Cooperation of Tim-3 and PD-1 in CD8 T-cell
exhaustion during chronic viral infection
Hyun-Tak Jina, Ana C. Andersonb, Wendy G. Tana, Erin E. Westa, Sang-Jun Hac, Koichi Arakia, Gordon J. Freemand,
Vijay K. Kuchroob, and Rafi Ahmeda,1
aEmory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322;bCenter for Neurologic
Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115;dDepartment of Medical Oncology, Dana-Farber Cancer Institute,
Department of Medicine, Harvard Medical School, Boston, MA 02115; andcDepartment of Biochemistry, College of Life Science and Engineering, Yonsei
University, Seoul 120-749, Korea
Contributed by Rafi Ahmed, July 8, 2010 (sent for review April 12, 2010)
Inhibitory receptors play a crucial role in regulating CD8 T-cell
containing molecule–3 (Tim-3) is well known to negativelyregulate
T-cell responses, but its role in CD8 T-cell exhaustion during chronic
infection in vivo remains unclear. In this study, we document core-
gulation of CD8 T cell exhaustion by Tim-3 and PD-1 during chronic
lymphocytic choriomeningitis virus infection. Whereas Tim-3 was
only transiently expressed by CD8 T cells after acute infection, vi-
rus-specific CD8 T cells retained high Tim-3 expression throughout
chronicinfection.Themajority (approximately65% to 80%)of lym-
of Tim-3 and PD-1 was associated with more severe CD8 T-cell ex-
haustion in terms of proliferation and secretion of effector cyto-
kines such as IFN-γ, TNF-α, and IL-2. Interestingly, CD8 T cells
expressing both inhibitory receptors also produced the suppressive
cytokine IL-10. Most importantly, combined blockade of Tim-3 and
and viral control in chronically infected mice. Taken together, our
dysfunction and for identifying virus-specific CD8 T cells that pro-
duce IL-10, and shows that targeting both PD-1 and Tim-3 is an
effective immune strategy for treating chronic viral infections.
functional exhausted state (1). These exhausted CD8 T cells are
characterized by the inability to produce immune-stimulatory
cytokines, lyse virally infected cells, and proliferate (1). After
CD8 T-cell exhaustion was initially characterized in the murine
lymphocytic choriomeningitis virus (LCMV), such a functional
impairment has been a common feature in human chronic viral
infections such as, HIV, hepatitis B virus, and hepatitis C virus
(HCV) (2). These functional defects in responding T cells are
probably a primary reason for failure of immunological control
of these persisting pathogens.
Recent studies have focused on the crucial role of inhibitory
receptors in regulating T-cell exhaustion during chronic viral
infections. Programmed death 1 (PD-1), an inhibitory receptor of
the CD28 superfamily, was shown to be highly expressed on
the LCMV system, and in vivo blockade of this pathway restored
the function of virus-specific CD8 T cells, resulting in enhanced
viral control (3). Involvement of the PD-1 pathway has also been
shown in various chronic viral infections including HIV, hepatitis
B virus, and HCV in humans (4, 5), and during simian immuno-
have suggested that PD-1 could be a major inhibitory pathway
during chronic infection and manipulation of this pathway may
have therapeutic potential. However, blockade of PD-1 pathway
involvement of other negative regulatory pathways in CD8 T-cell
exhaustion. Gene expression profiling studies have identified the
uring chronic viral infection, virus-specific CD8 T cells be-
come unresponsive to viral antigens and persist in a non-
presence of a number of other potential inhibitory receptors on
exhausted CD8 T cells suchas 2B4,LAG-3, CTLA-4, PirB,GP49,
and CD160 (8). Moreover, considerable evidence indicates that
the expression of these receptors is important for regulating
multiple functional aspects of CD8 T-cell exhaustion (7, 9).
Therefore, a more thorough understanding of the importance of
therapeutic targets leading to the restoration of CD8 T-cell
function and better viral control.
