4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection.
ABSTRACT Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.
- [show abstract] [hide abstract]
ABSTRACT: Immunity to viruses must be tightly controlled to avoid pathology. Receptors and ligands of the tumor necrosis factor (TNF) family play important roles in controlling lymphocyte activation and survival during an immune response. The role of specific TNF receptor (TNFR) family members in antiviral immunity depends on the stage of the immune response and can vary with the virus type and its virulence. Here, we focus on five members of the TNFR family that are prominently expressed on CD8(+) T cells during viral infections, namely, 4-1BB (CD137), CD27, OX40 (CD134), GITR, and TNFR2. 4-1BB, CD27, OX40, and GITR have primarily prosurvival roles for CD8(+) T cells during viral infection, although under some circumstances 4-1BB, GITR, or CD27 signals can limit immunity. Although TNFR2 can be costimulatory under some circumstances, its main role in CD8(+) T-cell responses during viral infection appears to be in contraction of the response. Several TNF family ligands are being explored as adjuvants for viral vaccines, and agonistic antibodies to TNFR family members are being investigated for immunotherapy of chronic viral infection alone and in combination with checkpoint blockade. Such therapies will require thorough and specific optimization to avoid pathology induced by hyperstimulation of these pathways.Immunological Reviews 09/2013; 255(1):125-48. · 12.16 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: High-level T cell expression of PD-1 during SIV infection is correlated with impaired proliferation and function. We evaluated the phenotype and distribution of T cells and Tregs during antiretroviral therapy plus PD-1 modulation (using a B7-DC-Ig fusion protein) and post-ART. Chronically SIV-infected rhesus macaques received: 11 weeks of ART (Group A); 11 weeks of ART plus B7-DC-Ig (Group B); 11 weeks of ART plus B7-DC-Ig, then 12 weeks of B7-DC-Ig alone (Group C). Continuous B7-DC-Ig treatment (Group C) decreased rebound viremia post-ART compared to pre-ART levels, associated with decreased PD-1(hi) expressing T cells and Tregs in PBMCs, and PD-1(hi) Tregs in lymph nodes. It transiently decreased expression of Ki67 and α4β7 in PBMC CD4(+) and CD8(+) Tregs for up to 8 weeks post-ART and maintained Ag-specific T-cell responses at low levels. Continued immune modulation targeting PD-1(hi) cells during and post-ART helps maintain lower viremia, keeps a favorable T cell/Treg repertoire and modulates antigen-specific responses.Virology 12/2013; 447(1-2):274-84. · 3.35 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: CD8 T cell activation and differentiation are tightly controlled, and dependent on the context in which naïve T cells encounter antigen, can either result in functional memory or T cell dysfunction, including exhaustion, tolerance, anergy, or senescence. With the identification of phenotypic and functional traits shared in different settings of T cell dysfunction, distinctions between such dysfunctional states have become blurred. Here, we discuss distinct states of CD8 T cell dysfunction, with an emphasis on: (i) T cell tolerance to self-antigens (self-tolerance); (ii) T cell exhaustion during chronic infections; and (iii) tumor-induced T cell dysfunction. We highlight recent findings on cellular and molecular characteristics defining these states, cell-intrinsic regulatory mechanisms that induce and maintain them, and strategies that can lead to their reversal.Trends in Immunology 11/2013; · 9.49 Impact Factor
The Journal of Immunology
4-1BB Signaling Synergizes with Programmed Death Ligand
1 Blockade To Augment CD8 T Cell Responses during
Chronic Viral Infection
Vaiva Vezys,*,1,2Pablo Penaloza-MacMaster,*,1Daniel L. Barber,* Sang-Jun Ha,*,3
Bogumila Konieczny,* Gordon J. Freeman,†Robert S. Mittler,* and Rafi Ahmed*
Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection.
Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically
infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with
an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of
LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and
timing of 4-1BB costimulation are carefully controlled. When PD-L1–blocking Abs are given together with a single low dose of
anti–4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs
to PD-L1 are given with a repetitive high dose of anti–4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells
by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7
posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However,
whereas the high dose of anti–4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of
anti–4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of
carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic
infection.The Journal of Immunology, 2011, 187: 1634–1642.
cells that are able to respond to a secondary challenge and express
IFN-g, TNF-a, and IL-2 (1–3). However, if the Ag persists, there
is a gradual loss of T cell function, resulting in progressive T cell
exhaustion and inability of T cells to respond to cognate Ag (1, 3).
This is the case with chronic infections such as HIV, hepatitis B
virus, and hepatitis C virus.
