The Journal of Experimental Medicine
Vol. 203, No. 10, October 2, 2006 2281–2292 www.jem.org/cgi/doi/10.1084/jem.20061496
Cellular immune responses play a pivotal role
in the ability of HIV-infected individuals to
control viral replication (1). The immune sys-
tem, however, ultimately fails to control the
virus and the majority of HIV-infected pa-
tients develop AIDS. Several mechanisms of
viral immune evasion have been proposed
(2–4). Accumulated data suggest that the func-
tional capacity of HIV-specifi c CD8+ T cells
is adversely aff ected by HIV infection. Un-
der chronic antigen stimulation HIV-specifi c
CD8+ T cells, which lack CD4+ T cell help,
express an impaired ability to produce cyto-
kines (termed “exhaustion”) (5). Part of the
CD8+ T cell profi le in HIV infection also
involves an impaired ability to survive and
proliferate (6) that could further contribute to
the overall impairment in cytokine production.
Therefore, therapeutic interventions that en-
hance the survival of these cells could also aug-
ment their proliferative and cytokine functions,
thereby leading to improved immune control
of HIV infection.
A variety of manipulations can augment
HIV-specifi c CD8+ T cell responses in vitro.
Treatment with cytokines (i.e., IL-2 and IL-
15) can increase survival and eff ector function
of these cells (7, 8). Furthermore, cross-linking
of costimulatory T cell receptors such as 4-
1BB, CD80, and OX40 has been previously
described to enhance HIV-specifi c CD8+ T
cell responses either directly (9, 10) or indi-
rectly (11). However, blocking of negative T
cell regulators off ers an alternative approach to
restoring T cell function. Emerging data indi-
cate that the outcome of a T cell response is, at
least in part, dependent on the interplay be-
tween positive and negative costimulatory
molecules expressed on T cells and antigen-
presenting cells (12, 13). Recently, restoration
of exhausted virus-specifi c CD8+ T cells in a
mouse model of chronic lymphocytic chorio-
meningitis virus infection was achieved by
blocking programmed death (PD)-1, but not
CTLA-4 (14), two negative regulators of T cell
activation and function (15). PD-1 is the newest
PD-1 is a regulator of virus-specifi c CD8+
T cell survival in HIV infection
Constantinos Petrovas,1 Joseph P. Casazza,1 Jason M. Brenchley,2
David A. Price,2,3 Emma Gostick,3 William C. Adams,1 Melissa L. Precopio,1
Timothy Schacker,4 Mario Roederer,5 Daniel C. Douek,2
and Richard A. Koup1
1Immunology Laboratory, 2Human Immunology Section, and 5ImmunoTechnology Section, Vaccine Research Center,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
3Nuffi eld Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
4Department of Medicine, University of Minnesota, Minneapolis, MN 55455
Here, we report on the expression of programmed death (PD)-1 on human virus-specifi c
CD8+ T cells and the effect of manipulating signaling through PD-1 on the survival, pro-
liferation, and cytokine function of these cells. PD-1 expression was found to be low on
naive CD8+ T cells and increased on memory CD8+ T cells according to antigen specifi city.
Memory CD8+ T cells specifi c for poorly controlled chronic persistent virus (HIV) more
frequently expressed PD-1 than memory CD8+ T cells specifi c for well-controlled persistent
virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of
maturational markers on memory CD8+ T cells and was not directly associated with an
inability to produce cytokines. Importantly, the level of PD-1 surface expression was the
primary determinant of apoptosis sensitivity of virus-specifi c CD8+ T cells. Manipulation of
PD-1 led to changes in the ability of the cells to survive and expand, which, over several
days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regula-
tor of apoptosis that can impact the frequency of antiviral T cells in chronic infections such
as HIV, and could be manipulated to improve HIV-specifi c CD8+ T cell numbers, but possibly
not all functions in vivo.
C O R R E S P O N D E N C E
Richard A. Koup:
Abbreviations used: mDC,
myeloid dendritic cell; PD,
programmed death; pDC,
plasmacytoid dendritic cell;
PD-L, programmed death ligand;
TLR, toll-like receptor; VV,
2282 PD-1 MEDIATES VIRUS-SPECIFIC CD8 T CELL SURVIVAL | Petrovas et al.
member of the CD28 family, expressed on activated CD4+
and CD8+ T cells, B cells, and macrophages (16). Although
there is some evidence showing delivery of positive signals by
the ligands of PD-1, programmed death ligand (PD-L)1 and
PD-L2, (17, 18) the vast majority of the data point to nega-
tive regulation of T cell activation, proliferation, and cyto-
kine production by these ligands (19–21). In support of this
negative role, in vitro studies using primary human T cells
have shown that PD-1 ligation can inhibit TCR-mediated
signaling by reducing phosphorylation of ZAP70, activation
of PKCθ (22), and activation of Akt (23). PD-1 was originally
cloned from cell lines exhibiting high sensitivity to apoptosis
(24). More recently, induction of apoptosis of tumor-specifi c
CD8+ T cells by PD-L1 was described (25), a process medi-
ated by other receptors in addition to PD-1 (25). Therefore,
induction of apoptosis could be a key mechanism used by the
PD-1–PD-L system to aff ect the outcome of a virus-specifi c
T cell response.
Importantly, blockade of PD-1 has been shown to be ef-
fective in enhancing virus-specifi c CD8+ T cell responses
Figure 1. PD-1 is highly expressed on HIV-specifi c CD8+ T cells.