T-cell Ig- and mucin-domain–containing molecule-3 (Tim-3)
was initially identified as a molecule expressed on T helper (Th)
1, but not Th2 (10). Interaction of Tim-3 with its ligand, galectin-
9, regulates Th1 responses by promoting the death of Th1 cells
and induces peripheral tolerance (11). Recently, it was reported
that Tim-3 was expressed by virus-specific T cells during HIV-1
and HCV infections, and the expression levels correlated with
the state of CD8 T-cell exhaustion (12, 13). In addition, blockade
of Tim-3 improved the responsiveness of the exhausted T cells in
vitro (12, 13), suggesting Tim-3 as another inhibitory marker of
exhausted T cells during chronic viral infection. However, it is
currently unclear whether Tim-3 regulates CD8 T cell exhaustion
in cooperation with PD-1 during chronic viral infection. Fur-
thermore, it will be important to explore the possibility of
a synergistic effect of blocking both the Tim-3 and PD-1 path-
ways for providing new opportunities in antiviral therapy.
In this study, we longitudinally investigated the expression of
Tim-3 on virus-specific CD8 T cells during acute and chronic
LCMV infection. We were especially interested in determining
the coexpression of Tim-3 and PD-1 on CD8 T cells to identify
populations with differential dysfunction during chronic viral
infection. In addition, we examined the effect of in vivo blockade
of Tim-3 and PD-1 regulatory pathways on the reversal of
exhausted CD8 T cells and viral control.
Tim-3 Expression Defines a Subpopulation of PD-1+Exhausted CD8 T
Cells During Chronic LCMV Infection. We examined expression of
Tim-3 on LCMV-specific effector, memory, and exhausted CD8
T cells during acute or chronic infection using the Armstrong
(Arm) or clone-13 (CL-13) strains of LCMV (14). First, we an-
alyzed Tim-3 expression on LCMV-specific CD8 (GP33+CD8)
T cells in lymphoid and nonlymphoid organs at various time
points after infection. During acute infection, the percentage of
Author contributions: H.-T.J., A.C.A., S.-J.H., V.K.K., and R.A. designed research; H.-T.J.,
W.G.T., E.E.W., and S.-J.H. performed research; A.C.A., G.J.F., and V.K.K. contributed new
reagents/analytic tools; H.-T.J., A.C.A., K.A., V.K.K., and R.A. analyzed data; and H.-T.J. and
R.A. wrote the paper.
Conflict of interest statement: R.A. received licensing fees from Genentech on using anti-
bodies to block the PD-1 inhibitory pathway. G.J.F. received patent royalties on the PD-1
and TIM-3 pathways. No other authors have any financial conflicts.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 17, 2010
| vol. 107
| no. 33
GP33+CD8 T cells expressing Tim-3 (Tim3+GP33+) was sig-
nificantly increased in spleen, lung, and liver at d 8 after infection
(Fig. 1A and Fig. S1A). However, this frequency rapidly de-
creased to approximately 10% in all tissues at day 30 after Arm
infection (Fig. 1B and Fig. S1A). On the contrary, during chronic
infection, the frequency of Tim3+GP33+T cells was markedly
elevated at day 8 in all tissues, which was sustained at approxi-
mately 70% to 80% for at least 90 d (Fig. 1A and Fig. S1A).
Although the frequency of Tim3+GP33+T cells was similarly
increased at day 8 in acute and chronic infection, we observed
significantly stronger mean fluorescence intensity (MFI) of Tim-
3 expression during chronic versus acute viral infections at day 8
(Fig. S1B). Moreover, this high intensity of Tim-3 expression on
virus-specific CD8 T cells was maintained during chronic viral
infection. These results indicate that Tim-3 is transiently up-
regulated at intermediate levels on activated T cells, and is
quickly down-regulated in acute infection, but continues to be
expressed at much higher levels during chronic infection.
Because it was not yet clear whether Tim-3 and PD-1 are
expressed by distinct or overlapping populations of CD8 T cells
during chronic infection (12, 13), we next analyzed the coex-
pression of Tim-3 and PD-1. During acute viral infection, only at
day 8 after infection was a small portion of GP33-specific CD8
T cells coexpressing Tim-3 and PD-1 (Tim3+PD1+) detectable
(Fig. 1C). In contrast, chronic viral infection resulted in much
higher frequency of Tim3+PD1+cells, which persisted even at
day 90 (Fig. 1C). We could also detect another dominant pop-
ulation expressing only PD-1 but not Tim-3 (Tim3−PD+) during
chronic infection (Fig. 1D). Thus, exhausted CD8 T cells found
duringchronic LCMV infection
Tim3+PD1+and Tim3−PD1+subsets. Notably, the distribution
of Tim3+PD1+or Tim3−PD+was slightly different depending
on the tissues analyzed, and a small percentage of CD8 T cells
detectable in the lung expressed only Tim-3 but not PD-1
(Tim3+PD1−; Fig. 1D), suggesting differential phenotypic char-
acteristics of exhausted CD8 T cells in varying anatomical sites.
