Published in Volume
123, Issue 6 (June 3, 2013)
J Clin Invest. 2013;123(6):2604–2615.
doi:10.1172/JCI67008.
Copyright © 2013, American Society for Clinical Investigation
Research Article
PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells
Erin E. West1, Hyun-Tak Jin1, Ata-Ur Rasheed1, Pablo Penaloza-MacMaster1, Sang-Jun Ha1, Wendy G. Tan1, Ben Youngblood1, Gordon J. Freeman2, Kendall A. Smith3 and Rafi Ahmed1
1Emory Vaccine Center and Emory University School of Medicine, Emory University, Atlanta, Georgia, USA.
2Department of Medical Oncology, Harvard Medical School, Boston, Massachusetts, USA.
3Department of Medicine, Division of Immunology, Weill Medical College of Cornell University, New York, New York, USA.
Address
correspondence to: Rafi Ahmed, 1510 Clifton Rd., Atlanta, Georgia
30322, USA. Phone: 404.727.4700; Fax: 404.727.3722; E-mail:
rahmed@emory.edu.
First published May 15, 2013
Received for publication September 24,
2012, and accepted in revised form March 21,
2013.
The
inhibitory receptor programmed cell death 1 (PD-1) plays a major role
in functional exhaustion of T cells during chronic infections and
cancer, and recent clinical data suggest that blockade of the PD-1
pathway is an effective immunotherapy in treating certain cancers. Thus,
it is important to define combinatorial approaches that increase the
efficacy of PD-1 blockade. To address this issue, we examined the effect
of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of
chronic lymphocytic choriomeningitis virus (LCMV) infection. We found
that low-dose IL-2 administration alone enhanced CD8+ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8+
T cells and increased expression of CD127 and CD44, resulting in a
phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy
had only a minimal effect on reducing viral load. However, combining
IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking
synergistic effects in enhancing virus-specific CD8+ T cell
responses and decreasing viral load. Interestingly, this reduction in
viral load occurred despite increased numbers of Tregs. These results
suggest that combined IL-2 therapy and PD-L1 blockade merits
consideration as a regimen for treating human chronic infections and
cancer.
Introduction
CD8
T cells play a key role in eliminating and intracellular infections and
tumors. However, in the setting of chronic antigen stimulation, such as
that seen in chronic infections and tumors, CD8 T cells undergo
exhaustion, causing them to become dysfunctional. This exhaustion is
characterized by decreased proliferative capacity, loss of cytokine
secretion, reduced cytotoxic killing abilities, and phenotypic changes,
including low expression of canonical memory markers, such as the IL-7
receptor α chain (CD127), and also an increase in inhibitory receptors (1–3).
While multiple mechanisms
contribute to the process of exhaustion, the inhibitory receptor
programmed cell death 1 (PD-1) has emerged as a major player in this
process. PD-1 is the most well-characterized inhibitory molecule
upregulated during chronic antigen stimulation and is associated with
disease progression and immune dysfunction (2).
Importantly, recent data from 2 clinical trials have highlighted the
role of PD-1 inhibition in human cancers and have shown that PD-1
blockade, by in vivo administration of humanized anti–PD-1 or anti–PD-1
ligand 1 (anti–PD-L1) antibodies, is an effective immunotherapeutic for
increasing tumor clearance. Notably, in vivo PD-1 blockade resulted in
durable tumor reduction or clearance in multiple cancers, including lung
cancer, which is highly refractory to any treatment (4–6).
These data correspond well with previous in vitro and in vivo animal
model data showing that PD-1 plays a central role in T cell dysfunction
during chronic infections and cancer and that PD-1 blockade can restore T
cell function (2, 3, 7–16).
Overall, these data indicate that PD-1 may be an important
immunotherapeutic for cancers and chronic infections and signify that it
is vital to find ways to increase the efficacy of PD-1 blockade.
Multiple inhibitory mechanisms regulate CD8 T cell exhaustion, and,
thus, combining PD-1 blockade along with other therapies, such as
simultaneous blockade of multiple inhibitory receptors or therapeutic
vaccination, results in enhanced reduction of viral loads and increased
CD8 T cell responses in animal models of chronic infection. However, it
is important to note that the mechanisms underlying the synergy of
combined treatments has not been well explored (17–19).
Overall, this suggests that combining strategies or treatments to
combat chronic infections and cancer may be a valid strategy to increase
efficacy.
IL-2 is a cytokine that
has a pleiotropic effect on multiple immune cell types and has been used
as a therapy for several human diseases/conditions. IL-2 has been used
to augment T cell responses against virus or tumor antigens in HIV and
patients with metastatic cancer. While high-dose intermittent IL-2
therapy has increased long-term survival for some patients with
metastatic renal cell carcinoma (20)
and IL-2 therapy alone or in combination with a peptide vaccine has
resulted in clinical improvement for patients with metastatic melanoma (21, 22),
it has shown very limited success when given during chronic human viral
infections, such as when it is combined with antiretroviral drugs
during HIV (23–28).
Greater improvement was seen in one trial, with IL-2 administration
combined with antiretroviral drugs and therapeutic vaccination during
HIV infection (29), although other small studies suggest that a long-term effect is not seen after antiviral therapy is discontinued (30–32).