(A) Representative histograms depicting PD-1, PD-L1, and PD-L2 expres-
sion in monocytes, mDCs, and pDCs from one subject tested. Cell prepa-
rations from at least three donors were tested. (B) The polychromatic
fl ow cytometry gating scheme for identifi cation of cell populations is
shown. Histograms depict the PD-1 expression in HIV- and CMV-specifi c
CD8+ T cells from the same sample. Memory subsets identifi ed by CD27
and CD45RO staining of total CD8+ T cells are presented. (C) Pooled data
showing the percentage of CD8+ T cells expressing PD-1+ phenotype in
total CD8+ T cells from healthy donors (n = 7) and HIV+ individuals
(n = 17), HIV-specifi c (n = 11), CMV-specifi c (n = 12), and EBV-specifi c
(n = 5) CD8+ T cells from HIV+ individuals, and VV-specifi c CD8+ T cells
from vaccinated healthy donors (n = 3). PD-1 expression in memory
subsets of total CD8+ T cells from HIV+ and healthy donors is also
shown. Horizontal lines depict mean values. The p values were calculated
using Student’s t test.
JEM VOL. 203, October 2, 2006
in mice lacking CD4+ T cell help (14), raising the possibil-
ity that, if such negative regulation of CD8+ T cell func-
tion were operative in HIV infection, blocking this pathway
might enhance HIV-specifi c CD8+ T cell function. Here,
we report on the expression of PD-1 on HIV-specifi c CD8+
T cells, and CD8+ T cells specifi c for other viruses, and the
consequences of manipulating the PD-1–PD-L system on
survival, proliferation, and cytokine production by these cells.
We found that HIV-specifi c CD8+ T cells express high levels
of PD-1 that may play a critical role for their survival, and
that the control of survival is the predominant mechanism
through which PD-1 aff ects HIV-specifi c T cell function.
HIV-specifi c CD8+ T cells express high levels of PD-1
independently of their maturational status
PD-1 has two known ligands, PD-L1 and PD-L2 (26). We
fi rst investigated the expression of PD-1 and its ligands on
antigen-presenting cells: monocytes, myeloid dendritic cells
(mDCs) (27), and plasmacytoid dendritic cells (pDCs) (28–
30). We purifi ed human CD11c+ mDCs and CD123+ pDCs
from elutriated monocytes (31, 32). In vitro activation of pu-
rifi ed monocytes with LPS resulted in up-regulation of PD-
L1, whereas PD-1 and PD-L2 were less aff ected (Fig. 1 A).
Cross-linking of toll-like receptor (TLR)7 or 8 (with the
TLR7/8 ligand Resiquimod) on mDCs substantially stimu-
lated the expression of both PD-L1 and PD-L2 and caused
a slight induction in PD-1 expression (Fig. 1 A). In marked
contrast, TLR7/8-mediated activation of pDCs had no eff ect
on either PD-1 or its ligands, suggesting that pDCs are not
involved in regulating CD8+ T cell function via PD-1–PD-L
interactions (Fig. 1 A). Our data indicate that mDCs and
monocytes, in addition to presenting antigen and costimula-
tory signals to virus-specifi c CD8+ T cells, may also serve to
regulate CD8+ T cell function through the expression of
PD-1–specifi c ligands.
We next assessed the expression of PD-1 on naive, mem-
ory, and virus-specifi c CD8+ T cells using polychromatic
fl ow cytometry. The gating scheme for identifi cation of the
various CD8+ T cell subsets is shown in Fig. 1 B. Our results
show that PD-1 expression on naive (CD27+CD45RO−)
CD8+ T cells is infrequent in both HIV− and HIV+ donors,
consistent with previous studies (19, 33) (Fig. 1 C). How-
ever, in HIV+ subjects, PD-1 expression was more frequent
on CD27+CD45RO+ memory CD8+ T cells than on the
other memory CD8+ subsets (P = 0.02 and 0.0001 versus
CD27−CD45RO+ and CD27−CD45RO− memory CD8+
T cells, respectively). This distinction was not present within
memory CD8+ T cells from HIV− subjects. We next assessed
PD-1 expression on virus-specifi c CD8+ T cells after staining
with HIV-, CMV-, EBV-, and vaccinia virus (VV)–specifi c
tetramers (example shown in Fig. 1 B). We found diff erent
levels of PD-1 expression on memory CD8+ T cells accord-
ing to their specifi city. In general, HIV-specifi c and EBV-
specifi c CD8+ T cells were found to express PD-1 more
frequently than CMV-specifi c CD8+ T cells, and VV-spe-
cifi c CD8+ T cells rarely expressed PD-1 (Fig. 1 C). Similar
patterns of PD-1 expression with respect to virus antigen
specifi city were obtained when PD-1 expression was ana-
lyzed by mean fl uorescence intensity, rather than the fre-
quency of cells that express a given level of PD-1 (unpublished
data). We did not see a correlation between plasma viral load
and PD-1 expression on HIV-specifi c CD8+ T cells in this
small group of subjects (unpublished data), although in larger
cohort studies such an association has been clearly demon-
strated (34, 35).
Figure 2. PD-1 expression is independent of the maturational sta-
tus of HIV- and CMV-specifi c CD8+ T cells. (A) Representative fl ow
cytometry showing the distribution of PD-1+ and PD-1− HIV- and CMV-
specifi c CD8+ T cells in memory CD8+ T cell subsets. Cells were gated as
in Fig. 1. The distribution of total CD8+ T cells is also shown. (B) Pooled
data showing the percentage of CD8+ T cells in memory populations for
PD-1+ and PD-1− subsets of HIV-specifi c (n = 8) and CMV-specifi c (n = 11)
CD8+ T cells from HIV-infected individuals. The p values were calculated
using Student’s t test.
2284 PD-1 MEDIATES VIRUS-SPECIFIC CD8 T CELL SURVIVAL | Petrovas et al.
Figure 3. No direct association between PD-1 expression and
cytokine production by virus-specifi c CD8+ T cells. (A) Pooled data
depicting the percentage of antigen-specifi c CD8+ T cells that produce
IFN-γ (left), TNF-α (middle), or IL-2 (right) after stimulation with HIV
Gag peptides, CMV pp65 peptides, or VV. Cells from HIV+ (n = 7), CMV+
(n = 10), and healthy individuals immunized with modifi ed vaccinia
virus Ankara (n = 25) were tested. (B) PBMCs from an HIV and CMV
coinfected subject were stimulated with A2-Gag or A2-CMV peptides,
and epitope-specifi c CD8+ T cells were gated according to their expres-
sion of tetramer and/or production of cytokine, and the production of
JEM VOL. 203, October 2, 2006
We asked whether PD-1 expression on antigen-specifi c
cells is related to their altered maturational status (6, 36). The
vast majority of HIV-specifi c CD8+ T cells was found to
express a CD27+ phenotype, in agreement with previously
published data (37). There was no diff erence in the matura-
tional phenotype of the PD-1+ and PD-1− fractions of HIV-
specifi c CD8+ T cells (Fig. 2, A and B). CMV-specifi c
CD8+ T cells were evenly distributed among the three
memory phenotypes defi ned by CD27 and CD45RO; fur-
thermore, PD-1+ and PD-1− CMV-specifi c CD8+ T cells
exhibited the same distribution of these three memory phe-
notypes (Fig. 2, A and B). We also found that there was
no diff erence in the expression of other homing and matura-
tional markers (CCR7 and CD57) between PD-1+ and
PD-1− antigen-specifi c CD8+ T cells (unpublished data).
Our data therefore indicate that PD-1 expression is indepen-
dent of the maturational state of antigen-specifi c memory
CD8+ T cells.
Expression of PD-1 does not directly affect the ability
of HIV-specifi c CD8+ T cells to produce cytokines
Knowing that sustained high levels of PD-1 are associated
with a diminished ability of LCMV-specifi c CD8+ T cells to
produce cytokine in chronically infected mice (14), we hy-
pothesize that PD-1 expression on human HIV-specifi c
CD8+ T cells would similarly result in impaired cytokine
production. We fi rst confi rmed that the ability of CD8+ T
cells to produce cytokines in response to antigen stimulation
was impaired in those T cells known to express high levels
of PD-1. We measured IFN-γ, TNF-α, and IL-2 produc-
tion by CD8+ T cells after HIV Gag, CMV pp65, and VV
stimulation, and assessed the proportion of the cells that
were able to produce any one of the three cytokines. As
shown in Fig. 3 A, there was no diff erence in the proportion
of the HIV Gag–, CMV pp65–, or VV-specifi c CD8+ T
cells that were able to produce IFN-γ, but there was lower
production of TNF-α and IL-2 by CD8+ T cells known to
express higher levels of PD-1 (HIV > CMV > VV). This
confi rmed that PD-1 expression is associated with impaired
cytokine (TNF-α and IL-2) production by antigen-specifi c
CD8+ T cells.
We next asked if the impaired cytokine function resulted
directly from the expression of PD-1, or whether the two
phenomena were only indirectly linked. The expression of
PD-1 in relation to production of cytokines under short (6 h)
stimulation was assessed in HIV- and CMV-specifi c CD8+ T
cells. Cells were stimulated with peptides corresponding to
HLA-A2–defi ned optimal HIV and CMV epitopes, and the
production of IFN-γ, TNF-α, and IL-2 was measured within
tetramer+ cells according to PD-1 expression. The data dem-
onstrate that IFN-γ and TNF-α are readily produced from
PD-1+ cells, whereas IL-2 production is rarely produced
from either PD-1+ or PD-1− CMV- or HIV-specifi c CD8+
T cells (Fig. 3 B). We next gated on the PD-1+ and PD-1−
subsets of antigen-specifi c cells (defi ned by cytokine produc-
tion and/or tetramer staining) and calculated the proportion
of the cells that were nonfunctional for cytokine production
after stimulation with a range of peptide concentrations (Fig.
3 C). For both HIV- and CMV-specifi c CD8+ T cells at all
levels of antigen stimulation, there was no diff erence in the
production (or lack thereof) of IFN-γ, TNF-α, or IL-2 be-
tween PD-1+ and PD-1− cells. Therefore, PD-1 expression
does not appear to be directly responsible for the inability of
some CMV- and HIV-specifi c CD8+ T cells to produce
We postulated, however, that PD-1 may not be ade-
quately engaged by its ligands during the ex vivo stimula-
tions. We therefore performed 6-h peptide stimulations in
the presence and absence of anti–PD-1 antibody to directly
engage the PD-1 pathway during the time of antigen stimu-
lation. Raw data for one subject and compiled data for three
subjects are shown in the left and right panels, respectively, of
Fig. 3 D, and show that engagement of PD-1 during antigen
stimulation had no eff ect on the ability of the antigen-specifi c
T cells to produce IFN-γ, TNF-α, or IL-2. Collectively,
these data demonstrate that PD-1 does not directly infl uence
the capacity of HIV- or CMV-specifi c CD8+ T cells to pro-
duce cytokines upon stimulation.
PD-1–PD-L1 manipulation alters virus-specifi c
CD8+ T cell proliferation and cytokine production
by regulating their survival
The major eff ect of PD-1 blockade in mice was on antigen-
specifi c CD8+ T cell proliferation (14). HIV-specifi c CD8+
T cells are characterized by limited proliferative capacity in
chronic HIV infection (38, 39), a defect that is linked to the
depletion of IL-2–secreting HIV-specifi c CD4+ and/or
CD8+ T cells (7, 39, 40). Therefore, we investigated whether
stimulation through, or interference with, the PD-1–PD-L1
pathway could aff ect the proliferation of HIV- and CMV-
specifi c CD8+ T cells. CFSE-labeled cells were stimulated
with HIV- or CMV-specifi c peptide pools in the presence
or absence of antibodies against either PD-1 or its ligand
PD-L1. After 6 d, the frequency of CFSElow cells, in either all
CD8+ or tetramer+ cells, was compared between cultures that
had received no blocking antibody, an anti–PD-1 antibody
cytokine in the PD-1+ and PD-1− subsets was assessed. The left panel
shows the PD-1 staining of CD8+ cells to demonstrate how the gating
of PD-1+ and PD-1− cells was chosen. (C) Pooled data showing the per-
centage of nonfunctional virus-specifi c CD8+ T cells from three HIV-
infected individuals off antiretroviral therapy [(tetramer+cytokine−) ×
100/(tetramer+cytokine−) + (tetramer+cytokine+)] at the different
peptide concentrations for both PD-1+ and PD-1− compartments are
shown. (D) Flow cytometry showing the production of IFN-γ, TNF-α, or
IL-2 from CD8+ T cells after stimulation with Gag or CMV peptides
for 6 h in the absence or presence of anti–human PD-1 antibody (left).
Pooled data are shown on the right. Samples from three HIV+ individuals
2286 PD-1 MEDIATES VIRUS-SPECIFIC CD8 T CELL SURVIVAL | Petrovas et al.
(that acts as a PD-1 agonist), or an anti–PD-L1 antibody
(clone MIH1; reference 41). Representative fl ow cytometry
plots are shown in the left panel of Fig. 4 A and composite
data from multiple experiments are shown in the right panel.
The data show that stimulation of PD-1, in both HIV- and
CMV-specifi c CD8+ T cells, resulted in a decrease in prolif-
erative capacity. Alternatively, blocking of the PD-1–PD-L1
interaction with an anti–PD-L1 antibody resulted in in-
creased proliferation of both HIV- and CMV- specifi c CD8+
T cells. There was variation in the amount of inhibition or
augmentation of proliferation that was not directly associated
with the simple level of PD-1 expression, suggesting that
PD-1 is not the only factor regulating the ability of antigen-
specifi c T cells to proliferate.
We postulated that the eff ect of PD-1 on proliferation
of antigen-specifi c T cells would aff ect the number of cytokine-
producing cells in a multiday assay. Therefore, the ability of
cells to produce cytokines after antigen stimulation and 6 d
of culture was examined. PD-1 cross-linking in the pres-
ence of HIV Gag peptide reduced the frequency of CD8+
T cells producing IFN-γ and had minimal eff ect on the
frequency of cells producing TNF-α (Fig. 4 B). On the
other hand, anti–PD-L1 treatment resulted in an increased
frequency of cells capable of producing both cytokines
(Fig. 4 B). Collectively, these data indicate that any eff ect
of PD-1 engagement on cytokine expression in a multiday
assay is secondary to enhanced proliferation of cytokine-
We next sought to determine if the previously described
eff ects of PD-1 on apoptosis were responsible for the ob-
served eff ects on proliferation. PD-1 expression was con-
sistently associated with higher sensitivity of total CD8+ T
cells to both spontaneous and CD95/Fas-induced apop-
tosis, estimated by annexin V surface exposure (Fig. 5 A,
representative data; Fig. 5 B, compiled data). Apoptosis sen-
sitivity was found to be signifi cantly higher in HIV-specifi c
compared with CMV-specifi c CD8+ T cells, in agreement
with previously published data (6, 42), and PD-1+ CD8+
Figure 4. PD-1–PD-L1 manipulation alters virus-specifi c CD8+
T cell proliferation and cytokine production. (A) Representative histo-
grams depicting the CFSE profi le of CD8+ T cells from an HIV+ donor.
Pooled data showing the fold change of the percentage of CFSE low cells
under treatment with aPD-L1 or aPD-1 antibody (right). The percentage
of cells that divided in the absence of antibody treatment was assigned a
value of 1. The ratio of the percentage in the presence and absence of
antibody treatment for each response was calculated. The fold change for
antigen-specifi c CD8+ (those that diluted CFSE in response to antigen;
● and ○) and gated antigen-specifi c tetramer+ CD8+ T cells (□ and ■)
is shown. The p values were calculated using Wilcoxon’s paired t test.
(B) Flow cytometry showing the CD8+ T cells secreting IFN-γ and TNF-α after
stimulation with Gag peptides in the absence or presence of anti–PD-1 or
anti–PD-L1 antibodies. Cells cultured for 6 d were restimulated with Gag
peptides for the last 6 h (left). Pooled data showing the percentage of
CD8+ T cells producing IFN-γ and TNF-α under these conditions (right).
At least four patients were tested for each treatment.
JEM VOL. 203, October 2, 2006
T cells were always more sensitive to apoptosis than PD-1−
CD8+ T cells irrespective of antigen specifi city (Fig. 5 B).
Of interest, PD-1− HIV-specifi c CD8+ T cells often were
more sensitive to apoptosis than PD-1+ CMV-specifi c
CD8+ T cells (Fig. 5 B). We therefore asked whether this
diff erence could be secondary to the diff erent maturational
phenotypes of HIV- and CMV-specifi c CD8+ T cells. In
fact we found that PD-1+CD8+ T cells were more suscep-
tible to both spontaneous and CD95/Fas-induced apoptosis
compared with PD-1−CD8+ T cells in all memory subsets
Figure 5. PD-1 regulates the in vitro survival of virus-specifi c
CD8+ T cells. (A) Representative histograms depicting the annexin V posi-
tivity in total, PD-1+ and PD-1− CD8+ T cells from an HIV+ donor under
spontaneous or CD95/Fas-induced apoptosis. (B) Pooled data showing the
percentage of annexin V+ cells in PD-1+ and PD-1− subsets of total (n = 8),
HIV- (n = 6), and CMV-specifi c (n = 5) CD8+ T cells from HIV+ individuals.
(C) Annexin V positivity in PD-1+ versus PD-1− CD8+ T cells in memory
subsets defi ned by CD27 and CD45RO markers. (D) PBMCs were cultured
in the absence or presence of plate-bound anti–PD-1, and the percentage
of annexin V+ cells was determined for CD8+ T cell subsets with different
levels of PD-1 expression. Flow cytometry showing the different levels
of spontaneous and anti–PD-1–induced apoptosis according to the level
of PD-1 expression (high, medium/high, medium/low, and low) in total,
Gag- and CMV-specifi c CD8+ T cells from the same HIV+ donor.
2288 PD-1 MEDIATES VIRUS-SPECIFIC CD8 T CELL SURVIVAL | Petrovas et al.
tested, but that sensitivity varied between maturational
phenotypes (CD27−CD45RO+ > CD27−CD45RO− >
CD27+CD45RO+; Fig. 5 C), suggesting that PD-1 expres-
sion augments apoptosis sensitivity upon the background de-
fi ned by maturation and activation status.
Another possible explanation for the greater apoptosis
sensitivity of HIV-specifi c CD8+ T cells than CMV-spe-
cifi c CD8+ T cells is the absolute level of PD-1 expression
on the cells. Although we had defi ned CD8+ T cells as be-
ing either PD-1 positive or PD-1 negative, it was appar-
ent that PD-1 expression was higher (as measured by mean
fl uorescence intensity) on HIV- than CMV-specifi c CD8+
T cells. We therefore asked if the absolute level of PD-1
expression on CD8+ T cells was the primary determinant
of apoptosis sensitivity while further determining whether
PD-1 ligation directly aff ected survival of CD8+ T cells. To
do this, we assessed apoptosis in the presence and absence of
plate-bound anti–PD-1 in samples from six HIV+ donors.
The results were consistent for all of the donors, and the
results from one are shown in Fig. 5 D. We found that HIV-
specifi c CD8+ T cells had higher expression of PD-1 than
total or CMV-specifi c CD8+ T cells, but that irrespective
of antigen specifi city, the CD8+ T cells with the highest
expression of PD-1 were the most sensitive to apoptosis and
were the cells that augmented their apoptosis to the great-
est extent upon PD-1 ligation. In fact, at medium/low and
low levels of PD-1 expression, there was low spontaneous
apoptosis that did not increase with PD-1 ligation, whereas
at high levels of PD-1 expression, PD-1 ligation augmented
the apoptosis to 80% or greater irrespective of antigen speci-
fi city. Therefore, the absolute level of PD-1 expression on
antigen-specifi c CD8+ T cells is the primary indicator of
apoptosis sensitivity and the major determinant of sensitiv-
ity to apoptosis upon PD-1 ligation. Collectively, our data
strongly support that PD-1 is a critical regulator of CD8+ T
cell survival in HIV infection.
The balance between positive and negative signals delivered
by costimulatory molecules to T cells appears to be critical
for the ultimate fate of cellular immune responses (13, 43).
Recent data (10, 11, 14) suggest that manipulation of T cell
costimulatory pathways may present a novel approach for en-
hancing and restoring virus-specifi c CD8+ T cell responses,
especially in the context of a chronic infection like HIV. We
report here on the role of PD-1, a negative costimulatory re-
ceptor of T cells, as a regulator of virus-specifi c CD8+ T cells
in HIV infection. To understand how PD-1 can aff ect the
function of CD8+ T cells, it is critical to identify which cells
provide the ligand(s) for PD-1. Our data indicate that mDCs
and monocytes may use the PD-1–PD-L system to regulate
adaptive antiviral immunity. Our fi ndings in this context are
very preliminary and much more needs to be investigated.
For instance, the relative kinetics of costimulatory molecule
expression on mDCs and monocytes after activation and the
impact of the “positive” and “negative” signals delivered by
these molecules to responding T cells remains to be eluci-
dated. In addition, it will be important to determine whether
there is redundancy between the various costimulatory sig-
nals aff ecting CD8+ T cells or if they act independently by
stimulating separate intracellular pathways after initiation of
virus-specifi c CD8+ T cell responses.
We found remarkably high expression of PD-1 on HIV-
specifi c CD8+ T cells. The frequency of PD-1 expression
on the diff erent virus-specifi c CD8+ T cells (HIV = EBV >
CMV > VV) is consistent with PD-1 regulation according
to antigen stimulation. Although it is unlikely that the level
of antigen in chronic EBV infection reaches that which
occurs in HIV infection, it has been shown that EBV is
continuously shed into saliva by induction of the lytic cycle
as B cells diff erentiate into plasma cells, thereby chronically
stimulating lytic cycle antigen-specifi c CD8+ T cells (44).
The mechanism by which CD8+ T cells control HIV and
EBV may diff er. Therefore, it may not follow that expres-
sion of PD-1 on EBV- and HIV-specifi c CD8+ T cells
will similarly impact CD8-mediated control of these two
diff erent virus infections. Further experiments are needed
to clarify the relative role of PD-1 in regulation of EBV-
specifi c CD8+ T cell responses and compare it to the regu-
lation of HIV-specifi c responses. In summary, although we
cannot conclude that chronic antigen stimulation is the sole
factor determining PD-1 expression, our data reveal that
HIV-specifi c CD8+ T cells, because of their high expres-
sion of PD-1, may be vulnerable to negative signals deliv-
ered by PD-1, potentially leading to functional consequences
Despite their diff erential expression of PD-1, no diff er-
ence in IFN-γ production was found between HIV-, CMV-,
and EBV-specifi c CD8+ T cells. On the other hand, PD-1
expression is associated with signifi cantly lower ability of
HIV-specifi c CD8+ T cells to produce TNF-α and even
lower production of IL-2. This is in agreement with the fi nd-
ing that PD-1–PD-L1 blockage has a substantial impact on
LCMV-specifi c CD8+ T cells producing both IFN-γ and
TNF-α, whereas it has only a slight eff ect on single IFN-γ
producers. However, we found that PD-1− and PD-1+ anti-
gen-specifi c CD8+ T cells were equally able to produce cy-
tokines upon antigen stimulation, indicating that PD-1
expression has no direct eff ect on cytokine production. This
was further supported by our fi nding that ligation of PD-1
during antigen stimulation had no eff ect on cytokine produc-
tion by virus-specifi c CD8+ T cells. Collectively these data
clearly demonstrate that PD-1 has no direct eff ect upon the
immediate ability of antigen-specifi c CD8+ T cells to pro-
duce IFN-γ, TNF-α, or IL-2.
Manipulation of the PD-1–PD-L system was found to
alter the proliferation of virus-specifi c CD8+ T cells. This
was accompanied by altered percentages of CD8+ T cells
producing cytokines. The change in proliferation could re-
sult from an altered ability of these cells to either survive or
divide. Importantly, we found no relationship between PD-1
expression and the degree of change in proliferative capacity
JEM VOL. 203, October 2, 2006
after manipulation of the PD-1–PD-L1 axis. In fact, in some
instances where the expression of PD-1 was very high on
HIV-specifi c CD8+ T cells, only minor eff ects of PD-1 liga-
tion on proliferation were observed. Therefore, although
PD-1 has a demonstrable eff ect on the ability of virus- specifi c
(CMV and HIV) CD8+ T cells to proliferate, it is not the
sole factor regulating this function. This is not surprising as
other factors (i.e., TCR activation threshold, relative expres-
sion of other costimulatory molecules, or levels of adaptor
proteins mediating the intracellular signaling delivered by
PD-1) could also contribute to the ability of PD-1 ligation to
aff ect proliferative capacity.
At least two interventions have now proven successful in
vitro in restoring the proliferation of HIV-specifi c CD8+ T
cells: the addition of IL-2 (or CD4+ T cells producing IL-2)
and the manipulation of costimulatory pathways such as PD-1.
This raises the question of whether these two diff erent ma-
nipulations aff ect proliferation through overlapping intracel-
lular mechanisms. In addition, whether they can act in a
synergistic mode remains to be elucidated. Since IL-2 cannot
overcome the proliferative defect in CD57+CD8+ T cells
(45), it is of particular interest to examine whether manipula-
tion of PD-1–induced pathways could specifi cally restore
their proliferative capacity.
Our data indicate that the primary mechanism by which
PD-1 aff ects CD8+ T cell function is by regulating the ability
of these cells to survive. This is in agreement with the origi-
nally described role of PD-1 as an apoptotic factor (24) and
the reduced survival that characterizes virus-specifi c CD8+ T
cells under conditions of chronic antigen stimulation (46,
47). We can speculate on how PD-1 expression could aff ect
HIV-specifi c CD8+ T cell survival. It is possible that stimula-
tion through PD-1 can direct cells into a cell cycle resting
state, as has been described for the PD-1–PD-L2 interaction
(21). We found that lack of PD-1 expression is associated
with similar levels of spontaneous and CD95/Fas-induced
apoptosis, whereas CD95/Fas-induced apoptosis is greatly
augmented in CD8+ T cells that express PD-1, indicating
that there may be cross-talk between the signals induced by
these two receptors. Previously published data have shown
that the Fas–FasL interaction impacts PD-L1–induced apop-
tosis of activated T cells (25). Although no direct link be-
tween PD-1 and CD95/Fas was described in that work (25),
the possibility that PD-1 could prime (especially under con-
ditions of chronic stimulation) CD8+ T cells to undergo
CD95/Fas-induced apoptosis cannot be excluded. There-
fore, clarifi cation of the intracellular mechanism(s) governing
the proapoptotic function of PD-1 is of particular interest.
Furthermore, the role of such a function on CD4+ T cell
survival in HIV infection would signifi cantly add to our un-
derstanding of HIV pathogenesis.
A clear conclusion from our results is that the absolute
level of PD-1 expression is a major determinant of spontane-
ous apoptosis and sensitivity to PD-1 ligation. We conclude
this despite our observation that PD-1− HIV-specifi c CD8+
T cells are often more susceptible to apoptosis than PD-1+
CMV-specifi c CD8+ T cells (Fig. 5 B). Although it is known
that sensitivity to apoptosis is also aff ected by other factors—
specifi cally the level of T cell activation (defi ned by CD38
expression; unpublished data) and maturational state, which
we have shown is independent of PD-1 expression (Fig. 2,
A and B and Fig. 5 C)—our data indicate that PD-1 is a pri-
mary determinant of apoptosis sensitivity over and above these
other factors. We conclude this because, within any popula-
tion of CD8+ T cells (defi ned by activation, maturation, or
antigen specifi city), the PD-1+ population is more sensitive
to apoptosis than the PD-1− population. In addition, al-
though we have described PD-1 expression as either positive
or negative in most of our data, expression really represents a
continuum, with high expression being associated with
greater impact upon PD-1–regulated functions (i.e., apopto-
sis; Fig. 5 D). When CD8+ T cells of diff erent antigen speci-
fi cities, but similar levels of PD-1 expression, are analyzed,
they exhibit similar levels of spontaneous apoptosis and sensi-
tivity to PD-1 ligation. In addition, cells with moderate to
low expression of PD-1 have low levels of spontaneous apop-
tosis and are not aff ected by PD-1 ligation. What this means
is that within any population of antigen-specifi c CD8+ T
cells, it is the absolute level of PD-1 that primarily dictates
the rate of spontaneous apoptosis and sensitivity to PD-1 li-
gation. Therefore, what is unique about HIV-specifi c CD8+
T cells is their high level of PD-1 expression leading to a
profound (but potentially reversible) survival defect. Al-
though the proliferative capacity of a CD8+ T cell is deter-
mined by more than just an ability to resist apoptosis, it is not
diffi cult to visualize how the level of PD-1 and associated
sensitivity to apoptosis would impact on the ability of a cell
Overall, our data demonstrate that PD-1 is preferentially
expressed on CD8+ T cells specifi c for chronic viruses, and
that PD-1 interaction with its ligands can regulate the ability
of these virus-specifi c CD8+ T cells to survive and proliferate.
Therefore, manipulation of this axis may lead to at least
partial restoration of antigen-specifi c cell numbers and func-
tion in chronic viral infections such as HIV. It is important
to remember that our data do not support the ability of
PD-1 manipulation to restore all of the T cell functions that
defi ne functional “exhaustion.” For instance, we have no
evidence that PD-1 blockade will restore absent cytokine
functions, and may only aff ect CD8+ T cell proliferation to
the degree possible in the context of other as yet undeter-
mined defects in HIV-specifi c CD8+ T cells. Therefore,
although our data identify PD-1 as a potential therapeutic
target for restoring functional capacity of HIV-specifi c CD8+
T cell responses, it may not be capable of fully restoring
function. In addition, it should be appreciated that the PD-
1–PD-L1 axis likely evolved to attenuate potentially harmful
CD8+ T cell responses to both self-antigens and chronic
pathogens. Given that many non–HIV-specifi c CD8+ T
cells express PD-1 (Fig. 1 C), it is likely that interventions to
release all CD8+ T cells from PD-1–mediated suppression
will have untoward eff ects.
2290 PD-1 MEDIATES VIRUS-SPECIFIC CD8 T CELL SURVIVAL | Petrovas et al.
MATERIALS AND METHODS
Patients. PBMCs were obtained from HIV+ and HIV− volunteers and
cryopreserved until use. None of the HIV-infected subjects in this study
were on antiretroviral therapy, and they had a range of viral loads from
<50 to 439,000 per ml. Signed informed consent approved by the rele-
vant Institutional Review Board was obtained. Samples from VV-naive
individuals, preimmunized with modifi ed vaccinia virus Ankara followed by
scarifi cation with Dryvax, were also used. Samples were taken within 3 mo
of Dryvax administration. Purifi cation of DC subsets has been previously
Antibodies. The following directly conjugated antibodies were used:
CD3-Cy7APC, IFN-γ–FITC, TNF-α–Cy7PE, CD14-FITC, CD11c-PE,
CD11c-APC, PD-1–PE, PD-L1–Cy7PE, PD-L2–APC (all from BD
Biosciences) and CD45RO-TexasRedPE (Beckman Coulter). Annexin
V-Cy5PE and the following antibodies were conjugated in our laboratory:
IL-2–FITC, IL-2–APC, CD14–Cascade blue, CD19–Cascade blue, CD8-
Qdot 705, and CD27-Cy5PE. The unconjugated mAbs were obtained from
BD Biosciences. Cascade blue was obtained from Molecular Probes. Cy5
was obtained from BD Biosciences. Quantum Dots were obtained from the
Quantum Dot Corporation. The following APC-labeled tetramers were
prepared as previously described (48): A2-Gag (S L Y N T V A T Y L ), A2-CMV
(NLVPVMTV), A2-Vaccinia (KVDDTFYYV), B8-Nef (FLKEKGGL),
and A2-EBV (GLCTLVAML).
Cell stimulation. PBMCs were thawed and rested overnight at 37°C.
Viability by Trypan blue exclusion was typically ≥90%. 2 × 106 PBMCs
were diluted to 1 ml with medium containing costimulatory antibodies
(αCD28 and αCD49d) (1 μg/ml) (Becton Dickinson), monensin (0.7 μg/ml;
BD Biosciences), brefeldin A (10 μg/ml; Sigma-Aldrich), in the absence
or presence of indicated amounts (μg/μl) of A2-Gag (SLYNTVATL) or
A2-CMV (NLVPVMTV) epitope peptides and incubated for 6 h. Cells
were washed and incubated with pretitrated amounts of APC-labeled A2-
gag or A2-Cmv tetramer for 15 min at 37°C. After washing, cells were
surface stained with PD-1–PE, CD8–Qdot 705, CD14– and CD19–cascade
blue, and 156 ng/ml violet amine reactive viability dye (vivid; In Vitrogen).
Following permeabilization (Cytofi x/Cytoperm kit; BD Biosciences),
cells were stained with IFN-γ–FITC or IL-2–FITC, TNF-α–PECy7, and
CD3-Cy7APC. Alternatively, cells were left untreated or preincubated
for 30 min at 37°C with an anti–human PD-1 antibody (AF 1086; R&D
Systems) (20 μg/ml) and subsequently stimulated with peptides (15mers
overlapping by 11) corresponding to full-length HIV-1 Gag (2 μg/ml each
peptide, 5 μl/ml; National Institutes of Health AIDS Research and Refer-
ence Reagent Program) or cytomegalovirus antigen (60 μl/ml; Microbix
Biosystems Inc.) for 6 h. After a washing step, cells were stained with vivid,
CD14– and CD19–cascade blue, CD8-Qd705, permeabilized, and stained
intracellularly with CD3-Cy7APC, IFN-γ–FITC, IL-2–APC, and TNF-
α–Cy7PE. For CFSE studies, PBMCs were washed thoroughly and labeled
with 0.25 μM CFSE (Molecular Probes). Cells were adjusted to 1.5 × 106
cells/ml and cultured in the presence of peptides (15mers overlapping by
11) corresponding to full-length HIV-1 Gag (2 μg/ml each peptide, 5 μl/ml;
National Institutes of Health AIDS Research and Reference Reagent
Program) or cytomegalovirus CF antigen (60 μl/ml; Microbix Biosystems
Inc.). aCD28/aCD49d (1 μg/ml) was used for costimulation. An unstimu-
lated and a positive control (culture with 1 μg/ml immobilized anti-CD3,
clone UCHT1; R&D Systems) was included in each assay. Antibodies
against human PD-1 (AF 1086; R&D Systems) (20 μg/ml) and human
PD-L1 (16–5983; eBioscience) (25 μg/ml) were used for cross-linking of
PD-1 and PD-L1, respectively. Cells were cultured for 6 d, harvested, and
stained fi rst with A2-Gag or A2-CMV tetramer–APC (15 min, 37°C)
and subsequently with annexin V–Cy5PE, CD3-Cy7APC, and CD8-PE.
2.5 mM CaCl2 was included in all staining steps. Alternatively, cells were cul-
tured under the same conditions, and on day 6 cells were stained with vivid
and CD8-Qd705 and intracellularly with CD3-Cy7APC, IFN-γ–FITC,
IL-2–APC, and TNF-α–Cy7PE.
Apoptosis studies. 1–1.5 × 106 PBMCs were cultured in 24-well plates
(BD Biosciences) in the absence or presence of plate-bound anti–human
CD95/Fas (IgM, CH11; Upstate Biotechnology) (5 μg/ml) or anti–human
PD-1 (AF 1086, R&D Systems) (20 μg/ml) for 12 h at 37°C. Cells were
harvested, washed, and surface stained with APC-labeled A2-Gag, A2-
CMV, or B8-Nef tetramers, and subsequently with annexin V–Cascade
blue, CD3-Cy7APC, CD8-Qd705, CD27-CyPE, CD45RO-TRPE, and
PD-1–PE and green amine reactive viability dye (grivid; InVitrogen).
2.5 mM CaCl2 was included in all staining steps.
Flow cytometry. Cells were analyzed with a modifi ed LSRII fl ow cy-
tometer (BD Immunocytometry Systems). Between 200,000 and 1 mil-
lion events were collected. Electronic compensation was conducted with
antibody capture beads (BD Biosciences) stained separately with individual
mAbs used in the test samples. Data analysis was performed using FlowJo
version 6.0 (TreeStar). Forward scatter area (FSC-A) versus forward scatter
height (FSC-H) was used to gate out cell aggregates. CD14+ cells, CD19+
cells, and dead cells were removed from the analysis to reduce background
staining. The cells were then gated through a FSC-A versus side scatter
height (SSC-H) plot to isolate small lymphocytes. Next, CD3+ cells were
selected and PD-1 expression was measured in gated total CD8+ T cells
and tetramer+ cells, and in relation to their diff erentiation level by using the
CD27-Cy5PE and CD45RO-TexasRedPE memory markers. Tetramer+
cells were selected and the percentage of PD1+ and PD1−, IFN-γ, and
TNF-α–producing cells was determined using gating criteria determined
using the total CD3+CD8+ population. For CFSE analysis, after initial gat-
ing (FSC-A versus FSC-H), apoptotic (annexin V+) cells were removed
and small lymphocytes were identifi ed. CD3+ cells were selected, and
the percentage of CFSE low cells was determined in gated total CD8+ T
cells and tetramer+ cells. For analysis of monocytes and DCs, the follow-
ing combinations of titrated antibodies were used: CD11c-APC/CD14-
and CD123-Cy5PE/PD-1–PE/PD-L1–Cy7PE/PD-L2–APC. PD-1, PD-
L1, and PD-L2 levels were determined in CD14+CD11c− (monocytes),
CD14−CD11c+ (mDCs), and high CD123 (pDCs) cells.
Statistical analysis. Statistical analysis was performed using Student’s t test
and Wilcoxon’s paired t test. P values < 0.05 were considered signifi cant.
The GraphPad Prism statistical analysis program was used.
The authors have no confl icting fi nancial interests.
Submitted: 17 July 2006
Accepted: 21 August 2006
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