The presence of this Tim3+PD1+cell could also be defined by
looking at total CD8 T cells (Fig. S1C).
Coexpression of Tim-3 and PD-1 Correlates with More Severe Ex-
haustion of Virus-Specific CD8 T Cells During Chronic Virus Infection.
We identified two dominant populations of virus-specific CD8
Tcellsexpressing PD-1alone(Tim3−PD1+)orcoexpressing Tim-
3 (Tim3+PD1+) during chronic LCMV infection. We further
assessed the phenotypic profile of virus-specific Tim3−PD1+and
Tim3+PD1+CD8 T cells by using a panel of markers, including
other inhibitory and differentiation markers. The Tim3+PD1+
virus-specific CD8 T cells displayed higher levels of other in-
hibitory receptors such as LAG3 and 2B4, along with lower levels
Considering the phenotypic signature of CD8 T-cell exhaustion
(8), this suggests that the virus-specific Tim3+PD1+T cells may
represent a more severely exhausted subpopulation.
We next directly compared the functional properties of puri-
fied population of Tim3−PD1+and Tim3+PD1+CD8 T cells. As
the majority of antigen-specific CD8 T cells in the spleen are
Tim3−PD1+or Tim3+PD1+during chronic infection (Fig. 1 and
Fig. S1), we focused on investigating the functionality of these
two subsets. To investigate the proliferation capacity, equal
numbers of GP33-specific Tim3−PD1+or Tim3+PD1+cells
were labeled with carboxyfluorescein succinimidyl ester (CFSE)
and stimulated with GP33-41 peptide or LCMV peptide pool.
When cell proliferation was assessed by CFSE dilution, some
division of Tim3−PD1+cells was observed, whereas Tim3+PD1+
cells hardly showed any proliferation after peptide stimulation
(Fig. 2A). These results show that the proliferative dysfunction is
greater in the Tim3+PD1+T cell population.
We then examined cytokine secretion by Tim3−PD1+or
Tim3+PD1+cells in response to LCMV peptide stimulation.
Consistent with our previous report (14), we confirmed the func-
tional exhaustion of CD8 T cells from chronically infected mice
characterized by a highly reduced ability to secret IFN-γ, TNF-α,
on antigen-specific CD8 T cells during chronic LCMV
infection. Expression of Tim-3 and PD-1 on GP33-
specific CD8 T cells during LCMV Arm (acute) or
Clone-13 (chronic) infections was analyzed by mul-
tiparameter flow cytometry. (A) Intensity of Tim-3
expression on GP33-specific CD8 T cells during in-
fection. (B) Representative data show the frequency
of GP33-specific CD8 T cells expressing Tim-3 on day
8 and 30 after infection. (C) The frequency of GP33-
specific CD8 T cell coexpressing Tim-3 and PD-1 is
shown over time. (D) Representative data showing
coexpression of PD-1 and Tim-3 by exhausted LCMV-
specific CD8 T cells on day 30 after infection. Num-
bers in quadrants indicate percent of each subset.
Error bars represent SEM. Data are representative
of three independent experiments with three mice
Sustained coexpression of Tim-3 and PD-1
| www.pnas.org/cgi/doi/10.1073/pnas.1009731107Jin et al.
and IL-2 compared with CD8 T cells from acutely infected mice
(Fig. S3A). When we further accessed the dysfunction of
Tim3−PD1+or Tim3+PD1+CD8 T cells in chronic infection, the
frequency of IFN-γ–secreting cells was similar between the two
populations, but the intensity of IFN-γ secretion (i.e., MFI) was
approximately 2-fold higher in the Tim3−PD1+subset compared
with the Tim3+PD1+subset (Fig. S3B). Furthermore, TNF-α and
IL-2 secretion from CD8 T cells in response to stimulation was
observed predominantly from the Tim3−PD1+population, with
minimal cytokine production observed in the Tim3+PD1+pop-
ulation (Fig. S3B). To more quantitatively determine the func-
tionality of these subsets, we examined cytokine secretion on
a “per-cell” basis by calculating the frequency of epitope-specific
CD8 T cells in each subset. The Tim3−PD1+population showed
T cell producing TNF-α or IL-2 than the Tim3+PD1+population
(Fig. 2B). These results therefore indicate that functional ex-
coexpression of Tim-3 and PD-1, and that Tim3+PD1+cells ex-
hibit a deeper exhaustion.
