Differential Association of Programmed Death-1 and CD57
with Ex Vivo Survival of CD8?T Cells in HIV Infection1
Constantinos Petrovas,2* Benjamin Chaon,* David R. Ambrozak,* David A. Price,†‡
J. Joseph Melenhorst,§Brenna J. Hill,†Christof Geldmacher,* Joseph P. Casazza,*
Pratip K. Chattopadhyay,¶Mario Roederer,¶Daniel C. Douek,†Yvonne M. Mueller,?
Jeffrey M. Jacobson,?Viraj Kulkarni,#Barbara K. Felber,#George N. Pavlakis,**
Peter D. Katsikis,?and Richard A. Koup2*
Recent studies have revealed the critical role of programmed death-1 (PD-1) in exhaustion of HIV- and SIV-specific CD8?T cells.
In this study, we show that high expression of PD-1 correlates with increased ex vivo spontaneous and CD95/Fas-induced apo-
ptosis, particularly in the “effector-memory” CD8?T cell population from HIV?donors. High expression of PD-1 was linked to
a proapoptotic phenotype characterized by low expression of Bcl-2 and IL7-R?, high expression of CD95/Fas and high mito-
chondrial mass. Expression of PD-1 and CD57 was differentially associated with the maturation status of CD8?T cells in HIV
infection. CD57 was linked to higher apoptosis resistance, with cells expressing a PD-1LCD57Hphenotype exhibiting lower levels
of cell death. The majority of HIV-specific CD8?T cells were found to express a PD-1HCD57Lor PD-1HCD57Hphenotype. No
correlation was found between PD-1 expression and ex vivo polyfunctionality of either HIV- or CMV-specific CD8?T cells.
Contrary to CD57, high expression of PD-1 was characterized by translocation of PD-1 into the area of CD95/Fas-capping, an early
necessary step of CD95/Fas-induced apoptosis. Thus, our data further support the role of PD-1 as a preapoptotic factor for CD8?
T cells in HIV infection. The Journal of Immunology, 2009, 183: 1120–1132.
cific immunity are defective in HIV infection (1). In particular,
HIV-specific CD8?T cells exhibit an exhausted phenotype char-
acterized by reduced ex vivo survival (2) and impaired cytokine
production (3). However, the molecular mechanism(s) leading to
this exhaustion remain undefined.
Our previous studies have shown that HIV-specific CD8?T
cells are characterized by 1) reduced Bcl-2 related anti-apoptotic
potential (4); 2) high binding of the mitochondrial-specific dye
Mitotracker Green FM (an index of mitochondrial mass) (5); and
3) increased expression of programmed death-1 (PD-1)3(6), a neg-
ative regulator of T cells (7). In terms of their ability to produce
cytokines directly ex vivo, an inverse correlation was found be-
espite a broad HIV-specific CD8?T cell response, the
immune system ultimately fails to control the virus. It is
now well recognized that some functions of virus-spe-
tween viral load and the polyfunctionality of HIV-specific CD8?
T cells in HIV progressors (3). Furthermore, the preservation of
polyfunctional HIV-specific CD4?and CD8?T cell responses
could be at least partially responsible for the good clinical outcome
observed in HIV-2, as compared with HIV-1, infection (8).
PD-1 is a negative regulator of T cells, originally identified as a
surface receptor involved in apoptosis (9). An increasing body of
evidence has revealed the critical role of PD-1 in regulating virus-
specific T cell responses both in vivo (10–12) and ex vivo (13–15).
Although this role is well established at the cellular level, the mo-
lecular pathways linking PD-1 to exhaustion of virus-specific T
cells in chronic infection are poorly understood. Some studies have
linked PD-1 expression to a reduced ability of T cells to produce
cytokines (10, 13, 16). More recently, a correlation was found
between PD-1 expression on effector CD8?T cells and the fre-
quency of IL-10?suppressor CD8?T cells in advanced HIV in-
fection (17). We, like others, have emphasized the predominant
role of this receptor in regulating the survival of T cells (6, 11,
18–21). PD-1-induced signaling suppresses PI3K/Akt activation
and reduces the expression of Bcl-xL, a potent anti-apoptotic fac-
tor (22), in human T cells (23, 24). Activation of the PI3K/Akt axis
is a critical mediator that transduces a plethora of extracellular
signaling events into multiple functional outcomes affecting me-
tabolism, survival, and proliferation of T cells (25, 26).
The surface molecule CD57 has been used as a marker of rep-
licative senescence in HIV infection (27, 28). Expression of CD57
was associated with an inability of HIV-specific CD8?T cells to
divide, and total CD57?CD8?T cells were prone to ex vivo death
*Immunology Laboratory,†Human Immunology Section and¶ImmunoTechnology Sec-
tion, Vaccine Research Center, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20814;‡Department of Medical Biochem-
istry and Immunology, Cardiff University School of Medicine, Cardiff, Wales, U.K.;
§Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of
Health, Bethesda, MD 20814;?Department of Microbiology and Immunology, and De-
partment of Medicine, Drexel University College of Medicine, Philadelphia, PA 19102;
#Human Retrovirus Pathogenesis Section and **Human Retrovirus Section, Vaccine Branch,
Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21701
Received for publication January 21, 2009. Accepted for publication May 20, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1The studies were supported in part by R01 AI46719 (to P.D.K.); D.A.P. is a Medical
Research Council (U.K.) Senior Clinical Fellow.