We, and many others, have previously shown that the pro-
grammed death 1 (PD-1) pathway plays an important role in
directing T cell exhaustion caused by chronic viral infection (4–8).
Decreased CD8 T cell proliferative potential and high viral loads
are major obstacles that limit the effectiveness of therapeutic
pon Ag challenge, naive T cells undergo a rapid phase of
If the Ag is cleared, T cells become bona fide memory
vaccination (9). Blockade of PD ligand 1 (PD-L1) results in an
increase of Ag-specific CD8 T cells, with enhanced functional
capacity, and this treatment improves viral control (4, 6). Addi-
tionally, blocking PD-1 inhibitory signals results in enhancement
of therapeutic vaccination during chronic infection (10). Thus, the
PD-1 pathway tightly regulates T cell responses during chronic
infection (7, 11, 12).
It is unclear, however, which other immune pathways may
synergize during PD-L1 blockade. Dual blockade of PD-1 with
other inhibitory molecules (e.g., LAG-3 and TIM-3) results in
additive effects on T cell restoration and viral reduction during
chronic infection (13, 14). We wanted to determine whether ag-
onistic costimulatory signals would synergize with PD-L1
blockade and result in a more robust rescue of exhausted virus-
specific CD8 T cells. 4-1BB (also known as CD137), a TNFR
family member (15), is expressed by activated T cells, NK cells,
NKT cells, mast cells, and neutrophils, whereas its ligand (4-
1BBL) is restricted mostly to APCs (16, 17). 4-1BB interactions
have been shown to be important for T cell responses to bacterial
and viral infections (18–20). Interestingly, the timing and dosing
amount of CD137 stimulation can result in different outcomes
during viral infections. During an acute lymphocytic choriome-
ningitis virus (LCMV) Armstrong infection, if agonistic anti-
CD137 Abs are given before viral priming, suppression of im-
munity occurs (21).
Conversely, if agonistic Abs to 4-1BB are administered a few
days postinfection, antiviral T cell responses are enhanced (21).
Such an increase in T cell responses could be beneficial during
persistent infections, where severe decreases in function and ab-
solute numbers of Ag-specific T cells are observed (1). Robertson
et al. (22) demonstrated that when mice chronically infected with
Friend virus are treated with an agonistic anti–4-1BB Ab, along
*Emory Vaccine Center, Emory University, Atlanta, GA 30329; and†Dana-Farber
Cancer Institute, Harvard Medical School, Boston, MA 02115
1V.V. and P.P.-M. contributed equally to this work.
2Current address: Department of Microbiology, Center for Immunology, University
of Minnesota, Minneapolis, MN.
3Current address: Department of Biochemistry, College of Life Science and Bio-
technology, Yonsei University, Seoul, Korea.
Received for publication January 12, 2011. Accepted for publication June 11, 2011.
This work was supported by National Institutes of Health Grant AI3004 (to R.A.).
Address correspondence and reprint requests to Dr. Rafi Ahmed, Emory Vaccine
Center, Emory University, 1510 Clifton Road, Atlanta, GA 30329. E-mail address:
The online version of this article contains supplemental material.
Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; LCMV, lympho-
cytic choriomeningitis virus; PD-1, programmed death 1; PD-L1, programmed death
with transfer of transgenic virus-specific CD8 T cells, there is
a 99% reduction of viral loads, as well as increased numbers of
transferred T cells. 4-1BB costimulation has also been shown to
be important for the proliferation of human CMV-specific CD8
T cells (23) and can also regulate immune responses to allo- and
autoantigens, as well as improve T cell-mediated antitumor effi-
cacy (24–28). Even though several reports demonstrate a positive
role for 4-1BB in regulating T cell responses, some evidence also
shows a negative role. 4-1BB2/2mice have reduced numbers of
memory CD8 T cells during latent murine CMV infection, but
paradoxically they show exaggerated primary CD8 T cell
responses to murine CMV, demonstrating a dual role of this co-
stimulatory pathway (29).
We wanted to determine whether synergistic effects could be
achievedbycombiningPD-L1blockadewith an anti–4-1BB regimen
during chronic LCMV infection. In this study, we show that com-
bining PD-L1 blockade with anti–4-1BB resulted in enhanced aug-
mentation of antiviral CD8 T cell responses during chronic LCMV
infection (compared with just PD-L1 blockade alone). Improved
expansion of exhausted LCMV-specific CD8 T cells was dependent
on the amount and duration of 4-1BB signaling. A high, constant
dose of agonistic anti–4-1BB given in combination with PD-L1
blockade resulted in a transient restoration of Ag-specific T cell
responses, whereas a low, one-time dose of anti–4-1BB resulted in
a sustained increase of Ag-specific CD8 T cell numbers, which
correlated with faster viral control early after initiation of treatment
(compared with PD-L1 blockade alone at day 7 posttreatment).
Materials and Methods
Mice and infections
Four- to 8-wk-old female C57BL/6 mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). Mice received 2 3 106PFU LCMV clone 13
i.v. via lateral vein. To establish a stringent chronic infection model with
life-long viremia, CD4 T cells were depleted by administration of 500 mg
GK1.5 Ab (BioXCell) given i.p. 1 d before and the day of infection with
LCMV clone 13. For a milder LCMV clone 13 infection model with
transient viremia lasting only 2–3 mo, mice were infected with clone 13
without performing CD4 depletion prior to infection. All mice were used
in accordance with National Institutes of Health and the Emory University
Institutional Animal Care and Use Committee guidelines.
Vero cells grown in MEM media supplemented with 10% FBS. Samples for
viral titration were diluted in 1% FBS DMEM and aliquoted on top of Vero
cell monolayers. Plates were then incubated for 1 h, rocking them every 10
min. Wells were overlayed with a 1:1 mixture of 1% agarose in 2x 199
media (20% FBS, penicillin/streptomycin) at 45˚C. Four days later, wells
were overlaid with a 1:1 mixture of 1% agarose in 23 199 media con-
taining 1:50 neutral red. Plaques were counted the day after.
blockade results in a transient in-
crease of virus-specific CD8 T cells.
A, Experimental set-up. Mice re-
ceived CD4-depleting Ab (GK1.5) the
day before and the day of infection
with LCMV clone 13. After 45 d,
mice received multiple doses of anti–
4-1BB agonistic Abs (200 mg) to-
gether with 200 mg PD-L1–blocking
Abs every 3 d (five times). B, Per-
centage of splenic CD8 T cells that
are specific for the LCMV immuno-
dominant Dbgp276–286 epitope at the
indicated times after start of Ab
treatment. C, Total numbers of sple-
nic CD8 T cells that are specific for
gp276–286 epitope at the indicated
times after start of Ab treatment. D,
Serum LCMV titers. *p , 0.05. Bars
indicate SEM; n = 15–21; experi-
ments were repeated five times.
High dose of 4-1BB
The Journal of Immunology1635
For PD-L1 blockade, 200 mg rat anti-mouse PD-L1 Ab (10F.9G2) or rat
IgG2b isotype control (BioXCell) was administered i.p. every 3 d (five
times). For the high-dose 4-1BB stimulation, 200 mg agonist rat anti-
mouse 4-1BB Ab (3H3) was administered i.p. together with PD-L1 Ab.
For low-dose 4-1BB stimulation, 50 mg agonist rat anti-mouse 4-1BB Ab
was delivered only once i.p. along with the first dose of anti–PD-L1 Ab.
T cell responses were analyzed at day 7 or 14 posttreatment.
Cell isolation and flow cytometry
Single-cell suspensions were made from spleens and nonlymphoid organs
via mechanical disruption. RBCs were lysed to isolate PBLs. MHC class I
tetramerswere producedand used asdescribed previously(5).AllAbs were
purchased from BD Biosciences, except granzyme B (Invitrogen). Surface
and intracellular staining protocols were followed as described previously
(1). LCMV peptide stimulations were performed at 37˚C for 5 h in a CO2
incubator in the presence of GolgiPlug and GolgiStop (BD Biosciences).
LCMV peptides were purchased from the Emory Microchemical Facility
(Atlanta, GA). Cells were acquired using a FACSCanto flow cytometer
(BD Biosciences) and analyzed using FlowJo (Tree Star).
Statitical analysis was performed using a nonparametric Mann–Whitney U
test on GraphPad Prism software.
High and repetitive doses of anti–4-1BB transiently augment
LCMV-specific CD8 T cell responses in chronically infected
animals during PD-L1 blockade
Our laboratory has previously reported that blockade of PD-1
signaling during chronic LCMV infection leads to the increase
and decreased viral loads (4, 10, 14). We sought to determine
whether further enhancement in immune function was possible by
combining blockade of the PD-L1 inhibitory pathway with an
agonistic regimen for the 4-1BB costimulatory pathway. Mice
chronically infected with LCMV were treated with a blocking Ab
to PD-L1, as previously published (4). As a combination modality,
these chronically infected animals also received anti–4-1BB (200
set-up. Mice received CD4-depleting Ab (GK1.5) the day before and the day of infection with LCMV clone 13. After 45 d, mice received a single dose of
anti–4-1BB agonistic Abs (50 mg) together with 200 mg PD-L1–blocking Abs at day 1. PD-L1 blockade alone was continued every 3 d (five times). B,
Percentage of splenic CD8 T cells that are specific for the LCMVimmunodominant Dbgp276–286 epitope at the indicated times after start of Ab treatment.