However, continuous IL-2 administration, along with therapeutic
vaccination and antiretroviral treatment, in macaques infected with
chronic SIV increases SIV-specific CD8 T cell responses and results in
decreased viral burden (33, 34).
Overall, a major
limitation of high-dose intermittent IL-2 therapy is that it can result
in severe toxicity issues, such as vascular leakage. By comparison,
daily, much lower doses of IL-2 can ameliorate these toxicity issues (35).
Recently promising human data indicate that daily low-dose IL-2 therapy
may be useful for increasing Treg numbers and reducing autoimmune
complications in patients with graft-versus-host disease as a result of
undergoing an allogeneic hematopoietic stem cell transplantation (36) and also in patients with hepatitis C–induced vasculitis (37). Importantly, these recent studies indicate that daily low-dose IL-2 therapy is well tolerated by patients (36, 37).
While daily low-dose IL-2 therapy increases Tregs in the context of
autoimmune complications, in contrast, our laboratory has previously
shown that daily low-dose IL-2 treatment during chronic mouse
lymphocytic choriomeningitis virus (LCMV) infection results in enhanced
virus-specific CD8 T cell numbers and function and slightly reduces
viral burden (38).
These data indicate that daily low-dose IL-2 therapy may be beneficial
during persistent infections, but a better understanding of the action
of low-dose IL-2 therapy on exhausted T cells and its role on Treg
numbers during chronic infection is needed.
Additionally, while the
clinical data on high-dose intermittent IL-2 therapy during chronic
viral infections has not been very promising, combining IL-2 therapy
with other immunomodulatory regimens may allow for the positive effects
of IL-2 to be enhanced, while diminishing the negative toxicity issues
associated with IL-2, through the use of a daily low-dose treatment of
IL-2. Therefore, in this study, we explored the idea of combining the
positive signal of IL-2 along with the blockade of an inhibitory
pathway, PD-1, as a therapy for chronic infection. Using the mouse LCMV
model of chronic infection, we determined the effect of daily low-dose
IL-2 treatment given alone, or as a combined immunotherapy in
conjunction with PD-1 blockade, on exhausted virus-specific CD8 T cells,
Tregs, and viral control. Herein, we show that when used as a
combination therapy, IL-2 administration strikingly enhances the
effectiveness of PD-1 blockade and this combined therapy may be an
important clinical therapeutic for fighting human cancer and chronic
infections.
Results
IL-2 therapy synergizes with PD-L1 blockade to enhance virus-specific CD8 T cell control of chronic LCMV infection.
To determine the effects of IL-2 therapy, PD-L1 blockade, and combined
IL-2 therapy and PD-L1 blockade on CD8 T cells and viral control during
chronic infection, we used the chronic LCMV mouse model. Mice were
infected with the clone-13 (cl-13) strain of LCMV, which results in a
protracted viral infection with >2 months of viremia. After chronic
infection was well established, beginning at day 23 to 27 after
infection, mice were treated with anti–PD-L1 blocking antibody once
every 3 days for a total of 5 treatments, and during the last 8 days of
anti–PD-L1 treatment 15,000 IU (1 μg) of recombinant human IL-2 was
administered once daily. This regimen was decided upon after
consideration of previously published data in this model (38)
and after some titration of the dose of IL-2, as similar LCMV-specific
CD8 T cell responses and viral reduction were seen when 15,000 IU IL-2
was injected twice daily and when it was injected once daily
(Supplemental Figure 1, A–C; supplemental material available online with
this article; doi:
10.1172/JCI67008DS1). Administration of IL-2 or anti–PD-L1 alone increased LCMV-specific CD8 T cells in both the blood and tissues (Figure 1,
A–C) that had an enhanced ability to produce both IFN-γ and TNF-α after
ex vivo restimulation with LCMV-specific peptides covering multiple
epitopes (Figure 1D
and Supplemental Figure 2, A and B). This increase in functional
LCMV-specific CD8 T cells after treatment with IL-2 or PD-L1 blockade
alone correlated with some reduction in viral loads, with a trend toward
PD-L1 blockade resulting in a greater reduction of viral burden than
that seen with IL-2 therapy alone (Figure 1E).
Strikingly, mice given combined IL-2 therapy and PD-L1 blockade had a
much larger expansion of LCMV-specific CD8 T cells in the blood and
tissues (~4-fold expansion for the DbGP33-41 epitope) (Figure 1,
A–C). In addition, after combined IL-2 therapy and PD-L1 blockade,
production of IFN-γ and coproduction of IFN-γ and TNF-α by CD8 T cells
after ex vivo stimulation with LCMV-specific peptides were greatly
increased in response to multiple epitopes, including the previously
undetectable NP396 epitope (Figure 1D).
These data indicate the expansion of CD8 T cells with a broader
response, which may help reduce the selection of viral escape mutants.
This effect was distinct compared with that seen with either treatment
given alone, as IL-2 or PD-L1 blockade alone did not enhance the NP396
response (Figure 1D).
Last, combined IL-2 therapy and PD-L1 blockade resulted in undetectable
viral loads in the majority of mice, indicating faster viral control
than that seen with either treatment given alone (Figure 1E).
These data indicate that IL-2 therapy synergizes with PD-L1 blockade,
resulting in a greatly expanded and functional LCMV-specific CD8 T cell
response to multiple epitopes and increased viral clearance during
chronic LCMV cl-13 infection.
IL-2
therapy and PD-L1 blockade have distinct effects on virus-specific CD8 T
cell expansion and viral loads during chronic LCMV infection.
Transient depletion of CD4 T cells in mice before infection with LCMV
cl-13, leads to a deeper exhaustion of LCMV-specific CD8 T cells and
high levels of viremia for the life of the mouse. We next tested the
ability of combined IL-2 therapy and PD-L1 blockade to rescue
LCMV-specific CD8 T cells in the absence of CD4 T cell help during
chronic LCMV infection (“unhelped” chronic infection model). To address
this, we transiently depleted mice of CD4 T cells, using an anti-CD4
depleting antibody, prior to infection with chronic LCMV cl-13.
Following 60 days after infection, when CD4 T cell numbers returned to
normal but no LCMV-specific CD4 T cells existed, we began treating the
mice with PBS/isotype control, IL-2 alone, PD-L1 blockade alone, or
combined IL-2 therapy and PD-L1 blockade. The appropriate groups were
given PD-L1 blocking antibody once every 3 days for 5 total treatments,
and 15,000 IU IL-2 was given every 12 hours i.p. continuously during the
12 days of PD-L1 blockade (Figure 2A).
This regimen of treatment was decided upon after determining that IL-2
administration given continuously during PD-L1 blockade resulted in
increased LCMV-specific CD8 T cells compared with IL-2 given early
after, in the middle of, or late after the start of PD-L1 blockade
(Supplemental Figure 3, A–D). Furthermore, administration of IL-2 every
24 hours had less effect on the LCMV-specific CD8 T cells than giving
IL-2 every 12 hours in this model of more severe CD8 T cell exhaustion
(data not shown).
Both
IL-2 treatment alone and PD-L1 blockade alone resulted in an increase
in the GP33- and GP276-specific cells in the blood and tissues (Figure 2,
B–E). However, combined IL-2 therapy and PD-L1 blockade led to a huge
expansion of LCMV-specific CD8 T cells, even as early as day 8 after the
beginning of treatment, resulting in up to approximately 30% of the CD8
T cells in the blood of some mice being specific for one LCMV epitope 2
days after the last treatment (Figure 2B) and an overall increase in both GP33- and GP276-specific CD8 T cells in the blood (Figure 2C).
In addition, combined IL-2 therapy and PD-L1 blockade resulted in a
large increase in the frequency and numbers of LCMV-specific CD8 T cells
in both lymphoid and nonlymphoid tissues at 2 days after the last
treatment (P < 0.01, P < 0.001, P < 0.5 for number of GP33+ CD8 T cells in the combined treatment group compared with PBS/isotype controls in the spleen, lung, and liver, respectively; P < 0.001, P < 0.001, P < 0.001, and P < 0.05 for number of GP276+ CD8 T cells in the spleen, lung, liver, and bone marrow, respectively) (Figure 2, D and E).
We next
determined whether these CD8 T cells that had expanded after combined
IL-2 therapy and PD-L1 blockade had also regained function. After ex
vivo restimulation with broad range of LCMV-specific peptides, we
assessed the ability of the CD8 T cells to produce cytokines after
treatment in order to address this question. While both IL-2 or PD-L1
blockade alone increased the number of CD8 T cells producing IFN-γ or
coproducing IFN-γ and TNF-α in response to a broad range of LCMV
epitopes, combined IL-2 therapy and PD-L1 blockade resulted in massive
increases (up to ~25%) in the frequency of CD8 T cells producing IFN-γ
in response to a broad range of LCMV epitopes (Figure 3A).
Notably, combined therapy resulted in striking responses to both
dominant (GP33 and GP276) and subdominant epitopes (GP118, NP205, and
NP235), including the previously undetectable epitope, NP396 (Figure 3A).
Interestingly, the distribution of IFN-γ–producing cells to different
LCMV peptides was altered in individual mice, with some mice making more
of a response to the dominant epitopes, (IL-2 + αPD-L1 no. 1 in Figure 3A) and other mice making a larger response to subdominant epitopes (IL-2 + αPD-L1 no. 2 in Figure 3A).
This difference in the breadth of the responses in individual mice may
be a reflection of the differential degree of exhaustion of T cell
subsets and/or differences in the precursor frequency in individual
mice. Furthermore, combined treatment resulted in immense increases in
the number of CD8 T cells producing IFN-γ, even up to an approximately
100-fold increase in cells responding to the GP33 epitope, and
multifunctional cells coproducing IFN-γ and TNF-α in response to a broad
range of dominant and subdominant LCMV epitopes (Figure 3, B and C).
Next,
we addressed whether the increased numbers of functional LCMV-specific
CD8 T cells seen after therapy correlated with viral reduction. First,
while IL-2 therapy alone resulted in a large expansion of LCMV-specific
CD8 T cells, there was no reduction in viral loads after treatment, but,
surprisingly, a slight increase in the levels of virus was observed
(Figure 3D).
In contrast, PD-L1 blockade alone, which results in less of an
expansion of LCMV-specific CD8 T cells compared with IL-2 therapy alone,
reduced viral loads (Figure 3D).
Importantly, combined IL-2 and PD-L1 therapy resulted in a more
significant decrease in viral load than that provided by PD-L1 blockade
alone (Figure 3D).
Therefore, IL-2 therapy synergizes with PD-L1 blockade in this model of
extreme exhaustion, resulting in a massive expansion of functional
LCMV-specific CD8 T cells and a greater reduction of viral load than
that seen with PD-L1 blockade alone.
Effectiveness of combined IL-2 therapy and PD-L1 blockade compared with that of other combination therapies. Multiple combinational strategies have been tested in the LCMV chronic infection model (17–19, 39). In the past, in our laboratory, we tested 3 other combined modalities: therapeutic vaccination combined with PD-L1 blockade (19), coblockade of IL-10R and PD-L1 (39), and combined Tim-3 and PD-L1 blockade (17).
Comparison of our new IL-2 and anti–PD-L1 combined therapy data with
these other previously published data indicate that IL-2 and anti–PD-L1
therapy has a more impressive effect on increasing the functional
LCMV-specific CD8 T cell response and decreasing viral titers than these
other treatments during chronic viral infection.
In addition,
to control for the variable of the treatments (mentioned above) not
being directly compared within the same experiment, we performed a new
experiment directly comparing IL-2 therapy and anti–PD-L1 blockade side
by side with the previously published combined blockade of Lag-3 and
PD-L1 (18).
We accomplished this by transiently treating CD4-depleted
cl-13–infected mice (after day 100 after infection) with these two
combination therapies. IL-2 plus anti–PD-L1 treatment resulted in
approximately 10- to 20-fold greater increase in virus-specific CD8 T
cells in the tissues, spleens, livers, and lungs of mice infected with
chronic LCMV compared with that in the group receiving combined Lag-3
and PD-L1 blockade (Supplemental Figure 4A). Furthermore, these
virus-specific CD8 T cells were highly functional in the IL-2 plus
anti–PD-L1 group, evidenced by a significant (P < 0.05)
increase in the number of IFN-γ–producing cells in the IL-2 plus
anti–PD-L1 group and increased double-producing IFN-γ+TNF-α+
cells compared with the anti–Lag-3 plus anti–PD-L1 group (Supplemental
Figure 4, B and C). Last, combined IL-2 therapy and PD-L1 blockade
resulted in an approximately 10-fold greater reduction of viral burden
than anti–Lag-3 plus anti–PD-L1 treatment (Supplemental Figure 4D).
Taken together, these data suggest that combined IL-2 therapy and
anti–PD-L1 blockade may be a more effective therapy than previously
published combined therapies during chronic antigen persistence.
IL-2 and combined therapy influences Treg numbers and activation markers.
Thus far, we have focused on the LCMV-specific CD8 T cell responses
after treatment; however, since Tregs express constitutively high levels
of the high-affinity IL-2Rα chain and IL-2 is known to expand their
numbers and increase their suppressive function (40, 41) and low-dose IL-2 treatment in humans has been shown to increase Treg numbers (36, 37), we assessed the Treg compartment (FoxP3+
CD4 T cells) after administration of IL-2 or PD-L1 blockade alone and
after combined IL-2 therapy and PD-L1 blockade during chronic LCMV
infection. IL-2 administration alone resulted in an increase in the
frequency of FoxP3+ CD4 T cells in the blood during ongoing
treatment (day 8 after the start of treatment) and also after the
completion of treatment (day 14 after the start of treatment) (Figure 4, A and B). Furthermore, upon the completion of treatment, there were increased FoxP3+ CD4 T cells in the tissues (P < 0.01 for spleen and liver; P < 0.05 for lung compared with PBS/isotype control group) (Figure 4C). In contrast, PD-L1 blockade alone did not significantly affect the frequency of FoxP3+ CD4 T cells in the blood or their numbers in the tissues (Figure 4, A–C). Combined IL-2 therapy and PD-L1 blockade resulted in an increase in the frequency of FoxP3+ CD4 T cells in the blood (Figure 4, A and B) and increased numbers in the tissues (P < 0.01 for spleen and P
< 0.05 in liver compared with PBS/isotype control); however, this
increase was very similar to that seen after administration of IL-2
therapy alone (Figure 4C). This indicates that IL-2 expands the FoxP3+ CD4 T cell population, but PD-1 blockade does not. To determine whether these FoxP3+
CD4 T cells had a more activated phenotype after treatment, we assessed
CD44, CD25, CD103, and GITR expression on these cells after treatment
by measuring the MFI of these markers using flow cytometry. CD44, CD25,
CD103, and GITR expression was increased on the FoxP3+ CD4 T cells after IL-2 or combined IL-2 plus PD-L1 blockade, as assessed by MFI, indicating that these FoxP3+ CD4 T cells had a more activated phenotype (Figure 4D). In contrast, PD-L1 blockade alone only very slightly affected the expression of these markers (Figure 4D). These data indicate that IL-2 therapy increases the number of “activated” Foxp3+
CD4 T cells during chronic infection, unlike PD-1 blockade. However, in
spite of the increase in activated Tregs after combined treatment,
virus-specific CD8 T cell numbers and function were augmented and virus
levels were reduced after treatment (Figures 1–3).
IL-2 modulates existing exhausted virus-specific CD8 T cells. Since exhausted CD8 T cells have low surface expression of the high-affinity IL-2Rα chain (42),
it is unclear how IL-2 is modulating these CD8 T cells. To better
understand whether IL-2 may modulate existing exhausted virus-specific
CD8 T cells, we transferred Thy1.1+ LCMV-specific DbGP33-41 transgenic TCR (P14) T cells into Thy1.2+ B6 mice, infected them with LCMV cl-13, and then assessed the P14 T cell numbers and IL-2 signal transduction in the Thy1.1+
P14 cells after establishment of chronic infection (day 23–27) by
measuring the level of STAT-5 phosphorylation (phospho-STAT-5) after in
vivo treatment with IL-2 (Figure 5A).
Following 6 days of daily IL-2 administration, the frequency of P14 T
cells in the blood was increased from approximately 0.3% of CD8 T cells
before treatment to approximately 13% after treatment, indicating a
large expansion of the P14 T cells in the blood after IL-2 therapy
(Figure 5B).
In addition, there was an approximately 4-fold increase in the number
of P14 T cells in the spleens of mice treated with IL-2 for 6 days
compared with those treated with PBS (Figure 5C).
This indicates that IL-2 causes the expansion of preexisting exhausted
CD8 T cells and does not act just on newly primed CD8 T cells coming
from the thymus during the ongoing infection. Second, to address whether
IL-2 may act directly on exhausted LCMV-specific CD8 T cells, we
assessed phospho-STAT-5 staining after IL-2 administration. Notably,
STAT-5 remained unphosphorylated 60 minutes after 1 injection of IL-2
(data not shown). However, when 15,000 IU IL-2 was administered i.p.
every 24 hours for 6 days and then on the seventh day either PBS or IL-2
was given to the mice followed by analysis of the P14 T cells 30
minutes after IL-2 injection, striking increases of phospho-STAT-5
expression were seen in the P14 T cells of mice given IL-2 30 minutes
prior (~40% are phospho-STAT-5+ after 30 minutes) (Figure 5,
D–F). These data are consistent with IL-2 signaling directly on the
LCMV-specific CD8 T cells; however, they do not exclude the effect that
IL-2 therapy may also have on other cell types during chronic infection
or a possible additional nondirect effect of therapy on the
LCMV-specific CD8 T cells. Notably, the IL-2 receptor components, IL-2Rβ
(CD122) and IL-2Rγ (CD132) were expressed by LCMV-specific T cells
(Figure 5G);
however, these cells did not express IL-2Rα, except for a small
population (8% or less) in some mice treated with IL-2 therapy (Figure 5G),
consistent with the idea that IL-2 may be able to act directly on
LCMV-specific CD8 T cells mostly via the lower-affinity IL-2 receptor
(IL-2Rβ plus IL-2Rγ). Taken together, these data indicate that IL-2 can
modulate existing exhausted virus-specific CD8 T cells.
IL-2 decreases inhibitory receptors and increases CD127 expression on exhausted virus-specific CD8 T cells.
We have shown that IL-2 can modulate existing exhausted CD8 T cells, so
we next determined whether IL-2 changes the expression of markers known
to be important in the exhaustion process. Multiple inhibitory
receptors are expressed on CD8 T cells during chronic infection and have
been shown to play a central role in inhibiting their function (2, 43);
so to begin, we first assessed the expression of the inhibitory
receptors PD-1, 2B4, Tim-3, and Lag-3 on exhausted CD8 T cells after
IL-2 therapy. LCMV-specific P14 TCR transgenic cells congenically marked
with Thy1.1 were transferred into mice that were subsequently infected
with LCMV cl-13. After chronic infection was established, at 27 days
after infection, the mice were treated with either PBS or 15,000 IU IL-2
every 24 hours for 8 days, and the following day, the expression of an
activation marker, CD44, and the inhibitory receptors on the P14 T cells
was assessed by flow cytometry. The P14 transgenic system was used to
show how IL-2 acts on preexisting exhausted cells (eliminating the
contribution of any newly primed CD8 T cells); however, similar results
were also seen with endogenous LCMV-specific CD8 T cells (data not
shown). Furthermore, IL-2 treatment was given alone, not in combination
with anti–PD-L1, so that virus loads would still be high, thereby
helping to eliminate the impact of viral load on inhibitory receptor
expression. The activation molecule CD44 was substantially increased on
the LCMV-specific CD8 T cells after IL-2 treatment (~2-fold increase in
MFI) (Figure 6A).
Interestingly, there was a large decrease in the inhibitory receptors
PD-1, 2B4, and Tim-3, as measured by MFI, after IL-2 treatment. In
contrast, Lag-3 was not altered by IL-2 treatment (Figure 6A).
In addition to this decrease in expression of the inhibitory receptors
on P14 T cells after IL-2 therapy, we found that these cells expressed
higher levels of the transcription factor T-bet (Figure 6B).
A recent paper by Kao et al. showed that LCMV-specific CD8 T cells that
expressed high levels of the transcription factor T-bet had decreased
expression of inhibitory receptors and increased functional capabilities
during chronic infection (44).
These data indicate that IL-2 can influence inhibitory receptor
expression on exhausted CD8 T cells during chronic LCMV infection, and,
thus, this may help lead to decreased inhibition of these cells.
Second,
we assessed the expression levels of 2 other markers, Bcl-2 and
granzyme B, which have been shown to be increased in CD8 T cells after
in vivo or in vitro IL-2 administration (45–49).
Bcl-2 is a known negative regulator of apoptosis and is important for T
cell survival, and while IL-2 has been shown to increase Bcl-2
expression in CD8 T cells (47–49),
we found that Bcl-2 expression was similar on LCMV-specific CD8 T cells
after IL-2 and PBS treatment during chronic infection (Figure 6C).
In contrast, granzyme B expression was increased in the LCMV-specific
CD8 T cells after IL-2 therapy during chronic infection (Figure 6C), indicating that these cells may have increased cytolytic potential.
Last, we
looked at the expression of the IL-7 receptor α chain (CD127) on
exhausted CD8 T cells after treatment with IL-2 or PD-L1 blockade or
combined IL-2 therapy and PD-L1 blockade. CD127 is a CD8 T cell marker
that helps define functional memory cells in acute infection, as the
responding CD8 T cells that reexpress CD127 are preferentially destined
to become memory CD8 T cells (50). However, in contrast, exhausted CD8 T cells do not reexpress this memory marker during chronic infection (51),
and as of yet there is not a reported treatment that increases CD127
expression on CD8 T cells during chronic infection. Interestingly, we
found that CD127 was upregulated on the LCMV-specific CD8 T cells after
IL-2 therapy alone or after combined IL-2 therapy and PD-L1 blockade
treatment during chronic LCMV infection (Figure 6D).
This increase in CD127 expression was not due to viral clearance, as
mice treated with only IL-2 alone (which does not result in viral
clearance) also had increased expression of CD127 (Figure 1E and Figure 6D).
In addition, in the “unhelped” model of chronic LCMV infection, in
which viremia is maintained at high levels for the life of the animal,
only combined IL-2 therapy and PD-L1 blockade resulted in increased
CD127 expression (data not shown). This increased expression of CD127
may indicate a reprogramming away from the exhausted state and may lead
to increased cell survival.
Discussion
Herein
we show that combined daily low-dose IL-2 therapy and PD-L1 blockade
enhance CD8 T cell responses and function during chronic LCMV infection
and result in decreased viral burden. The effects of the combined
therapy were greater than those of either treatment given alone. To
better understand the way in which IL-2 affected the T cell responses,
we treated mice solely with daily low-dose IL-2 therapy. IL-2
administration alone was shown to modulate CD8 T cells, causing the
phosphorylation of STAT-5. Furthermore, daily low-dose IL-2 therapy
resulted in a decrease in the expression of the inhibitory receptors
Tim-3, PD-1, and 2B4 (CD244). This reduction in inhibitory receptor
expression was seen, even though IL-2 treatment alone does not clear
virus, indicating that IL-2 therapy can reduce their expression, even in
the presence of high levels of virus. In addition to a reduced
expression of inhibitory receptors, we found that the activation
molecule CD44 was upregulated after IL-2 therapy. CD8 T cells that have
increased CD44 expression and intermediate PD-1 expression have been
shown to have greater proliferative potential, increased ability to
control infections, and are more responsive to PD-1 blockade than their
counterparts with intermediate CD44 and high PD-1 high expression (52).
This suggests that IL-2 administration may result in the generation of a
pool of CD8 T cells that are more responsive to PD-L1 blockade.
Furthermore, a recent report showed that antigen-specific CD8 T cells
expressing high levels of T-bet were more functional during chronic LCMV
infection and had reduced inhibitory receptor expression (44),
and our data show that IL-2 treatment increases T-bet expression. Last,
IL-2 increased granzyme B expression, consistent with enhanced
cytolytic potential.
Importantly, IL-2
treatment alone increased IL-7 receptor α expression (CD127), which is a
marker of long-lived highly functional memory CD8 T cells that has not
previously been shown to be upregulated on CD8 T cells during chronic
infection. This increase in CD127 expression was seen even in cases in
which antigen persisted, such as in the mice treated only with IL-2
alone, therefore it is reexpressed even in the presence of high viral
loads. Additionally, during normal chronic LCMV infection, CD127 was
induced by IL-2 alone or by combined IL-2 therapy and PD-L1 blockade;
however, in the more severe “unhelped” chronic LCMV infection only the
combined IL-2 therapy and PD-L1 blockade was able to induce CD127
expression. This interesting difference between the 2 models may be due
to the fact that the CD8 T cells are exhausted more extremely during
“unhelped” LCMV infection and therefore need more immunomodulatory
stimulus to “revive” them and allow for reexpression of CD127.
Expression of CD127 may indicate that these cells are more functional
and have increased survival capacity compared with the exhausted CD8 T
cells found during chronic infection in untreated mice.
Our result of IL-2
increasing CD127 expression on exhausted CD8 T cells during chronic
infection is the opposite result seen when early effector CD8 T cells
are given IL-2 either in vitro or in vivo during an acute infection, in
which IL-2 sways the cells toward a CD127-negative and more terminally
differentiated state (53–55).
This distinct effect of IL-2 on CD127 expression in cases of limited
antigen stimulation and persistent stimulation during chronic infection
may be due to the different programming/state of exhausted cells
compared with that of early effector CD8 T cells in acute infection. It
has been well documented that exhausted cells are molecularly distinct
from naive, effector, or memory CD8 T cells (2, 42).
Moreover, IL-2 has been shown to have differential effects on CD8 T
cells when administered in the presence of inflammatory signals (53).
Therefore, IL-2 may act differentially on these exhausted CD8 T cells
during a chronic infection that has a distinct inflammatory milieu
compared with that seen by CD8 T cells during a quickly resolved acute
infection. Overall, IL-2 appears to play a unique role on exhausted CD8 T
cells, compared with CD8 T cells during acute infection.
While IL-2 therapy, PD-L1
blockade, or IL-2 therapy combined with PD-L1 blockade greatly increased
antigen-specific CD8 T cell numbers and increased their function, there
was not a similar effect on total or LCMV-specific CD4 T cell numbers
or LCMV-antibody production (data not shown). In contrast, IL-2 therapy
alone or combined therapy resulted in an increase of FoxP3+
CD4 T cells (Tregs). These data are consistent with previous work, which
has shown that IL-2 is important for Treg development and function and
IL-2 can increase FoxP3 expression (56, 57).
Notably, combined treatment did not enhance Treg numbers above those
seen with IL-2 therapy alone and PD-1 blockade alone did not increase
Treg numbers, thus indicating that IL-2 itself is responsible for
increasing Tregs in this system. These data highlight the fact that
low-dose IL-2 therapy increases Tregs during chronic infection in mice,
similar to what has been shown recently with low-dose IL-2 treatment in
humans for chronic graft-versus-host disease and hepatitis C
virus–induced vasculitis (36, 37).
However, even in mice in which Tregs were increased, LCMV-specific CD8 T
cells were greatly expanded, regained function, and were able to reduce
viral loads, showing that the CD8 T cells are still functional, even in
the presence of increased Treg numbers. This emphasizes the distinct
roles that IL-2 therapy can have on multiple cell types in differing
environments, in which, during an autoimmune manifestation, low-dose
IL-2 therapy can increase Tregs, resulting in clinical improvement (36, 37);
in the mouse, therapy increases Tregs during chronic infection, but
virus-specific CD8 T cells are expanded to a greater extent and
reacquire functional capabilities.
IL-2 treatment alone
resulted in a reduction of viral loads during chronic cl-13 infection
(in which mice are viremic for ~2 months after infection), as previously
reported (38).
However, surprisingly, even though IL-2 treatment alone resulted in a
large expansion of LCMV-specific CD8 T cells, it did not result in
significant viral reduction in the more stringent “unhelped” chronic
LCMV infection (in which mice are highly viremic for life). One possible
explanation for the inability of these cells to reduce viral loads in
these highly viremic mice is that while IL-2 therapy alone expands
LCMV-specific CD8 T cells that are capable of cytokine secretion, these
cells still express PD-1, albeit at lower levels than CD8 T cells from
untreated mice. Therefore, while IL-2 therapy effectively increases the
number of functional CD8 T cells, these cells still may be unable to
kill their target cells due to inhibition by PD-1 binding to it’s widely
expressed ligand, PD-L1, on target cells. Accordingly, nonhematopoietic
cells are a major source of viral burden, and PD-L1 expression on
nonhematopoietic cells has been shown to impair the ability of CD8 T
cells to clear virus during chronic LCMV infection (58).
This indicates that PD-1 blockade therapy works at the level of the
cells to allow for effective CTL killing of infected target cells.
Importantly, when IL-2 therapy and PD-L1 blockade are combined,
LCMV-specific CD8 T cells are expanded, thereby increasing the
effector-to–infected target ratio, and blockade of the inhibitory signal
PD-1 then results in increased ability of the cells to effectively
reduce viral burden. This implies a distinct and synergistic effect of
IL-2 therapy and PD-L1 blockade on CD8 T cell responses and viral
control.
Overall these data
indicate that combined IL-2 and PD-1 blockade therapy may be a promising
therapy for increasing CD8 T cell function and reducing viral loads
during chronic infections and possibly cancers. In addition, since both
IL-2 therapy alone and PD-1 blockade alone have been used clinically,
combined therapy is something that could be implemented for in vivo use
in human clinical studies. Moreover, combining these two therapies may
allow for titration of the IL-2 dose, allowing for the positive effects
of IL-2 to be gained while using a low enough dose to minimize toxicity
issues. Additional studies will need to be performed to determine
whether IL-2 or combined IL-2 therapy helps “reprogram” exhausted CD8 T
cells in humans, making them more like the functional memory CD8 T cells
that express high levels of CD127 found after acute infections.
Moreover, since IL-2 therapy or combined IL-2 therapy and PD-L1 blockade
resulted in increased expression of the IL-7 receptor α chain (CD127),
it may render these cells more responsive to IL-7 therapy. While IL-7
therapy has recently been shown to enhance CD8 T cell responses and
decrease viral loads when given for long periods of time in the less
stringent model of chronic LCMV infection (59, 60),
in our hands, we found little effect of IL-7 therapy alone in the more
stringent model of chronic LCMV infection (in which CD4 T cell help is
absent) (data not shown). However, since IL-2 therapy increases
expression of the IL-7 receptor α chain on CD8 T cells during chronic
infection, combining IL-2 therapy (plus or minus PD-L1 blockade) along
with IL-7 therapy may have enhanced therapeutic benefit during chronic
infection, especially in cases of very high viral loads and extreme
exhaustion.
Last, a major concern for
the use of in vivo IL-2 therapy as a treatment for cancers and
infections is that IL-2 can increase Tregs, which may dampen the
effector T cell response (36, 37, 40, 41).
However, herein, we have shown that although low-dose IL-2 therapy does
increase Treg numbers, it still results in a highly augmented and
functional antiviral CD8 T cell response and decreased viral burden.
In conclusion, combining
daily low-dose IL-2 therapy with blockade of the inhibitory receptor
PD-1 may be a useful clinical strategy for reversing CD8 T cell
exhaustion during chronic infections, leading to an enhanced reduction
in viral burden or viral control. Further, since PD-1 also plays an
important regulatory role in some cancers, combined IL-2 therapy and
PD-1 blockade might be an important clinical tool for treating cancer.
Overall, this work may help us to design rational strategies for
developing immunotherapies for chronic infections and cancer.
Methods
Mice, infections, and cell transfers.
Six-week-old female C57BL/6 mice were purchased from The Jackson
Laboratory. P14 TCR transgenic mice were bred in house. Mice were
infected with 2 × 106 PFU of LCMV cl-13 i.v. by tail vein. Viral titers were determined by plaque assay on Vero E6 cells as described previously (1).
For chronic infection in an “unhelped” environment, mice were given 500
μg of the CD4-depleting antibody GK1.5 i.p. (BioXcell) 1 day prior to
infection and again on the day of infection. For experiments using P14 T
cells, 2 × 103 Thy1.1+ P14 T cells were transferred into mice i.v. 1 day prior to infection.
Lymphocyte isolation and flow cytometry. Lymphocytes were isolated from the blood, spleen, liver, lungs, and bone marrow as previous described (3, 61).
All antibodies were purchased from BD except CD44, Thy1.1, and Thy1.2
(Biolegend); anti–PD-1 and anti-FoxP3 (ebioscience); and anti–Tim-3
(R&D Systems). MHC class I tetramers were prepared and used as
previously described (1). Intracellular cytokine staining was performed as previously described (1).
Phospho-STAT-5 staining was done following the manufacturer’s protocol
(BD Biosciences). Cells were analyzed on a LSR II or Canto flow
cytometer (BD Immunocytometry Systems). Dead cells were excluded by
gating on Live/Dead NEAR IR (Invitrogen).
In vivo blockade and IL-2 therapy.
For blockade of the PD-1 pathway, 200 μg rat anti-mouse PD-L1 antibody
(10F.9G2 prepared in house) or rat IgG2b isotype control was
administered i.p. every 3 days for 5 total treatments beginning on the
day after infection, as indicated in the appropriate figure or figure
legend. For blockade of Lag-3, 200 μg rat anti-mouse Lag-3 antibody
(C9B7W, Biolegend) was administered i.p. every 3 days for 5 total
treatments beginning on the day after infection, as indicated in the
appropriate figure or figure legend. For IL-2 therapy, 15,000 IU (1 μg)
of recombinant human IL-2 (Amgen) diluted in PBS with 0.1% normal mouse
serum was given i.p. to the mice either every 12 or 24 hours for 8 to 12
consecutive days (as indicated in the appropriate figure legend)
beginning after chronic infection was established (at the time point
indicated in the appropriate figure legend).
Statistics. Statistical analysis was performed using 2-tailed unpaired Student’s t tests (when comparing 2 groups) or 1-way ANOVA (when comparing >2 groups) using Prism 5.0 (GraphPad) software. P values of less than 0.05 were considered statistically significant.
Study approval.
Mice were maintained and used according to institutional and NIH
guidelines in a specific pathogen–free facility. All animal studies were
approved by the IACUC of Emory University.
Supplemental data
View Supplemental data
Acknowledgments
We
thank Sue Kaech and the members of the Ahmed laboratory for helpful
discussions. This work was supported by NIH grants AI030048 and AI080192
(to R. Ahmed) and AI056299 (to G.J. Freeman and R. Ahmed) as well as
Gates Foundation Grand Challenge in Global Heath grant AHMED05GCGH0 (to
R. Ahmed). R. Ahmed, S.-J. Ha, and G.J. Freeman have patents and receive
patent royalties related to the PD-1 pathway.
Footnotes
Conflict of interest:
Rafi Ahmed, Sang-Jun Ha, and Gordan J. Freeman have patents and receive
patent royalties related to the PD-1 pathway. Gordon J. Freeman is a
scientific founder and scientific advisory board member of CoStim
Pharmaceuticals.
Citation for this article:J Clin Invest. 2013;123(6):2604–2615. doi:10.1172/JCI67008.
Erin E. West’s present address is: Laboratory of Immunology and Immunology Center, NHLBI, NIH, Bethesda, Maryland, USA.
Hyun-Tak Jin’s present address is: Research Institute, Genexine Co., Seongnam-si, Republic of Korea.
Pablo
Penaloza-MacMaster’s present address is: Beth Israel Deaconess Medical
Center, Viral Pathogenesis Division, Boston, Massachusetts, USA.
Sang-Jun
Ha’s present address is: Department of Biochemistry, College of Life
Science and Engineering, Yonsei University, Seoul, Republic of Korea.
Wendy G. Tan’s present address is: Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, USA.
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