2Address correspondence and reprint requests to Drs. Constantinos Petrovas and
Richard A. Koup, Vaccine Research Center, NIAID, National Institutes of Health, 40
Convent Drive, Bethesda, MD 20892. E-mail addresses: email@example.com and
3Abbreviations used in this paper: PD-1, programmed death-1; mDC, myeloid den-
dritic cell; MFI, mean fluorescence intensity; BDS-R3, bright detailed similarity R3;
DISC, death-inducing signaling complex; H, high; D, dim; L, low; HAART, Highly
Active Antiretroviral Therapy.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
upon stimulation with PMA for 48 h (27). Furthermore, high ex-
pression of adhesion molecules, cytotoxic potential, and low ex-
pression of cell-cycle related genes was recently described for
CD8?CD57?T cells from both HIV infected and uninfected sub-
jects, suggesting that such cells migrate to nonlymphoid tissues
without further cycling (29). A recent study, however, described
that CD8?CD57?T cells from healthy donors do have a capacity
for rapid expansion and production of IL-5, an anti-apoptotic cy-
tokine (30, 31), upon TCR stimulation (32), thereby challenging
the use of CD57 as a marker of terminal differentiation.
In this study, we further investigated the role of PD-1 in regu-
lating HIV-specific CD8?T cell survival. Our data show that PD-1
and CD57 reach maximal expression on CD8?T cells at opposing
ends of the memory maturation spectrum. HIV-specific CD8?T
cells predominantly express a PD-1H/DCD57Lphenotype. CD8?T
cells expressing only CD57 are resistant to spontaneous and CD95/
Fas-induced apoptosis. PD-1HCD8?T cells express a proapoptotic
phenotype, characterized by reduced levels of Bcl-2, increased mi-
tochondrial mass and high levels of CD95/Fas. Finally, copolar-
ization of PD-1 and CD95/Fas was observed during experimen-
tally induced CD95/Fas capping.
Materials and Methods
Peripheral blood was collected from HIV?individuals (n ? 32) and HIV?
donors (n ? 5). Signed informed consent was obtained in accordance with
the Declaration of Helsinki and approved by the relevant Institutional Re-
view Board. All HIV?individuals were infected for at least 1 year (range,
1–24 years), the median CD4 count was 395 cells/?l (range, 4–1401 cells/
?l), and the median viral load was 460 RNA copies/ml plasma (range, ?50
to 1325850 RNA copies/ml blood); 10 individuals were asymptomatic and
21 were on antiretroviral treatment. The vast majority of experiments were
conducted using freshly isolated PBMC; in other cases, cells were cryo-
preserved until use. RPMI 1640 (Life Technologies) supplemented with
10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ?g/ml strep-
tomycin-sulfate was used for culturing PBMC or Jurkat cells. Fresh my-
eloid dendritic cells (mDC) were prepared, cultured, and stimulated with a
TLR-7/8 agonist as previously described (6).
The following directly conjugated mAbs were used: 1) CD3-Cy7
allophycocyanin, CD8-allophycocyanin, IFN-?-FITC, TNF-?-Cy7PE,
PD-1-FITC, active capsase 3-PE, Bcl-2-FITC, CD95/Fas-PE, CD95/Fas-
allophycocyanin, CD57-FITC, CD70-FITC, CD11c-allophycocyanin (BD
Biosciences) and 2)CD45RO-TexasRedPE,
Coulter). The following mAbs were conjugated in our laboratory (http://
drmr.com/abcon/index.html): IL-2–allophycocyanin, CD14–Pacific blue,
CD19–Pacific blue, CD8-Qdot 705, CD8-Qdot 585, CD57-Qdot 545,
CD57-Qdot 705, CD27-Cy5PE, CCR7-Cascade blue. The unconjugated
mAbs were obtained from BD Biosciences. Cascade blue was obtained
from Molecular Probes and Cy5 from BD Biosciences. Quantum dots were
obtained from Invitrogen. The violet (vivid), aqua and green (gravid)
amine reactive viability dyes were obtained from Invitrogen. Annexin V-
FITC and annexin V-allophycocyanin were obtained from BD Bioscience.
Biotinylated anti-PD-1 Ab was obtained from R&D Systems (BAF 1086)
and streptavidin (Cy7PE or Qdot 655) was obtained from Molecular
Probes. The mitochondria specific dye MitoTracker Green FM was ob-
tained from Molecular Probes.
Cells were analyzed with a modified LSRII flow cytometer (BD Immuno-
cytometry Systems) as previously described (6). Between 200,000 and 1 ?
106events were collected and electronic compensation was conducted with
Ab capture beads (BD Biosciences) stained separately with individual
mAbs used in the test samples. Data were analyzed using FlowJo version
8.0 (TreeStar). Forward scatter area vs forward scatter height was used to
gate out cell aggregates. CD14?, CD19?, and dead cells were removed
from the analysis to reduce background staining. For mitochondrial mass
evaluation, PBMC were stained for surface markers using CD3-Cy7 allo-
CD45RO-TRPE, and vivid (or aqua in combination with annexinV-Cas-
cade blue) then incubated with 100 nM of MitoTracker Green FM for 45
min at 37°C, 5% CO2. Cells were fixed with 1% paraformaldehyde and
events were collected immediately after the assay was completed. The
combination Grivid/CD3-Cy7 allophycocyanin/CD8-Qd705/CD27-
was used for further characterization of cells located in the CD27H
CD45ROHcompartment. CD70 expression was analyzed in ?CD3/?CD28
stimulated PBMC from HIV?donors and sorted, live mDC (CD14low
CD11chigh) stimulated through TLR 7 and 8.
PBMC (1–1.5 ? 106) 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 ?g/ml) for 12–14 h at 37°C. Cells were har-
vested, washed, and surface stained with annexin V, CD3, CD8, CD27,
CD45RO, PD-1, CD57, CD14, CD19 and violet amine reactive viability
dye. In some experiments surface staining was followed by fixation/per-
meabilization (Cytofix/CytoPerm kit; BD Biosciences) and intracellular
staining with anti-active caspase 3. When MitoTracker Green FM was
used, cells were incubated for 45 min at 37°C in the presence of 100 nM
MitoTracker followed by surface staining. In all staining steps 2.5 mM
PBMC (2 ? 106) were diluted to 1 ml with medium containing the co-
stimulatory mAbs (?CD28 and ?CD49d) (1 ?g/ml each; BD Biosciences),
monensin (0.7 ?g/ml; BD Biosciences), and brefeldin A (10 ?g/ml; Sigma-
Aldrich), in the absence or presence of peptides (15mers overlapping by 11
residues) corresponding to full-length HIV-1 Gag (2 ?g/ml each peptide, 5
?l/ml; National Institutes of Health AIDS Research and Reference Reagent
Program) or CMV pp65 Ag (2 ?g/ml, 5 ?l/ml; Microbix Biosystems) for
6 h. After washing, cells were surface stained for PD-1, CD57, CD8,
CD27, CD45RO, CD14/CD19, and violet amine reactive viability dye.
Following permeabilization (Cytofix/Cytoperm kit; BD Biosciences), cells
were stained for CD3, IFN-?, IL-2, and TNF-?.
Plasmids and transfection
PD-1 was amplified from human-activated T cell cDNA using Phusion
DNA polymerase (New England Biolabs). The 5? primer included a Kozak
sequence for higher expression in eukaryotic cells (33). The amplified open
reading frame was cloned into the mammalian expression vector pCI (Pro-
mega). Insert integrity was confirmed by sequencing. PD-1 was fused to
the enhanced GFP mutant FRED143 (34) cloned into the pCMVkan (35)
that provided the CMV promoter and growth hormone polyadenylation
signal. Cultured Jurkat cells (American Type Culture Collection) were
transfected with PD-1-GFP or empty parental vector using the Amaxa sys-
tem (kit V, program X-001, Amaxa). Following overnight culture, live
(vivid-) cells were sorted and subjected to further analysis.
Imaging experiments: confocal analysis
CD95/Fas capping was induced in live-sorted transfected Jurkat cells as
previously described (5, 36). In brief, 1–2 ? 106cells were incubated with
anti-CD95/Fas Ab (clone CH-11; final concentration 10 ?g/ml) for 30 min
on ice, transferred to 37°C for 2–3 min and transferred again on ice. After
incubation with a Rhodamine Red-X-conjugated goat anti-mouse IgM Ab
(Jackson Immunoresearch Laboratories), cells were transferred onto poly-
L-lysine-coated slides (Sigma-Aldrich), mounted with mounting medium
(Gold anti-FADE with 4?, 6-diamidine-2?-phenylindole dihydrochloride,
Invitrogen), and visualized using a confocal microscope (Leica TCS SP2;
Leica Microsystems) equipped with a 63?/1.4 oil-immersion objective
lens (Leica). The Z-stack feature of the software was used to obtain a
library of images of various sections of cells; three-dimensional images
were acquired using Leica Confocal Software (Leica Microsystems), and
Adobe Photoshop 6.0 software (Adobe Systems) was used to process them.
Multitracking mode was used to eliminate spillover between fluorescence
Imaging experiments: ImageStream analysis
Untransfected Jurkat cells were stained with anti-CD95/Fas-PE, phalloidin-
TRPE (Molecular Probes), and Draq5 (for nuclear localization, Biostatus)
or anti-CD3?-PE, anti-TCR??-FITC, and Draq5. Alternatively, Vivid?
PD-1-GFP transfected Jurkat cells were stained with an anti-CD95/Fas-PE
Ab and Draq5 following CD95/Fas capping and fixed with 1% parafor-
maldehyde. Primary Vivid?CD3?CD8?CD27?T cells were sorted from
HIV infected donors under sterile conditions by using a FACS Aria system
in a BSL-3 facility and incubated for 1 to 3 h at 37°C with CH-11 Ab. Cells
1121 The Journal of Immunology
were washed and stained with anti-PD-1-FITC (or anti-CD57-FITC), anti-
CD95/Fas-PE, and Draq5. Primary or Jurkat cells were analyzed on an
ImageStream Imaging Flow Cytometer (Amnis Corporation) using 488 nm
laser excitation. Classifiers were used to eliminate collection of debris
based on low area in the brightfield imagery while camera saturating
events, based on the presence of peak intensities greater than 1022, were
also excluded. Typical files contained imagery for 10,000 to 60,000 cells
with each cell imaged with side scatter, brightfield, and three channels of
fluorescence. Images of fixed cells were analyzed using ImageStream Data
Exploration and Analysis Software. Spectral compensation was digitally
performed on a pixel-by-pixel basis before data analysis. Following com-
pensation, similarity analysis was conducted on in-focus single cell images
(supplementary Fig. 2B, lower panel)4(37).
Experimental variables were analyzed using the nonparametric Mann-
Whitney U test. Spearman rank correlation analysis was used when clini-
cal/demographic data and experimental variables were analyzed. The effect
of antiretroviral treatment on measured parameters was analyzed by ap-
plying the Mann-Whitney U test. Bars depict median values. When “boxes
and whiskers” graphs are used, the box size represents the limits of the data
for the second and third quartiles. p values ?0.05 were considered signif-
icant. The GraphPad Prism statistical analysis program (GraphPad Soft-
ware) was used throughout. Analysis and graphical representation of cy-
tokine production in relation to PD-1 expression was conducted by using
the data analysis program Simplified Presentation of Incredibly Complex
Evaluations (SPICE version 2.9; provided by M. Roederer, National Insti-
tutes of Health, Bethesda, MD) (38).
PD-1HCD27LCD45ROH/DCD8?T cells from HIV-infected
donors have the greatest sensitivity to ex vivo spontaneous and
Our previous studies have shown that PD-1 expression is associ-
ated with increased susceptibility to ex vivo apoptosis of total and
virus-specific CD8?T cells from HIV-infected donors, irrespec-
tive of Ag specificity (6). Furthermore, the absolute level of PD-1
expression was the primary indicator of apoptosis sensitivity of
virus-specific CD8?T cells (6). In this study, we sought to further
investigate the role of PD-1 in the ex vivo apoptosis of CD8?T
cells from HIV positive donors. Polychromatic flow cytometry was
used to quantify the expression level of PD-1 (high/dim/low)
within multiple memory and naive CD8?T cell compartments
(Fig. 1A). Naive (CD27HCD45ROL) CD8?T cells were used to
set the gates for PD-1 expression on memory CD8?T cell popu-
lations (Fig. 1A, lower panel). Apoptosis sensitivity was measured
either using annexin V or an Ab against activated caspase 3 or both
(Fig. 1B). Positivity for annexin V was consistently accompanied
by activation of caspase 3 (Fig. 1B, upper panel), indicating that
the apoptosis is caspase-mediated, in agreement with previous
studies (4, 39–42). There was increased spontaneous and CD95/
Fas-induced apoptosis with progressive maturation of CD8?T
cells (Fig. 1B, lower panel). Specifically, CD27LCD45ROHand
CD27LCD45RODwere the populations expressing the highest ap-
optosis sensitivity (Fig. 1B). CD27HCD45ROHcells expressed the
lowest sensitivity among memory populations, although this sen-
sitivity was still significantly higher (p ? 0.0001, for both spon-
taneous and CD95/Fas-induced apoptosis) than of naive CD8?T
cells (Fig. 1B). When apoptosis sensitivity was analyzed in relation
to PD-1 expression, ex vivo survival was found to be significantly
reduced in cells expressing a PD-1highphenotype in all memory
populations tested (Fig. 1C). A similar trend, although not statis-
tically significant, was observed between PD-1Dand PD-1Lcells
(Fig. 1C). In addition, CD8?T cells from HIV uninfected indi-
viduals showed similar characteristics. PD-1HCD27LCD45ROH
was the population exhibiting the highest sensitivity to both spon-
taneous (5.4 ? 4.4 vs 0.5 ? 0.2 (%)Vivid?AnnexinV?CD8?T
cells for PD-1Hand PD-1Lpopulations, respectively) and CD95/
Fas-induced apoptosis (5 ? 1.9 vs 0.84 ? 0.2 for PD-1Hand
PD-1Lpopulations, respectively). No correlation was found be-
tween age, CD4 T cell counts, and the percentage of CD8?T cells
expressing a PD-1Hphenotype in memory populations. A negative
correlation, however, was found between CD8 T cell counts and
this percentage in the CD27LCD45RODpopulation (p ? 0.0023).
In contrast, a strong positive correlation was found between viral
load and the percentage of PD-1HCD8?
CD27HCD45ROH(p ? 0.0007), CD27LCD45ROH(p ? 0.0001)
and CD27LCD45ROD(p ? 0.0029) populations. The percentage
of PD-1HCD27LCD45ROHCD8?T cells was significantly lower
in Highly Active Antiretroviral Therapy (HAART)-treated donors
(7.1 ? 9) compared with untreated ones (31.9 ? 13, p ? 0.006).
No correlation was found between age, CD4 T cell counts, CD8 T
cell counts, viral load, and ex vivo spontaneous or CD95/Fas-in-
duced apoptosis within total CD8?T cell memory populations.
PD-1Hcells, however, in the CD27LCD45ROHcompartment from
treated donors were more sensitive to CD95/Fas-induced apoptosis
compared with treatment naive individuals (p ? 0.0231). In agree-
ment with our previous data, these data show that high expression
of PD-1 is a strong indicator of poor ex vivo survival of memory
CD8?T cells from HIV-infected donors (6). This is most evident
in highly differentiated memory populations.
T cells in the
High expression of PD-1 in CD8?T cells from HIV-infected
donors is accompanied by a proapoptotic phenotype
The molecular mechanism(s) governing the increased ex vivo ap-
optosis sensitivity of CD8?T cells in HIV-infected donors is not
well understood. Our previous studies have revealed a potential
role for Bcl-2 family molecules in regulating this phenotype (4). In
addition, we have also shown that “mitochondrial mass” is in-
creased, especially in HIV-specific CD8?T cells, suggesting a role
for mitochondria in this phenotype (5). Therefore, the relation be-
tween PD-1 and apoptosis-related factors was analyzed in CD8?T
cells from HIV-infected donors. To minimize variations due to
comparison of mean fluorescence intensity (MFI) values obtained
from different experiments all parameters were expressed as fold
change over values in naive CD8?T cells. Similar data, however,
were obtained when raw MFI values were analyzed (data not
shown). Bcl-2 levels were found to be reduced in PD-1Has com-
pared with PD-1DCD8?T cells in all memory compartments, with
the exception of CD27LCD45ROLcells (Fig. 2A, upper left panel).
The difference in Bcl-2 expression was most evident in the
CD27LCD45ROHpopulation (p ? 0.05 and p ? 0.027 for com-
parison between PD-1H/PD-1Dand PD-1H/PD-1L, respectively)
(Fig. 2A, upper left panel). Similarly, PD-1Hcells were found to
express higher levels of CD95/Fas surface receptor than PD-1D
cells, reaching statistical significance in CD27HCD45ROH(p ?
0.014 for PD-1Hvs PD-1D) and CD27LCD45ROHcells (p ?
0.0062 for PD-1Hvs PD-1D) (Fig. 2A, upper right panel). Previ-
ously, MitoTrackerGreen FM has been used as a marker of
“mitochondrial mass” (5, 43, 44). PD-1Hcells showed increased
binding of MitoTracker in all memory CD8?T cell compartments
with the exception of CD27LCD45ROLcells (Fig. 2A, lower left
panel). This binding was significantly higher compared with 1)
PD-1D(p ? 0.03) and PD-1L(p ? 0.01) cells in the
CD27HCD45ROHcompartment; 2) PD-1L(p ? 0.04) cells in the
CD27LCD45ROHcompartment; and 3) PD-1L(p ? 0.03) cells in
CD27LCD45RODmemory populations. In parallel, apoptosis sen-
sitivity was measured in relation to Mitotracker binding in memory
populations. In agreement with our previous data (5) annexin V
positivity was almost exclusively detected in the MitotrackerH
4The online version of this article contains supplementary material.
1122PROAPOPTOTIC STATUS OF PD-1HCD8?T CELLS
compartment (Fig. 2B). Next, the expression of IL-7R? (CD127),
a major survival factor for memory CD8?T cells (45), was ex-
amined in relation to PD-1 expression in all memory compart-
ments. CD127 was found to be predominantly expressed in the
CD27HCD45ROHcompartment (Fig. 2A, lower right panel). This
expression was significantly reduced in PD-1Hcells (p ? 0.015 for
PD-1Hvs PD-1Dand 0.002 for PD-1Hvs PD-1L, respectively).
Surface expression of PD-1, CD127, and CCR7 was then as-
sessed in the memory CD8?T cell compartments (supplemen-
tary Fig. 1). A significant proportion of CD27HCD45ROHcells
from HIV-infected donors were found to express a CCR7Hphe-
notype, although this expression was lower compared with
CCR7 in naive cells (supplementary Fig. 1). Our analysis
revealed that this memory compartment also contains a population
expressing a PD-1LCD127HCCR7Hphenotype (supplementary Fig.
1). This population exhibited a Bcl-2HCD95LMitotrackerLpheno-
type and minimum levels of ex vivo spontaneous as well as
CD95/Fas-induced apoptosis (Fig. 1C, annexin V positivity for
Differential association of PD-1 and CD57 with survival ability
of CD8?T cells from HIV-infected donors
CD57 expression is increased on T cells from HIV-infected donors
and under conditions associated with immune activation and in-
creasing age (46, 47). HIV-specific CD8?T cells expressing CD57
were found to exhibit replicative senescence and are characterized
by increased sensitivity to ex vivo death upon stimulation with
PMA (27). We therefore investigated the relative expression of
PD-1 and CD57 on total and virus-specific CD8?T cells from
HIV-infected donors as well as their association with ex vivo spon-
taneous and CD95/Fas-induced apoptosis.
CD57 levels gradually increased during maturation of CD8?
T cells with the CD27LCD45ROLmemory compartment ex-
pressing the highest levels (Fig. 3A). Analyzing both PD-1 and
CD57, we observed that the two surface receptors reach max-
imal expression levels at opposing ends of the maturation spec-
trum (Fig. 3A, lower panel). The ex vivo sensitivity to sponta-
neous and CD95/Fas-induced apoptosis was examined in
memory populations expressing all possible combinations of
PD-1 and CD57 receptors (Fig. 3B, lower panel). The PD-
1HCD57Lpopulation exhibited the highest sensitivity to apo-
ptosis for both types of treatments in all but the CD27H
CD45ROHmemory populations (Fig. 3B, lower panel). In
contrast, cells expressing CD57 in the absence of PD-1 were the
most resistant to ex vivo death (Fig. 3B, lower panel). Interest-
ingly, the apoptosis sensitivity of CD8?PD-1LCD57Hcells was
significantly lower than that of CD8?PD-1HCD57Hcells. Fur-
thermore, spontaneous and CD95/Fas-induced apoptosis was
comparable within the CD8?PD-1LCD57Hcompartment across
all memory populations (Fig. 3B, lower panel).
Next, the expression of PD-1 and CD57 on virus-specific CD8?
T cells from HIV infected donors was investigated. Virus-specific
CD8?T cells were identified by intracellular staining for IFN-?,
TNF-? and IL-2 production upon ex vivo stimulation with appro-
priate peptide pools (Fig. 4A, upper panel). Our gating strategy
enables detection of CD8?T cells exhibiting each and every com-
bination of these three cytokines. No correlation between PD-1
expression and ex vivo cytokine production was found for either
HIV- or CMV-specific CD8?T cells (Fig. 4B); this was true for all
of the cytokine combinations. The majority of HIV-specific CD8?
T cells were found to be either PD-1HCD57Lor PD-1HCD57H
while PD-1LCD57Hwas the least frequent population in all groups
tested (Fig. 4A, lower panel). In contrast, CMV-specific CD8?T
cells were found to exhibit a PD-1LCD57Lor PD-1LCD57Hphe-
notype in IFN-? and IFN-?/TNF-? groups (Fig. 4A, lower panel).
Interestingly, CMV-specific CD8?T cells producing both IFN-?
and IL-2 expressed mostly a PD-1LCD57Lor PD-1HCD57Lphe-
notype (Fig. 4A, lower panel). Overall, our data show opposing
expression of PD-1 and CD57 during CD8?T cell maturation with
PD-1 expression having the greatest impact upon ex vivo sensi-
tivity to apoptosis.
PD-1 but not CD57 copolarizes with CD95/Fas upon ex vivo
induction of CD95/Fas capping
CD95/Fas capping is an essential early molecular event upon ex
vivo cross-linking of the receptor (36, 48, 49). Recent studies,
however, have shown that recruitment of other surface receptors in
the proximal area of CD95/Fas capping can modulate CD95/Fas-
induced apoptosis (50–52), possibly by affecting events at the
level of the cytoplasmic membrane (50, 51). Therefore, we sought
to examine the localization of PD-1 under ex vivo conditions pro-
moting CD95/Fas capping. We started by using Jurkat cells, a cell
line that does not express PD-1 (data not shown) and have been
extensively used in CD95/Fas capping experiments (36, 49). Cells
were transiently transfected with an expression vector coding for
human PD-1 tagged with GFP. Primary CD8?T cells transfected
with this vector revealed that an anti-PD-1-PE Ab recognized PD-
1-GFP, thereby confirming the proper surface expression of PD-1
by the transgene (supplementary Fig. 2A). The relative localization
of PD-1-GFP and CD95/Fas under capping conditions was ana-
lyzed in live transfected Jurkat cells by using the Image Stream
System. The parameter bright detailed similarity R3 (BDS-R3)
was used to estimate the proximity of these two surface receptors
in cells induced to cap CD95/Fas (Fig. 5, A and B). Jurkat cells
were also stained either for TCR?? and CD3 or CD95/Fas and
actin (Fig. 5A). BDS-R3 values ?2 indicate proximal localization
of the two surface receptors under investigation (Fig. 5A). PD-1
was localized in the proximal area of CD95/Fas capping in 51 ?
7.6% (n ? 3 experiments) of cells expressing a high PD-1-GFP
phenotype (Fig. 5B). Similar localization was found in 41 ? 1.5%
and 6 ? 1% of the cells with dim and low PD-1-GFP expression,
respectively (Fig. 5B). Control experiments using the parental-
empty vector show a diffused distribution of GFP even in CD95/
Fas-capped cells (supplementary Fig. 2C). We further analyzed
colocalization using confocal microscopy. Similar to the previous
analysis, capped cells were characterized by copolarization of
PD-1 and Fas (Fig. 5C).
The relative distribution of PD-1 (or CD57) and CD95/Fas was
next examined in primary sorted CD27?CD8?T cells (the mem-
ory compartment that shows maximum sensitivity to ex vivo ap-
optosis) from HIV-infected donors under ex vivo conditions in-
ducing CD95/Fas-capping. Again, endogenous PD-1 was found to
cotranslocate to the area of capping (Fig. 6A). Similar data were
obtained when sorted CD3?CD8?CD27LPD-1Lcells were trans-
fected with PD-1-GFP vector (supplementary Fig. 2D). In sharp
contrast, CD57 was “excluded” from this area in the majority of
capped cells, especially in cells expressing a CD57Hphenotype
(Fig. 6B). Taken together, our data reveal an orchestrated move-
ment of PD-1 and CD95/Fas during the early steps of CD95/Fas-
induced apoptosis, thereby further supporting an active role of
PD-1 in this process.
In this study, we report on the differential association of PD-1 and
CD57 with ex vivo sensitivity to spontaneous and CD95/Fas-in-
duced apoptosis in CD8?T cells from HIV-infected donors. Fur-
thermore, we provide data showing that PD-1 expression is linked
to a proapoptotic phenotype of CD8?T cells characterized by low
1123 The Journal of Immunology
cytometry gating scheme for identification of CD8?T cell populations expressing low, dim, and high levels of PD-1 is shown. Histograms depict the PD-1
expression in naive and memory populations of CD8?T cells from the same sample. Memory subsets identified by CD27 and CD45RO staining of total
The absolute expression of PD-1 is a primary indicator of ex vivo apoptosis of CD8?T cells in HIV infection. A, The polychromatic flow
1124PROAPOPTOTIC STATUS OF PD-1HCD8?T CELLS
expression of Bcl-2 and IL-7R?, increased levels of CD95/Fas,
and high mitochondrial mass. In agreement with our previous data
(6), no correlation was found between PD-1 levels and ex vivo
cytokine production analyzing either single or combinations of
multiple cytokines (IFN-?, TNF-?, and IL-2). Prior studies have
revealed an increased sensitivity of CD45ROHCD8?T cells from
HIV-infected donors to ex vivo CD95/Fas-induced apoptosis (53,
54), a phenomenon associated predominantly with “effector mem-
ory” cells (2). In line with our previous data (6), ex vivo apoptosis
was primarily observed in the CD27LCD45ROHand CD27L
CD45RODcompartments followed by lower levels in the highly
differentiated CD27LCD45ROLpopulation. Although apoptosis in
the CD27HCD45ROHmemory compartment was significantly
higher compared with the naive compartment (CD27HCD45ROL),
this population still exhibits relatively high resistance to both spon-
taneous and CD95/Fas-induced apoptosis compared with other
memory groups. Still, the majority of apoptotic cells are charac-
terized by high expression of PD-1 even in this memory compart-
ment. A hierarchy was found when apoptosis was analyzed in re-
lation to PD-1 levels, indicating that the absolute level of PD-1 is
a primary determinant of apoptosis sensitivity in memory CD8?T
cells (6), especially CD27LCD45ROHand CD27LCD45RODcells.
This is further supported by the finding of a similar trend in CD8?
T cells from HIV uninfected donors. The lower ex vivo apoptosis
sensitivity of CD8?T cells from HIV uninfected donors compared
with HIV donors, even within the PD-1Hcompartment, indicates
that additional mechanism(s) contribute to the high ex vivo apo-
ptosis of CD8?T cells in HIV infection. A strong correlation
between viral load and PD-1 expression in memory CD8?T cells
was found. We have previously described the lack of such corre-
lation when HIV-specific CD8?T cells were analyzed (6). We
hypothesize that chronic Ag-specific TCR stimulation, a major
mechanism leading to high sustained PD-1 levels in virus-specific
CD8?T cells (20, 55), probably overrides non-TCR stimuli that
potentially affect the expression of PD-1 in total CD8?T cell
populations in a non-Ag specific manner (56). Furthermore, the ex
vivo lower percentage of PD-1HCD27LCD45ROHin HAART-
treated donors is associated with a higher sensitivity to CD95/Fas-
induced apoptosis compared with cells from untreated donors.
Whether this is a reflection of higher in vivo turnover of PD-1H“ef-
fector memory” cells upon HAART treatment needs further
The survival ability of a mammalian cell is determined by nu-
merous extracellular factors, which can induce death signals as
well as the intracellular pathways that control the transduction of
such signals. We analyzed several parameters that have been as-
sociated with the survival of CD8?T cells from HIV?donors.
PD-1 was found to be consistently associated with a preapoptotic
phenotype; specifically, that of high mitochondrial mass and low
Bcl-2 expression. These data indicate that these cells are potentially
more susceptible to mitochondria-mediated cell death. Whether PD-1
induces a direct signal affecting mitochondria or if this process could
be mediated by other cellular pathways such as Akt-mediated signals
(24) is not known and warrants further investigation.
A somewhat unexpected finding was that although CD27H
CD45ROHCD8?T cells express the highest levels of both PD-1
and CD95/Fas, they are relatively resistant to ex vivo apoptosis.
These cells, however, express high levels of CD27, a receptor that
delivers positive signals in vivo for CD8?T cells which we re-
cently reported can rescue virus-specific CD8?T cells from CD95/
Fas-induced death during the development of an anti-viral re-
sponse (57). Our data show that ex vivo stimulated CD4?and
CD8?T cells as well as mDC treated with a TLR7/8 agonist ex-
press high levels of CD70, the ligand for CD27 (supplementary
Fig. 3), indicating that an extended in vivo cellular network could
potentially provide survival signals to CD27Hcells. Furthermore,
mitochondrial mass was higher
CD27HCD45RODcells compared with CD27HCD45ROHcells
while IL-7R? was significantly higher on CD27HCD45ROHcells,
particularly the ones expressing low levels of PD-1. The presence
of CCR7 on many of them could also indicate a “central memory”
phenotype that is associated with high resistance to apoptosis. Al-
though the balance of surface receptors inducing pre- or proapop-
totic signals may be important, execution of apoptosis is largely
dependent on the relative expression/function of intracellular me-
diators of CD95/Fas-induced signaling. The relative expression of
cellular caspase-8-like inhibitory protein (anti-apoptotic) and Fas-
associated death domain (preapoptotic) could critically affect the
CD95/Fas-induced apoptosis of T cells. More recently, the critical
role of Rac-mediated signaling in this process was described (58).
Investigation of the relative expression of such factors would be
very informative regarding the sensitivity of memory CD8?T cell
populations to ex vivo apoptosis.
For CD95/Fas-induced apoptosis, two types of cell lines have
been described based on apoptosis signaling pathways (59): 1)
type I cells, in which apoptosis is dependent on the integrity of the
actin network and high levels of CD95/Fas results in massive
death-inducing signaling complex (DISC) formation and mito-
chondria-independent death; and 2) type II cells that express lower
levels of CD95/Fas and DISC-induced signaling, in which apopto-
sis requires a mitochondria-dependent amplification process. This
latter pathway of cell death is independent of actin. All of the
CD8?T cells we tested were positive for CD95/Fas, in agreement
with our previous data (2). Our previous data have shown that
Bcl-2-related molecules and mitochondria may play a critical role
in the survival of HIV-specific CD8?T cells (4, 5) while destruc-
tion of actin abolishes the CD95/Fas-induced apoptosis (60). Over-
all, our data indicate that primary PD-1Heffector memory CD8?T
cells from HIV infected patients exhibit a mixed phenotype where
the integrity of actin is necessary but the intracellular signal in-
duced by the lower expression of CD95/Fas expression (compared
with CD27HCD45ROHcells) can be amplified through an ex-
tended mitochondria network. This hypothesis is in agreement
with recent reports showing that both CD95/Fas-induced and mi-
tochondria-mediated pathways cooperate to shut down antiviral
immune responses during chronic infection (61, 62).
The percentage of CD8?T cells expressing CD57, a marker of
replicative senescence in CD8?T cells from HIV?donors (27),
CD8?T cells are also presented. B, Representative flow cytometry plots showing simultaneous measurement of annexin V binding and active caspase
3 levels in naive and memory CD8?T cell from an HIV?donor cultured for 12–14 h at 37°C (upper panel). Pooled data showing the percentage
(%) of apoptotic naive and memory CD8?T cells from HIV?donors (n ? 26) cultured in the absence or presence of anti-CD95/Fas Ab for 12–14
h (lower panel). C, Pooled data showing the percentage (%) of spontaneous and CD95/Fas-induced apoptosis in naive and memory CD8?T cell
compartments from HIV?donors (n ? 26) and in relation to expression of PD-1. Apoptosis sensitivity was evaluated based on annexin V binding
or the simultaneous measurement of annexin V binding and active caspase 3 expression. Bars depict median values. p values were calculated using
Mann-Whitney U test.
1125The Journal of Immunology
was found to increase with differentiation reaching maximum lev-
els in the CD27LCD45ROLpopulation. We have previously shown
that CD57HCD8?T cells from HIV?donors are susceptible to
activation-induced cell death upon ex vivo treatment for 48 h (27).
In this study, we report on the lack of association between CD57
and spontaneous or CD95/Fas-induced apoptosis. Previous studies
CD95/Fas (upper right), mitochondrial mass (lower left), and IL-7R? (CD127) (lower right) in memory CD8?T cell populations from HIV?individuals
in relation to PD-1 expression. MFI values for the parameters tested were expressed as fold increase (decrease) over those of naive cells. Representative
flow cytometry plots are also shown. Horizontal lines depict median values. p values were calculated using Mann-Whitney U test. B, Representative flow
cytometry plots showing annexin V binding in relation to mitochondrial mass (binding of Mitotracker Green FM) of memory CD8?T cells from HIV?
donor cultured for 12–14 h at 37°C.
CD8?PD-1HT cells are characterized by a proapoptotic phenotype. A, Pooled data showing the ex vivo expression of Bcl-2 (upper left),
1126 PROAPOPTOTIC STATUS OF PD-1HCD8?T CELLS
donors. A, Representative flow cytometry plots showing the expression of PD-1 vs CD57 (upper left) as well as the expression of CD57 in memory CD8?
T cell populations (upper right). Pooled data showing the relative expression of PD-1 and CD57 in relation to maturation status of CD8?T cells from HIV?
donors (n ? 26) are shown in the lower panels. Bars depict median values. p values were calculated using the Mann-Whitney U test. B, Representative
plots showing the expression of PD-1 in relation to CD57 one in naive and memory CD8?T cell populations (upper panel). