additional authentication was performed. The chemically-induced murine bladder carcinoma
MB49 cell line (15) was kindly provided by Dr. Ashish Kamat (MDACC) in 2008 and used
without further authentication. CT26.iICOS-L cells with stable integration of a tet-inducible
mouse ICOS-L expression cassette were generated with standard lentiviral transfection of
ATCC-derived CT26.wt cells as described in the Supplementary Materials and Methods. ICOS-
L expression was induced in vivo by oral gavage of doxycycline (2 mg / dose, in H2O), or
through doxycycline chow (Harlan Teklad, TD.01306).
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Tumor growth and treatment
For CT26 experiments, Balb/c mice were anesthetized and inoculated with 5 or 10 x 104
CT26.wt or CT26.iICOS-L cells as indicated by subcutaneous injection. Mouse health was
monitored by weight assessment and visual inspection. Tumor volumes were assessed by digital
calipers twice weekly (V = 0.5 L x W2), and mice were euthanized when tumors reached the
primary endpoint of 2000 mm3; date of euthanasia was used to generate survival plots.
Antibodies and dosing are further described in the Supplementary Materials and Methods.
Flow Cytometry
Spleens were prepared by manual dissociation followed by ACK lysis of erythrocytes.
Tumor-infiltrating lymphocytes were isolated by mincing tumors followed by digestion with an
enzyme cocktail (Miltenyi, 130-096-730) using a Miltenyi OctoDissociator. Single cell
suspensions were stained with LIVE/DEAD fixable Blue kit (L-23105, Life Technologies),
followed by addition of 2.4G2 Fc receptor block and surface antibodies as described in the
Supplementary Materials and Methods. Stained cells were fixed and permeabilized with the
eBioscience Foxp3 Buffer Set (00-5523-00), followed by intracellular staining for Foxp3 and/or
Ki67, data collection on a BD LSRII, and analysis by FlowJo (TreeStar Inc.).
Statistical Analysis
Dot plots show mean +/- SD; tumor growth curves display mean +/- SEM. All analyses
were performed on GraphPad Prism 6, and statistical significance was determined by unpaired
parametric t-tests with Welch’s correction unless otherwise indicated; p values less than 0.05
were considered significant.
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Results and Discussion
Agonist antibodies against TNF family receptors are known to promote tumor rejection in
mouse syngeneic tumor models (16), but the primary cellular targets of such therapies vary
between receptors: Evidence suggests that anti-OX40 therapy requires direct interaction with
CD4+ T cell populations to mediate its anti-tumor efficacy (17), while anti-4-1BB therapy
depends only on CD8+ T cell responses (18). Prior work has shown that, in addition to driving
signaling through the OX40 receptor, the anti-mouse OX40 clone OX86 also exerts its anti-
tumor effects in part through FcR engagement and Treg depletion (19). We obtained the OX86
sequence, re-formatted the anti-OX40 antibody to contain either a mouse IgG1 (OX86-g1) or
mouse IgG2a (OX86-g2a) Fc region, and confirmed that strong FcR engagement by OX86
enhances tumor regression, as OX86-g2a promoted more frequent tumor rejection than OX86-g1
in CT26 syngeneic tumor models (Fig. 1A). Furthermore, dramatic tumor Treg depletion was
observed following treatment of tumor-bearing mice with the mouse IgG2a variant of OX86,
while OX86-g1 did not significantly affect Treg frequencies within the tumor (Fig. 1B,
Supplementary Fig. S1). Lastly, we confirmed that CD4+ T cells play a critical role in driving
the anti-tumor efficacy of anti-OX40 by showing that CD4+ depletion (Supplementary Fig. S2)
significantly impaired the ability of anti-OX40 to promote survival of CT26 (Fig. 1C) and MB49
(Fig. 1D) tumor-bearing mice. An important role for CD8+ T cells was also observed in these
experiments. However, while CD8+ T cells may respond directly to OX40 signaling (20), a role
for CD4+ T cells is consistent with prior studies which have demonstrated that CD8+ T cell anti-
tumor responses require anti-OX40-driven CD4+ T cell help to carry out optimal anti-tumor
responses (21).
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We next sought to determine whether a combination immunotherapy strategy targeting
ICOS and OX40 might drive enhanced tumor efficacy, as ICOS is known to have a key role in
supporting the efficacy of Treg-depleting anti-CTLA-4 therapy (8, 9, 22, 23). We first examined
patterns of ICOS expression in mouse syngeneic tumors, and found that ICOS surface expression
was highly upregulated within the tumor microenvironment (Fig. 2A). Among tumor-resident T
cells, ICOS was expressed most highly by Tregs, but was also upregulated across CD8+ and CD4+
effector populations at the protein and transcript level (Fig. 2A, 2B) when compared to splenic T
cells. Anti-CTLA-4 can drive increased ICOS expression on T cells in clinical trials (24, 25),
and the upregulation of ICOS on peripheral T cells correlates with clinical responses to anti-
CTLA-4 (25). We therefore examined expression of ICOS on peripheral and CT26 tumor-
derived T cells from mice that had received treatment with either PBS or anti-OX40, and found
that OX40 stimulation alone was sufficient to drive an increase in the frequency of ICOS
expression among all peripheral T cell subsets (Fig. 2C, Supplementary Fig. S3). While ICOS
was already expressed at a high frequency in baseline CT26 tumors, we were also occasionally
able to observe an increase in ICOS expression among CD4+ effector T cells within the tumor as
well (Fig. 2D). Taken together, these results suggest that targeting the ICOS pathway may
further boost the efficacy of anti-OX40 therapy.
We next sought to understand the effects of modulating ICOS-dependent signaling. To
selectively perturb ICOS:ICOSL signaling we generated and used a novel rat anti-mouse ICOS-L
antibody reformatted to contain a mouse IgG1 Fc. Treatment of tumor-bearing mice with our
ICOS-L-blocking antibody resulted in a significant increase in ICOS surface expression across
all tumor-derived T cell subsets (Fig. 3A), suggesting that ICOS signaling is tightly regulated
within the tumor microenvironment. We found minimal evidence that blockade of ICOS-L alone
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was sufficient to alter the progression of the tumor immune response (Supplementary Fig. S4A),
although modestly lower tumor volumes were observed across multiple experiments
(Supplementary Fig. S4B). However, when we combined ICOS-L blockade with anti-OX40
therapy, we observed a significant inhibition of the ability of anti-OX40 treatment to promote
expansion of proliferating, ICOS+ CD4+ effector T cells (Fig. 3B), while the effect on Foxp3+
Tregs was less robust (Supplementary Fig. S4C). Both high and low FcR-binding variants of the
OX86 clone promoted ICOS-dependent expansion of ICOS+ Ki67+ Teff (Supplementary Fig.
S4D), suggesting that OX40 agonism was sufficient to drive CD4+ Teff expansion.
We next addressed whether the ability of ICOS-L blockade to inhibit CD4+ Teff
expansion would impair anti-OX40 therapy, and found that co-treatment of CT26 tumor-bearing
mice with anti-OX40 and ICOS-L blockade resulted in a significant reduction in overall survival
when compared with anti-OX40 alone (Fig. 3C). Importantly, ICOS-L blockade alone was
sufficient to drive reduction of tumor Tregs (Supplementary Fig. S4E), which were further
depleted by co-treatment with OX86g2a, suggesting that inhibition of anti-OX40 efficacy by
ICOS-L blockade is not a result of enhanced Treg-based immunosuppression, but instead derives
from impaired ICOS-dependent CD4+ Teff responses. In contrast to anti-OX40, an agonist
antibody against TNF family member 4-1BB which is able to drive extensive tumor regression in
the CT26 syngeneic tumor model (Supplementary Fig. S4F, S4G), did not promote expansion of
ICOS+ CD4+ effectors in either the presence or absence of ICOS-L blockade (Fig. 3B).
Furthermore, CD8+ T cell-dependent anti-4-1BB-mediated tumor rejection was not impaired by
ICOS-L blockade (Supplementary Fig. S4F, S4G), and ICOS-L blockade alone did not impair
IFN-γ production by tumor-derived CD8+ T cells (Supplementary Fig. S4H), suggesting that
ICOS signaling may be dispensable for CD8+ T cell-directed immunomodulators but required to
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support the function of CD4+ Teff dependent tumor rejection. Finally, the importance of ICOS
signaling in supporting anti-OX40-mediated anti-tumor efficacy led to us to address whether
provision of additional ICOS signaling could enhance the function of anti-OX40 therapies.
Towards this end, we created a modified version of the CT26 cell line with doxycycline-
inducible ICOS-L expression (CT26.iICOS-L) and confirmed that these modified cells expressed
ICOS-L in a doxycycline-controlled manner (Fig. 3D). While we had previously found that
ICOS-L blockade inhibited the activity of anti-OX40, we observed that provision of additional
ICOS ligand within the tumor microenvironment together with anti-OX40 promoted enhanced
tumor rejection and survival when compared with anti-OX40 treatment alone (Fig. 3D).
Importantly, doxycycline administration to mice bearing wild-type CT26 tumors did not alter the
effects of anti-OX40 treatment (Supplementary Fig. S4I), and rejection of tumors by dual OX40
treatment and ICOS-L induction continued to be mediated by both CD4+ and CD8+ Teff
responses (Supplementary Fig. S4J).
In summary, these results show that ICOS is highly upregulated within the tumor
microenvironment, and expressed across T cell subsets with opposing functions. Thus, simple
manipulation of ICOS signaling will likely have opposing effects of suppressive and activating
arms of the T cell response, and helps to explain the lack of anti-tumor efficacy by ICOS-L
blockade alone. However, our data suggests that depletion of Tregs in conjunction with ICOS
agonism may remove the potential for ICOS signaling to promote immunosuppressive Tregs
responses and allow ICOS agonism to act solely in promoting activity of CD4+ Teff. It should be
noted that within the tumor, ICOS is expressed most highly by tumor Tregs, and thus a single
ICOS agonist with strong Fc engagement may be sufficient to drive simultaneous ADCC-
mediated depletion of Tregs and agonist-based enhancement of Teff responses, a pattern of activity
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which seems to be achieved by OX86 therapy (19). Additionally, while the potential for
combination therapy of anti-ICOS and anti-CTLA-4 has been well described (8, 9), the results
from these studies also support combination of ICOS agonism with other Treg-depleting therapies
such as those targeting OX40 and GITR (26).
Author’s Contributions
Conception and design: T. Metzger, J. Lin, R. Feldman
Development of methodology: T. Metzger, R. Feldman, H. Long
Acquisition of data (provided animals, acquired and managed patients, provided facilities,
etc.): T. Metzger, H. Long, T. Pertel, T. Fu
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational
analysis): T. Metzger, H. Long, S. Potluri, J. Lin, T. Fu, J. Allison, R. Feldman
Writing, review, and/or revision of the manuscript: T. Metzger, H. Long, S. Bailey-
Bucktrout, J. Lin, T. Fu, P. Sharma, R. Feldman
Administrative, technical, or material support (i.e., reporting or organizing data,
constructing databases): T. Metzger, T. Fu
Study Supervision: R. Feldman, S. Bailey-Bucktrout, P. Sharma, J. Allison, J. Lin
Acknowledgements
The authors thank Orla Cunningham at Pfizer Global Biotherapeutics (Dublin, Ireland) for the
generation and provision of in-house anti-ICOS-L antibodies.
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Figure Legends
Figure 1. Anti-OX40 tumor immunotherapy requires both CD4+ and CD8+ T cell
responses. (A) Growth curves showing individual tumor volumes over time of CT26-bearing
mice randomized at day 9 post-inoculation and treated on days 9, 13, and 16 as indicated.
Representative of three independent experiments. (B) Frequency of Foxp3+ events among tumor-
derived CD4+ T cells at 17 days post-inoculation, taken from CT26-bearing mice treated 9, 12,
and 15 days post-inoculation as indicated. (C) Treatment schematic (top) and survival plots
(bottom), based on euthanasia when CT26 tumor volumes reached 2000 mm3 endpoint. p-
values for Tx (OX40) vs. all other curves are <0.01 by Log Rank (Mantel-Cox) tests. (D)
Treatment schematic (top) and survival plots (bottom) for MB49-bearing mice treated as
indicated. Tx vs. NK depl, ns; Tx vs. CD8 depl, p = 0.135; Tx vs. CD4 depl, p = 0.0026; Tx vs.
CD4 + CD8 depl, p = 0.0015.
Figure 2. Anti-OX40 treatment induces ICOS expression on CD4+ effector T cells. (A)
Flow cytometric analysis of ICOS expression among indicated populations isolated from the
spleen or tumor of CT26 tumor-bearing mice at two weeks post-implantation. Representative of
more than 5 independent experiments. (B) RNA seq analysis of Icos mRNA transcripts from T
and NK cell populations isolated from spleens and tumors of CT26-bearing Foxp3-GFP mice
implanted 2.5-3 weeks before tissue harvest. Tconv = CD4+Foxp3-, Treg = CD4+Foxp3+. (C)
Frequency of ICOS+ events among the indicated T cell subsets isolated from the spleen of CT26
tumor-bearing mice following one week of pre-treatment with either PBS alone or the indicated
anti-OX40 antibodies. (D) Frequency of ICOS+ events among tumor Tconv (left) and CD8+ T cells
(right) from mice following the treatment regimen in (C).
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Figure 3. ICOS signaling promotes anti-OX40-mediated anti-tumor efficacy. (A) Mean
fluorescent intensities (MFI) of ICOS staining on the indicated tumor-derived T cell subsets from
mice pre-treated with an isotype control (MOPC-21) or anti-ICOS-L. (B) Frequency of ICOS+
Ki67+ events among Foxp3- CD4+ Teff from the spleens of tumor-bearing mice 15 days post-
inoculation following i.p. treatment with the indicated antibodies at days 9 and 13.
Representative of two to four experiments per condition. (C) Pooled data from two separate
experiments showing overall survival of mice with indicated treatments. p = 0.0482 by two-
tailed chi2 contingency test of survival at 37 days post-inoculation between OX86g2a +/- isotype.
(D) Flow cytometric analysis of ICOS-L expression by the indicated cell populations after 16
hours of culture in vitro (left) and survival curves for CT26.iICOS-L-bearing mice (right)
randomized at day 10 post-inoculation and treated on days 10, 13, and 17 i.p. with isotype
control or OX86-g2a, and treated with doxycycline as indicated beginning on day 10. Results
are pooled from three independent experiments. *p<0.05,**p <0.01, ***p<0.001, ****p<0.0001.
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Figure 1
B
Days 0
anti-T cell subset, i.p.
9 12 15
Monitor tumors
anti-OX40 (OX86-mIgG2a), i.p.
CT26
s.c.
100
20
40
60
80
100
Perc
ent S
urvi
val
20 30 40
Un Tx
Tx + CD8 DeplTx + CD4 Depl
Tx (OX40)
days post inoculation
A
10 20 300
500
1000
1500
2000
days post inoculation
tum
or v
olum
e (m
m3 )
OX86g1
10 20 30 40days post inoculation
0
500
1000
1500
2000
tum
or v
olum
e (m
m3 )
OX86g2a
0
500
1000
1500
2000
tum
or v
olum
e (m
m3 )
10 20 30 40days post inoculation
4/10
CDays 0
anti-NK and -T cell subset, i.p.
1 2 5 8 9 Monitor tumors
anti-OX40 (OX86-mIgG2a), i.p.
0 20 40 600
20
40
60
80
100
Perc
ent S
urvi
val
days post inoculation
Un Tx
Tx + NK DeplTx + CD8 DeplTx + CD4 Depl
Tx (OX40)
Tx + CD4/8 Depl
MB49 s.
c.
D
PBS6g
1
OX86g2a
0
20
40
60
% F
oxp3
+ o
f CD
4+
8XO
Control
***ns
**
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Figure 2A
C
PBS
OX86g1
OX86g2a
0
10
20
30
40
50
% IC
OS+
of S
pl T
regs
PBS
OX86g1
OX86g2a
0
5
10
15
20
25
% IC
OS+
of S
pl T
conv
PBS
OX86g1
OX86g2a
0
1
2
3
4
% IC
OS+
of S
pl C
D8+
T c
ells
CD4+ Foxp3+
CD4+ Foxp3-
CD8+
ICOS
% M
axSpleen Tumor
D
B
NKCD8+
Tconv
Treg NKCD8+
Tconv
Treg102
103
104
105
Spleen Tumor
Icos
rela
tive
expr
essi
on
***
**ns
****
*ns
*ns
ns
*ns
ns
010
50
60
70
80
90
100
% IC
OS+
of T
IL T
conv
PBS
OX86g1
OX86g2a
PBS
OX86g1
OX86g2a
0
20
40
60
80
100
% IC
OS+
of T
IL C
D8+
nsns
ns
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Figure 3
0
5
10
15
20
25
% IC
OS+ K
i67+ o
f Fox
p3-
Iso ICOS-L Iso ICOS-L Iso ICOS-L+ 4-1BB + OX86-g2a
****
**
nsns
**
Std IsotypeStd OX86g2aDox IsotypeDox OX86g2a
BA
C
CT26.iICOS-L 1 ug/mL DoxCT26.iICOS-L control mediaCT26.wt control media
% M
ax
ICOS-L
D
0
5000
15000
10000
Treg Tconv CD8+
Iso ICOS-L Iso ICOS-L Iso ICOS-L
*
***
**
ICO
S M
FI
0 20 400
50
100
Perc
ent s
urvi
val
*
0 20 40 600
50
100
days post tumor inoculation
Perc
ent s
urvi
val
OX86g2a +IsotypeOX86g2a +ICOS-L blockade
Isotype
ICOS-L blockade
*
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Published OnlineFirst May 4, 2016.Cancer Res Todd C Metzger, Hua Long, Shobha Potluri, et al. anti-OX40 mediated tumor rejectionICOS promotes the function of CD4+ effector T cells during
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