C, Total numbers of splenic CD8 T cells that are specific for the LCMV immunodominant Dbgp276–286 epitope at the indicated times after start of Ab
treatment. D, Serum LCMV titers. *p , 0.05. Bars indicate SEM; n = 15–21; experiments were repeated six times.
Low dose of 4-1BB stimulation together with PD-L1 blockade results in a permanent increase in virus-specific CD8 T cells. A, Experimental
16364-1BB AND PD-L1 BLOCKADE BOOST EXHAUSTED CD8 T CELLS
mg) every 3 d, five times (Fig. 1A). During this high-dose regimen,
both anti–PD-L1 and anti–4-1BB Abs were administered on the
same days. Seven days after treatment, the percentage of LCMV
gp276–286-specific CD8 T cells increased .3-fold in the dual-
treated mice, as compared with control (Fig. 1B). In contrast, the
anti–PD-L1 alone group only increased by 2-fold by day 7 post-
treatment. We continued to treat groups of mice using the same
high Ab dosing protocol for another week to determine whether
this constant stimulation could keep inflating the percentages of
antiviral CD8 T cells. After day 14 posttreatment, however, the
percentages of Ag-specific cells in the dually treated animals
returned to original pretreatment values (Fig. 1B).
The percentages of LCMV-specific CD8 T cells correlated with
their absolute numbers at all the days tested. The total numbers
of LCMV-specific CD8 T cells in anti–4-1BB– plus anti–PD-L1–
treated mice increased ∼8-fold by day 7 posttreatment, as com-
pared with pretreatment numbers (Fig. 1C). This is a 2.5-fold
increase over anti–PD-L1 alone at day 7 posttreatment. The
continuous addition of anti–4-1BB and anti–PD-L1 resulted in
a dramatic loss of cells, recoiling to original T cell numbers by
day 14 (Fig. 1C). Because the number of antiviral T cells after
14 d was essentially the same as the starting time point, this in-
dicated that this stimulation protocol only transiently increased
numbers of antiviral T cells, whereas a stable population was
generated with PD-L1 blockade alone. Spleen data are shown, and
similar results were obtained from liver, lung, and blood (data not
We next addressed whether such a transient increase in LCMV-
specific CD8 T cell numbers affected viral titers. At day 7, there
was a 5-fold drop in viral titers in animals treated with the dual Ab
treatment (greater viral control than that by PD-L1 blockade alone
at day 7) (Fig. 1D). However, by day 14, virus rebounded to
starting levels in the dual-treated mice, mimicking the recoiled
kinetics of the antiviral CD8 T cell response. Thus, the transient
together with PD-L1 blockade increases cyto-
kine production from LCMV-specific CD8
T cells. Experimental set-up as in Fig. 2A. A,
Percentage of CD8 T cells from spleen-pro-
ducing cytokines after 5 h stimulation with
several LCMV peptides. B, Total numbers of
IFN-g cytokine-producing CD8 T cells after 5
h stimulation with several LCMV peptides. C,
Total numbers of dual IFN-g and TNF-a cy-
tokine-producing CD8 T cells after 5 h stimu-
lation with LCMV peptides. *p = 0.05, **p =
0.0016, ***p = 0.0010. Experiments were re-
peated four times.
Low dose of 4-1BB stimulation
The Journal of Immunology1637
increase in antiviral CD8 T cell numbers by day 7 due to 4-1BB
costimulation and PD-L1 blockade contributes to quick viral
control, but this viral control is only transient. Viral titration was
performed in several tissues with similar results (data not shown).
Reducing the dose and frequency of anti–4-1BB treatment
improves T cell longevity
We wanted to determine whether modulating the dosing of 4-1BB
costimulation would enhance T cell longevity during combined
treatment with anti–PD-L1. We had the same experimental PD-L1
blockade regimen as before, but we decreased the dosing of 4-1BB
agonistic Ab. The new treatment consisted of only one injection of
anti–4-1BB (50 mg) along with the first dose of anti–PD-L1 Ab
(Fig. 2A). At day 7 after treatment, the percentages of LCMV-
specific CD8 T cells were increased in the dual-treated (low-dose
anti–4-1BB) group, similar to the high dose of anti–4-1BB plus PD-
L1 blockade (Fig. 2B), and there was an ∼8-fold increase compared
with untreated mice. Interestingly, at day 14, animals receiving low-
dose 4-1BB costimulatory regimen plus PD-L1 blockade were able
to maintain the high percentages of LCMV-specific CD8 T cells that
were seen at day 7 (Fig. 2B). Absolute numbers of Ag-specific CD8
T cells were also higher in the dual-treated group (Fig. 2C). Spleen
data are shown, and similar results were obtained from liver, lung,
and blood (data not shown).
Acceleration of viral control with a reduced dose of anti–
4-1BB together with PD-L1 blockade
A significant drop in viral titer was detected in dual-treated ani-
mals at day 7 (Fig. 2D), similar to what was seen in the high-dose
protocol. This control of viremia was maintained to day 14
posttreatment, correlating well with sustained numbers of antiviral
CD8 T cells. By day 14, both sera and tissue viral titers were also
similar to PD-L1 blockade alone (Supplemental Fig. 1). Thus, the
amount of stimulation is an important factor in longevity of T cells
rescued from exhaustion. Activating signals should be carefully
controlled during chronic infection to ensure T cell viability.
Functional and phenotypic changes in viral-specific CD8
T cells after a reduced 4-1BB stimulation paired with PD-L1
Functional changes occurring in mice treated with combined 4-
1BB and PD-L1 therapy were examined to determine how this
stimulation was affecting T cell physiology. We evaluated the
ability of virus-specific T cells to produce cytokines by stimulat-
ing cells with different LCMV CD8 T cell epitopes. For several T
cell epitopes tested, the low-dose 4-1BB stimulation with PD-L1
blockade enhanced cytokine expression of antiviral CD8 T cells
above the level of rescue seen with anti–PD-L1 alone (Fig. 3A).
Also, the percentage of CD8 T cells producing IFN-g and TNF-a
increased compared with control or PD-L1 blockade alone (anti–
4-1BB alone was similar to IgG control animals). In most cases,
the percentage of dual cytokine-producing T cells increased in the
double-treated group. Therefore, a reduced agonistic anti–4-1BB
regimen given concomitantly with PD-L1 blockade acts syner-
gistically to augment CD8 T cell responsiveness and function
during chronic LCMV infection. Absolute numbers of functional
cells were significantly enhanced for some LCMVepitope-specific
responses (Fig. 3B). The effect was more noticeable for sub-
dominant responses (NP235 and NP166), which had significantly
more coexpression of antiviral cytokines IFN-g and TNF-a (Fig.
3C). This suggested qualitative differences between dual regimen
and PD-L1 blockade alone. No generation of viral-specific CD4
T cell responses was observed with any of these regimens (data
not shown). In this model of stringent chronic LCMV infection,
mice are devoid of LCMV-specific CD4 T cells and cannot be
rescued by any treatment.
One differencebetweenPD-L1 aloneandthecombined(low-dose
anti–4-1BB) regimen was the large increase in total activated CD8
T cells. There is a doubling in the percentages of lymphocytes that
are CD8 T cells as well as increased percentages of activated
(CD44hi) CD8 T cells in the dual-treated mice, compared with
control mice (Fig. 4A, first row). Additionally, the percentages of
CD8 T cells that were PD-1 positive was dramatically increased in
the dual treatment, suggesting enhanced expansion of exhausted
CD8 T cells of several specificities (Fig. 4A, second row).
The dual-treated group had a marked increase of granzyme
B production in CD8 T cells (Fig. 4B, first row). Indeed, 50% of
all CD8 T cells after dual treatment now produced granzyme B,
compared with 8% in the control group or 20% in the PD-L1 alone
group. Also, CD8 T cells from the dual group had lower CD62L
blockade results in marked phenotypic and functional differences in
LCMV-specific CD8 T cells. Experimental set-up as in Fig. 2A. A, First
row, Flow plots are gated on total lymphocytes in spleen. Bottom numbers
represent percentage CD8+T cells within the total lymphocyte population.
Top numbers represent the percentage of CD8+T cells that are activated
(CD44hi) within the total lymphocyte population. Second row, Flow plots
are gated on CD8 T cells. Numbers indicate the percentage of cells that are
PD-1hiCD44hiwithin the CD8 population (representing total expanded
CD8 T cells). B, Granzyme B, CD62L, Ki67, and CD127 expression on Db
gp276–286-specific CD8 T cells in spleen. Plots are gated on CD8 T cells.
Experiments were repeated at least three times.