Tim3+PD1+Exhausted CD8 T Cell Produces the Inhibitory Cytokine
IL-10. A novel population of HIV-specific CD8 T cells has been
shown to express IL-10 during HIV-1 infection (15, 16), sug-
gesting that this specific population may have an important
regulatory role in immune dysfunction in chronic infections.
However, no phenotypic marker has been defined to identify the
IL-10–positive CD8 T cells. In this regard, we examined whether
Tim3−PD1+or Tim3+PD1+cells secrete IL-10 in response to
stimulation with LCMV peptides. In contrast to effector cyto-
kines (IFN-γ, TNF-α, and IL-2), IL-10 production was signifi-
cantly observed only by the Tim3+PD1+but not the Tim3−PD1+
populations (Fig. 2C and Fig. S3C). These results indicate that
Tim-3 coexpression with PD-1 may provide an immunopheno-
typic marker for determining the IL-10–producing CD8 T cells as
well as severely exhausted CD8 T cells.
Simultaneous in Vivo Blockade of Tim-3 and PD-1 Pathways Syner-
gistically Restores CD8 T Cell Function and Enhances Viral Control.
Our results show a correlation between coexpression of Tim-3
with PD-1 and deeper CD8 T cell exhaustion. However, it is not
clear whether the Tim-3 and PD-1 pathways have overlapping
functions or contribute independently to T cell dysfunction
during chronic viral infection. To address this issue, we blocked
the Tim-3 and PD-1 pathways with Tim-3-Ig fusion protein
(Tim3Ig) and blocking antibody to PD-L1 (αPDL1), either alone
or in combination during chronic LCMV infection. We first
measured the frequency of GP33-specific CD8 T cells in the
blood before and after treatment (Fig. 3). Tim-3 blockade alone
(Tim3Ig) induced almost no change in GP33-specific CD8
(GP33+CD8) T-cell population. PD-1 blockade alone (i.e.,
αPDL1) resulted in a 5.3-fold increase of GP33+CD8 T-cell
population (P = 0.0013), and dual blockade of Tim-3 and PD-1
(Tim3Ig + αPDL1) led to the 6.8-fold increase of GP33+CD8
T cell pool (P = 0.001). We then analyzed virus-specific CD8
T cells responses in lymphoid and nonlymphoid tissues. Treat-
ment with Tim3Ig alone showed only marginal effects on the
absolute number of GP33+CD8 T cells, whereas blocking the
PD-1 pathway significantly augmented the GP33+CD8 T cell
response compared with isotype control (Fig. 3). More in-
terestingly, the simultaneous administration of Tim3Ig and
αPDL1 further increased the number of GP33+CD8 T cells even
when compared with αPDL1 alone (Fig. 3). It is worth noting
that, when treatments started at 200 d after infection, dual
blockade still increased the quantity of virus-specific CD8 T cells,
but αPDL1 alone was only minimally effective (Fig. S4).
To determine the effect of dual blockade of Tim-3 and PD-1
pathways on the function of virus-specific CD8 T cells during
chronic viral infection, we measured the ability of LCMV epi-
tope-specific CD8 T cells to produce the cytokine IFN-γ and to
degranulate by monitoring CD107a/b expression. The treatment
with Tim3Ig alone showed only a modest increase of CD107a/
b expression and IFN-γ production, whereas treatment with
cells responding to multiple LCMV epitopes (Fig. 4A and Fig.
a much greater increase in the degranulation and IFN-γ–pro-
ducing CD8 T cell population, particularly to GP33-41, GP276-
286, NP396-404 peptides, and a pooled peptide mixture, com-
pared with either treatment alone or isotype control (Fig. 4A and
Fig. S5A). Furthermore, only dual blockade elevated the per-
centage of polyfunctional CD8 T cells coproducing IFN-γ and
TNF-α (Fig. 4B). Next, to test the effect of the blockade treat-
haustion of LCMV-specific CD8 T cells during chronic infection. Functions of
Tim3+PD1+or Tim3−PD1+CD8 T cells were analyzed using splenocytes at d 50
after infection. (A) The isolated Tim3+PD1+or Tim3−PD1+CD8 T cells were
labeled with CFSE and stimulated with GP33 peptide or a pool of LCMV
peptides for 3 d. Proliferation was determined by dilution of CFSE, and the
number in flow cytometry plot indicates the frequency of proliferating cells.
(B) Frequency of GP33- or GP276-specific CD8 T cells producing cytokine after
stimulation for 5 h with GP33-41 or GP276-286 peptides. (C) Frequency of
Tim3+PD1+, Tim3−PD1+, or Tim3−PD1−CD8 T cells producing IL-10 was ana-
lyzed after stimulation for 5 h with the LCMV peptide. Data are represen-
tative of three independent experiments. Error bars represent SEM. LCMV
peptide pool consists of GP33-41, GP276-286, GP70-77, GP92-101, NP166-175,
NP205-212, NP235-249, and NP396-404.
Coexpression of Tim-3 and PD-1 correlates with more severe ex-
Jin et al.PNAS
| August 17, 2010
| vol. 107
| no. 33
ments on the proliferation of virus-specific CD8 T cells, we ana-
lyzed the frequency of Ki67-positive GP33+CD8 T cells. Tim-3
blockade alone slightly increased the frequency of Ki67-positive
GP33+CD8 T cells compared with isotype control (Fig. 4C and
Fig. S5B). PD-1 blockade alone showed a significant increase in
the frequency of Ki67-positive GP33+CD8 T cells compared with
indicate that dual blockade of Tim-3 and PD-1 pathways sub-
stantially enhanced virus-specific CD8 T cell responses in quality
as well as quantity during chronic viral infection.
We next assessed if the heightened CD8 T-cell response in-
duced by dual blockade of these inhibitory receptors also en-
hanced viral control. The viral load of mice treated with isotype
control or Tim3Ig alone in the serum was not significantly al-
tered after treatment (Fig. 5). In contrast, αPDL1 alone or dual
blockade (Tim3Ig + αPDL1) treatments led to 4.2-fold and
a 6.3-fold reductions, respectively, in virus titer of serum (Fig. 5).
Similarly, treatment with Tim3Ig alone resulted in slightly lower
viral loads in the spleen, liver, and lung, whereas treatment with
αPDL1 alone showed significant reduction of viral load in all
tissues compared with the isotype control group (Fig. 5). More
impressively, dual blockade with Tim3Ig and αPDL1 led to fur-
ther reduction in viral loads in all tissues compared with block-
ade with αPDL1 alone (Fig. 5).
In this study, we found that, although Tim-3 was transiently
expressed by CD8 T cells after acute LCMV infection, it was
rapidly down-regulated, whereas CD8 T cells retained high Tim-3
was mainly coexpressed with PD-1 on virus-specific CD8 T cells
during chronic infections. Importantly, this subset of CD8 T cells
coexpressing Tim-3 and PD-1 (Tim3+PD1+) showed the pheno-
typic and functional characteristics of more severely exhausted
CD8 T cells than did those expressing only PD-1 (Tim3−PD1+).
Finally, simultaneous in vivo blockade of Tim-3 and PD-1 path-
ways had synergistic effects in restoring antiviral immunity and
viral control compared with blockade of either pathway alone.
Collectively, these results indicate that Tim-3 and PD-1 pathways
may cooperate and independently contribute to negatively regu-
late CD8 T cell responses during chronic viral infections.
CD8 T-cell responses during chronic viral infection. Chronically infected
C57BL/6 mice (80 d after infection) were treated every third day or every
other day for 2 wk with αPDL1 or Tim3Ig, respectively. Frequency of GP33-
specific CD8 T cells before and after treatment of individual mouse is shown
in the blood. Total number of GP33-specific CD8 T cells in the indicated
tissues at 2 wk after treatment. Data are representative of three in-
dependent experiments with five to six mice per group in each experiment.
In vivo blockade of Tim-3 and PD-1 pathways enhances virus-specific
ces function in exhausted virus-specific CD8 T cells. (A) IFN-γ
production and degranulation by CD8 T cells in treated mice
at 2 wk after therapy. The percentage of IFN-γ+CD107+CD8
T cells specific for each of the LCMV peptides are summa-
rized. (B) Polyfunctional (TNF-α+IFN-γ+) CD8 T cells in treated
mice at 2 wk after therapy. (C) The proliferation of antigen-
specific CD8 T cell after dual blockade is shown as the per-
centage of Ki67+on LCMV GP33-specific CD8 T cells. Data
are representative of three independent experiments with
five to six mice per group in each experiment. *P < 0.05;
**P < 0.01.
Dual blockade of Tim-3 and PD-1 pathways enhan-
| www.pnas.org/cgi/doi/10.1073/pnas.1009731107Jin et al.
The subset of CD8 T cells coexpressing Tim-3 and PD-1
(Tim3+PD1+) also showed high expression of other inhibitory
receptors, such as 2B4 and Lag3 (Fig. S2). 2B4 and Lag3 are still
not clearly characterized for their inhibitory function on CD8 T
cells. 2B4 expression is up-regulated and remains high on virus-
specific exhausted CD8 T cells (8), and Lag3 is known to play
a negative role in T cell homeostasis and expansion (17, 18). In
addition, a recent study found that coexpression of multiple
these receptors was associated with greater T-cell exhaustion (9),
which is consistent with our result showing that Tim3+PD1+
subset exhibits a deeper exhaustion than the Tim3−PD1+subset.
We identified subsets of exhausted CD8 T cells based on Tim-3
and PD-1 expression during chronic LCMV infection. A majority
of virus-specific CD8 T cells expressed both Tim-3 and PD-1
(Tim3+PD1+), indicating that Tim-3 and PD-1 mark overlapping
subsets of exhausted CD8 T cells. In our study, the frequency of
the CD8 T cell subset expressing only Tim-3 (Tim3+PD1−) was
too low to analyze the phenotype and functionality of this subset.
However, given that there is a hierarchical process of T cell ex-
haustion—thus, diverse inhibitory receptors may regulate distinct
aspects of T-cell exhaustion—this Tim3+PD1−subset could also
be one of the stages in differentiation of exhausted T cells during
chronic infection. Contrary to our study, the Tim3+PD1−subset
was shown in another study to be one of the major populations of
CD8 T cells in peripheral blood of HIV-1 infection (12). This
difference may be explained by the different type and/or severity
of infection. Many studies have demonstrated the relationship
between severity of infection and the extent of CD8 T-cell ex-
haustion (14, 19, 20). Moreover, these exhausted CD8 T cells
segregated into a series of discrete subsets that expressed differ-
ent numbers and combinations of inhibitory receptors (9). Al-
ternatively, the precise phenotypic characteristic of exhausted
CD8 T-cell subsets may vary in different anatomical sites. During
chronic infection, there was substantial redistribution of virus-
specific CD8 T cells to nonlymphoid tissues (14). Following this
redistribution, expression of inhibitory receptors on virus-specific
CD8 T cells might be differentially regulated by different tissue
microenvironments. Indeed, during chronic LCMV infection,
virus-specific CD8 T cells in the liver, brain, and bone marrow
expressed higher PD-1 than those in the blood, which was de-
pendent on the differential levels of virus antigen load and PD-1
we also observed compartmental differences in Tim-3 and PD-1
expression on CD8 T cells during chronic LCMV infection (Fig.
1D and Fig. S1C). Thus, together with previous reports (14, 21),
our results suggest that analysis of one compartment during
chronic viral infection may not predict the phenotypic character-
istics as well as the number of antigen-specific CD8 T cells present
in other tissues.
Recently, a novel population of HIV-specific suppressor CD8
T cells has been shown to express IL-10 during HIV infection (15,
16). However, as a surface marker has not yet been identified, the
use of functional characterization by IL-10 production is required
to discriminate suppressor from effector CD8 T cells during
chronic infections. In the present study, we demonstrated that the
subset of CD8 T cells producing IL-10 was phenotypically iden-
PD-1. These results suggest that coexpression of Tim-3 and PD-1
may be used as a phenotypic marker for defining antigen-specific
suppressor CD8 T cells. In addition, it is possible that therapeutic
vaccine-derived antigenic stimulation may drive these suppressor
CD8 T cells to produce IL-10 during chronic infection, further
limiting the magnitude of CD8 T-cell responses. However, con-
sidering that many therapeutic vaccines occur in the setting when
viral load is substantially reduced by antiviral drug treatment, it
should be first addressed whether this IL-10–producing CD8
subsetwouldexistwhen antigen load isdecreased.Interestingly, it
production from monocytes and led to reversible CD4 T cell
how PD-1 or Tim-3 pathways are involved in IL-10 production on
Tim3+PD1+CD8 T cells, which might provide new insights in
developing immune therapies against chronic infections.
notonTh2 cellsand plays animportant roleininducingperipheral
tolerance (11). This suppressive effect of Tim-3 was mediated by
the apoptosis of Th1 cells (23). However, in the present study,
exhausted CD8 T cells expressing Tim-3 were sustained without
elimination during chronic viral infection. Although our results
indicate that Tim-3 pathway is a central negative regulator of the
T cell response, it is not clear why these exhausted CD8 T cells
expressing Tim-3 were not killed by apoptosis. One possible ex-
planation is that the expression of galectin-9 may be limited in
concentration in the chronically infected hosts. Another possibility
CD8 T and Th1 cells, resulting in differential susceptibility to
cells expressing Tim-3 were more resistant to galectin-9–induced
apoptosis than CD4+effector T cells (24).
Although blockade of the Tim-3 pathway alone had only
minimal effect on T-cell function and viral control, combined
blockade of Tim-3 and PD-1 pathways resulted in substantially
better reversal of exhaustion and control of virus than did
blockade of PD-1 pathway alone. Similarly, in another study (9),
blockade of Lag3 alone had minimal to no effect on the severity
of exhaustion but combined blockade of Lag3 and PD-1 syner-
gistically improved T-cell responses and diminished viral load in
vivo. Conversely, in vitro blockade of Tim-3 alone restored the
function of HIV-1–specific T cells in which the Tim3+PD1−
subset was one of the major populations of CD8 T cells (12).
Considering that Tim3−PD1+and Tim3+PD1+subsets were
major populations of exhausted CD8 T cells during chronic
LCMV infection, it appears that the PD-1 pathway is the major
regulator in promoting and maintaining exhausted CD8 T cells
expressing PD-1. However, during late stage of chronic LCMV
infection (>200 d), single blockade of PD-1 pathway also showed
Viral titer was determined by plaque assay in the blood before and after
treatment. Viral load in spleen, liver, and lung at 2 wk after treatment is
shown. Data are representative of three independent experiments with five
to six mice per group in each experiment. Error bars represent SEM.
Dual blockade of Tim-3 and PD-1 pathways enhances viral control.
Jin et al. PNAS
| August 17, 2010
| vol. 107
| no. 33
only modest effect on antiviral immunity, whereas dual blockade Download full-text
still expanded the virus-specific CD8 T cells responses (Fig. S4).
Although the precise role of Tim-3 and PD-1 pathways in CD8 T
cell exhaustion should be further investigated, these results in-
dicate that the Tim-3 and PD-1 pathways may play independent
roles in CD8 T-cell exhaustion during chronic viral infection.
This can be further supported by our data showing that the
population of CD8 T cell coexpressing Tim-3 and PD-1 exhibited
a more severe defect in proliferation and production of cyto-
kines, such as IFN-γ, TNF-α, and IL-2, than did those cells
expressing only PD-1. Thus, dual blockade of Tim-3 and PD-1
pathways may allow for a more comprehensive reversal of T-cell
exhaustion, potentially leading to potent combination therapies.
Materials and Methods
Mice and Infections. Six-week-old female C57BL/6 mice were purchased from
Jackson Laboratory. LCMV strains were propagated, titered, and used as
previously described (14). For acute infection, mice were intraperitoneally
infected with 2 × 105pfu of LCMV Armstrong strain. For chronic infection,
mice were infected i.v. with 2 × 106pfu of LCMV clone-13 strain, and were
intraperitoneally injected with 500 μg anti-CD4 antibody (GK 1.5; BioExpress)
on day −1 and day +1 relative to infection with LCMV clone-13 on d 0. All
mice were used in accordance with National Institutes of Health and the
Emory University Institutional Animal Care and use Committee guidelines.
Flow Cytometry and Intracellular Cytokine Staining. Lymphocytes were iso-
lated from tissue including spleen, liver, lung, and blood as previously de-
scribed (3). The antibodies to CD8 (53-6.7), Ki67 (B56), IFN-γ (XMG 1.2), TNF-α
(MP6-XT22), IL-2 (JES6-5H4), IL-10 (JES5-16E3), CD107a (1D4B), and CD107b
(ABL093) were purchased from BD Bioscience. Anti-CD44 (IM7), anti–Tim-3
(215008), and anti–PD-1 (RMP 1–30) were obtained from eBioscience, R&D
Systems, and BioLegend, respectively. MHC class I tetramers or multimers
conjugated with APC and Qdot565 (Invitrogen) were generated and used as
previously described (14, 25). Surface and intracellular cytokine staining was
performed as described (14). To detect degranulation, splenocytes were
stimulated with individual LCMV peptides or a pool of eight LCMV epitopes
for 5 h in the presence of brefeldin, monensin, anti–CD107a-FITC, and anti–
CD107b-FITC. Cells were then analyzed on an LSR II flow cytometer (BD
Immunocytometry Systems). Data were analyzed with FlowJo v.8.8 (TreeS-
tar). Dead cells were removed by gating on Live/Dead NEAR IR (Invitrogen).
In Vitro Proliferation Assay. CD8 T cells were purified to more than 90% purity
by using magnetic beads (Miltenyi Biotec). Tim3+PD1+and Tim3−PD1+CD8 T
cells were isolated by FACS sorting. CFSE-labeled Tim3+PD1+and Tim3−PD1+
CD8 T cells were cocultured with splenocytes from Thy1.1+C57BL/6 mice in
the presence of LCMV peptides for 3 d. To analyze proliferation, loss of CFSE
in Thy1.2+-gated cells was determined by flow cytometry.
In Vivo Blockade and Virus Titration. For blockade of PD-1 pathway, 200 μg of
rat antimouse PD-L1 antibody (10F.9G2; prepared in house) or rat IgG2b
isotype control were administered intraperitoneally every 3 d for 2 wk. For
blockade of Tim-3 pathway, 100 μg of Tim-3-Ig fusion protein (prepared in
house) were injected intraperitoneally every 2 d for 2 wk. The ability of Tim-
3-Ig and anti–PD-L1 to block the Tim-3 and PD-1 pathways, respectively, has
been previously demonstrated (3, 26). Titers of virus from serum or ho-
mogenized tissue sample were determined by plaque assay on Vero cells as
previously described (27).
Statistical Analysis. Data were analyzed using Prism 5.0 software (GraphPad).
Experiments were repeated two or three times. The data presenting the
differences between the groups were assessed using two-tailed unpaired
Student t tests. P < 0.05 indicated that the value of the test sample was
significantly different from that of the relevant controls.
ACKNOWLEDGMENTS. We thank B. T. Konieczny and H. Wu for technical
assistance and members of the R.A. laboratory for helpful discussion. This
work was supported by grants from the Gates Foundation Grand Challenge
in Global Health (R.A.); National Institutes of Health Grants AI054456,
AI08080192, AI056299 (to G.J.F.), AI73748, NS038037, NS045937, NS030843
(to V.K.K.), and NS054096 (to A.C.A.); Korean Health Technology Research
and Development Project A091204 from the Ministry for Health, Welfare
and Family Affairs, Republic of Korea (to S.-J.H.); National Multiple Sclerosis
Society (V.K.K.); Juvenile Diabetes Research Foundation Center for Immu-
nological Tolerance at Harvard University (V.K.K.); and an Innovation Award
from the Ragon Institute of Massachusetts Institute of Technology, Massa-
chusetts General Hospital, and Harvard University (V.K.K.). V.K.K. is a re-
cipient for the Javits Neuroscience Investigator Award from the National
Institutes of Health.
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