Low dose of 4-1BB stimulation together with PD-L1
1638 4-1BB AND PD-L1 BLOCKADE BOOST EXHAUSTED CD8 T CELLS
and CD127 expression, suggesting higher overall activation
compared with PD-L1 blockade alone (Fig. 4B, second and fourth
The dual treatment also induced more T cell proliferation, as
measured by expression of Ki67 (Fig. 4B, third row) (25). This
correlates with the increase of T cell numbers seen in the dually
treated mice, indicating that at least part of T cell rescue is cou-
pled to T cell division.
CD8 T cell viability and apoptosis in either high- or low-dose
4-1BB stimulation combined with PD-L1 blockade
We wanted to determine how changing the amount and duration of
4-1BB stimulation during standard PD-L1 blockade affected Ag-
specific CD8 T cell viability. Therefore, we examined apoptosis
of LCMV-specific CD8 T cells in animals given a high (Fig. 1A) or
low (Fig. 2A) dose of anti–4-1BB together with PD-L1 blockade.
We measured apoptosis by annexin V/7-aminoactinomycin D
(7AAD) staining at days 7 and 14 after treatment (Fig. 5). As
expected, a proportion of Ag-specific CD8 cells are apoptotic in
untreated animals, in agreement with previous reports (30).
At day 7, there was a 2-fold increase in the percentages of viral-
specific CD8 T cells that were apoptotic in either of the combined
regimens (Fig. 5A, first row). This suggested that even though
there is expansion of viral-specific cells, there is also apoptosis
occurring at the same time in the dual-treated groups. By day 14,
however, the low-dose combined regimen equilibrated and had
very few percentages of apoptotic cells, whereas the high com-
bined regimen resulted in increased percentages of apoptotic cells
(Fig. 5A, second row, 5B). Our results show that the amount and
duration of 4-1BB signaling is crucial, as overstimulation will lead
to apoptotic death, whereas an optimal (lower) level of costimu-
lation leads to a larger, more viable T cell population. The per-
centages of annexin V2/7AAD2cells, which represented viable
cells, were examined (Fig. 5C). By day 7, most groups had similar
percentages of live (annexin V2/7AAD2) viral-specific CD8
T cells. However, by day 14, the low-dose anti–4-1BB plus PD-L1
blockade group had a statistically higher percentage of live cells,
compared with the high-dose anti–4-1BB plus PD-L1 blockade
group (Fig. 5C). This suggested a survival advantage after a low
dose of anti–4-1BB and anti–PD-L1. We have also looked at later
time points (after day 30), and the low-dose anti–4-1BB plus PD-
L1 blockade group still has increased levels of viral-specific cells
after interruption of treatment (data not shown).
Synergism of 4-1BB signaling and PD-L1 blockade is optimal
during an infection resulting in chronic viremia
All experiments presented so far are with mice that were depleted
of CD4 T cells prior to infection with LCMV clone 13 (we waited
after cotreatment with high or low dose of anti–4-
1BB together with PD-L1 blockade. Experimental
set-up as in Figs. 1A or 2A. A, FACS plot showing
the percentage of splenic Dbgp276–286-specific
CD8 T cells that are apoptotic or alive by annexin
V/7AAD staining in spleen at days 7 and 14
posttreatment. B, Percent of Dbgp276–286-specific
CD8 T cells that are positive for both annexin V
and 7AAD (apoptotic population). C, Percentage of
Dbgp276–286-specific CD8 T cells that are nega-
tive for both annexin Vand 7AAD (42). *p , 0.05.
Experiments were repeated three times.
LCMV-specific CD8 T cell viability
The Journal of Immunology1639
.45 d to allow for full CD8 T cell exhaustion). This infection
protocol provides a stringent infection, which results in lifelong
viremia (31). We were interested in understanding how CD8
T cells in a less stringent chronic infection model would respond
to 4-1BB stimulation with PD-L1 blockade. To test this, mice
were not CD4 T cell depleted before infecting with LCMV clone
13, and they were then treated with the low-dose 4-1BB
stimulation/PD-L1 blockade starting anytime between day 15 or
21 postinfection (Fig. 6A). In this less stringent model of chronic
infection, this is the optimal time window to start PD-L1 blockade.
The percentage (Fig. 6B) and total numbers (Fig. 6C) of
cytokine-expressing cells were similar by day 14 posttreatment.
Viral control was moderately impaired in the dual-treated group
compared with PD-L1 blockade alone (Fig. 6D) and was similar in
tissues (Supplemental Fig. 2). Thus, during a less stringent chronic
LCMV infection, additional 4-1BB costimulation during PD-L1
blockade does not result in additional increase of viral-specific
CD8 T cell responses or viral control.
We hypothesized that the rescue of exhausted CD8 T cells achieved
with PD-L1 blockade could be enhanced by additional costimu-
lation. So far, the combinatorial effects of blocking Abs against
multiple inhibitory receptors during chronic infection have been
explored (13, 14). However, to our knowledge, no one has tested
the effect of blocking the PD-1 pathway and inducing additional
T cell costimulation (in the form of 4-1BB) at the same time. We
have tested the effect of dual treatment with an agonistic anti–4-
1BB Ab 200 mg every 3 d, along with anti–PD-L1 Ab in mice
chronically infected with LCMV.
Our initial experiments showed that the inclusion of 4-1BB
stimulation boosted numbers of LCMV-specific CD8 T cells 8-fold
at a time point when PD-L1 blockade alone only increased cell
numbers ,3-fold. This also resulted in a drop in viral titers in
dual-treated mice at an early time point, indicating the usefulness
of this therapeutic protocol to quickly control virus. However, the
large numbers of LCMV-specific CD8 T cells that were seen at
day 7 were not maintained to day 14 when excessive costimulation
was provided (Fig. 1), indicating that this extra push of costimu-
lation through 4-1BB was acting to overstimulate CD8 T cells,
leading to apoptosis (Fig. 5). No significant differences in viral-
specific CD8 T cell frequencies or viral control were observed
between the single treatments with the high-dose anti–4-1BB
alone and IgG-treated animals. A slight increase in total numbers
was seen by day 7, but it was not statistically significant.
We reasoned that lowering the dose of 4-1BB costimulation may
14 posttreatment. Thus, we adapted our protocol by decreasing the
amount and duration of anti–4-1BB treatments to a single dose of
50 mg given together only with the first day of our standard PD-L1
blockade. This combination modality with a low dose of anti–4-
1BB resulted in significant expansion of LCMV-specific CD8
blockade during a milder chronic
infection does not result in enhanced
CD8 T cell rescue compared with
PD-L1 blockade alone. A, Experi-
mental set-up. Mice were infected
with LCMV clone 13 (without prior
CD4 depletion). Around days 15–21
postinfection, mice received a single
dose of anti–4-1BB agonistic Abs (50
mg) together with PD-L1 blockade
on day 1. PD-L1 blockade alone was
continued every 3 d (five times). B,
Percentage of CD8 T cells from
5 h stimulation with several LCMV
peptides. C, Total numbers of cyto-
kine producing CD8+splenocytes
after 5 h stimulation with LCMV
peptides. D, LCMV serum titers. IgG
treatment was similar to anti–4-1BB
alone. Experiment was repeated twice;
n = 12.
Low dose of 4-1BB
together with PD-L1
1640 4-1BB AND PD-L1 BLOCKADE BOOST EXHAUSTED CD8 T CELLS
T cells at day 7 (similar to mice receiving combination modality
with high-dose 4-1BB signaling). In contrast to the high dose of
anti–4-1BB combined regimen, antiviral CD8 T cell numbers
were stable with the reduced costimulatory regimen (Fig. 2). No
significant differences were observed between the low-dose anti–
4-1BB alone and IgG-treated animals.
Therefore, there is a delicate balance between a sufficient level
of costimulation to potentiate T cell rescue and overstimulation
leading to cell death. The connection between excessive immune
activation and exhaustion has been widely documented (32–34).
Caution should be taken with therapies based on costimulatory
regimens to ensure optimal cell longevity and function.
The dual low-dose 4-1BB combined therapy also potentiated the
function of LCMV-specific CD8 T cells in the face of a stringent
chronic infection. Both the numbers of LCMV-specific CD8 T cells
producing IFN-g (single producers) and both IFN-g and TNF-a
(dual producers) were increased compared with PD-L1 blockade
alone (Fig. 3). This was statistically significant for subdominant
responses. Even though we observed a permanent increase in the
viral-specific CD8 T cell response in the low anti–4-1BB plus
anti–PD-L1 blockade group, viral control was only accelerated by
day 7 (after day 14, viral titers were similar to anti–PD-L1 alone).
It is possible that LCMV variants may appear as a result of in-
creased T cell-mediated cytotoxic control, resulting in similar
viral loads despite increased rescue of T cell responses. Another
possibility may be that increased T cell rescue above a threshold
(dictated by just PD-1 blockade) may not directly correlate to
enhanced viral control. It is possible that viral control is a mea-
surement determined not only by the magnitude of viral-specific
T cell responses, but also by immunoregulatory pathways that act
concertedly with PD-1/PD-L1 to modulate target cell killing. We
are currently investigating the reasons for these results. Our
findings, however, may be important in therapeutic vaccination
regimens that require fast clinical intervention resulting in accel-
erated decline in viral titers.
There were also significant phenotypic changes after co-
administration of anti–4-1BB (low dose) and anti–PD-L1 Abs.
CD8 T cells downregulated CD62L and CD127, which may cor-
relate with their overall activation status, and immediate cytotoxic
capability (35–37). This phenotype may be a result of an expanded
effector cell population (35). Anti–4-1BB treatment also resulted
in an increase of Ki67+antiviral CD8 T cells, as well as a large
change in the frequency of CD8 T cells producing granzyme B.
There were also increased percentages of PD-1+CD44+CD8
T cells after dual treatment. This last subset seems to represent the
total increase in Ag-specific cells after treatment, showing ex-
pansion of exhausted cells from different specificities that are
contained within the PD-1+CD44+subset. This cell population
contains exhausted, Ag-specific T cells with different proliferative
capacities after PD-1 blockade, with PD-1intCD44hihaving a pro-
liferative advantage over PD-1hiCD44hi(38).
We then wanted to know what would be the outcome of the
immune response after dual treatment in the less stringent model of
T cell exhaustion (LCMV clone 13 infection without CD4 de-
pletion). In this less stringent model of chronic infection, viremia
lasts only 2–3 mo and CD8 T cell exhaustion is less pronounced
compared with the CD4-depleted clone 13 infection model. Dual
treatment with PD-L1 blockade and a low dose of 4-1BB agonistic
signaling resulted in rescue of Ag-specific T cell responses similar
to that seen with PD-L1 alone (Fig. 6). However, viral control was
slightly delayed compared with anti–PD-L1 alone. Hence, under
less stringent conditions, the threshold of T cell rescue and viral
control achieved by PD-L1 blockade alone may obviate the need
for any additional costimulation, and additional activating signals
may hamper antiviral control. Additionally, it is possible that
lower viral Ag loads in this less stringent clone 13 infection model
may be responsible for the reduced expansion of exhausted CD8
T cells after PD-L1 blockade (compared with the expansion by
PD-L1 blockade in the more stringent infection model). This
could be due to reduced TCR signaling in the less stringent in-
fection, highlighting the importance of “Ag sensing” as a driver
for T cell proliferation during T cell rescue regimens.
One lesson from these studies is that excessive T cell responses
produced by providing additional costimulation do not always
case determined by just PD-L1 blockade). Administration of ad-
ditional activating signals may hamper antiviral function and
overall T cell fitness if not carefully controlled and tailored to
a particular infection status.
The dual nature of 4-1BB costimulatory pathway is well
documented. If agonistic Abs to 4-1BB are given after Ag priming,
this results in augmentation of T cell responses (21, 39). Con-
versely, administration of Abs agonistic for 4-1BB before or
during priming results in suppression of Ag-specific responses
(21, 40, 41). This blunting of pathogen-specific responses has been
reported to be TNF-a dependent (21). Even though our treatment
started late after Ag challenge, and after chronic infection has
ensued, we hypothesize that a similar mechanism of TNF-a–de-
pendent suppression may be occurring between day 7 and day 14
posttreatment in the mice that received the high-dose anti–4-1BB
plus PD-L1 blockade. One of our future directions is to test anti–
TNF-a therapy to see whether it prevents the drastic decline of the
expanded cells after day 7 with the high-dose dual regimen in the
stringent infection LCMV model.
By further manipulating the kinetics of T cell responses fol-
lowing blockade of the inhibitory PD-1 pathway, it may be possible
to accelerate viral clearance during established chronic infections.
However, careful tailoring of the amount of activating signals and
the status of infection should be taken into consideration to prevent
overt stimulation above a physiologically accepted level. In con-
clusion, in a more stringent model of chronic infection, PD-1
blockade together with a low dose of 4-1BB costimulation ac-
celerates T cell restoration and viral control compared with PD-1
blockade alone. This may be important in clinical settings where
immediate T cell restoration is wanted.
We thank Hong Wu for technical assistance and Hyun-Tak Jin for discus-
R.A., G.J.F., D.L.B., and S.-J.H. have patents and receive patent royalties
related to the PD-1 pathway. The other authorshave no financial conflicts of
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16424-1BB AND PD-L1 BLOCKADE BOOST EXHAUSTED CD8 T CELLS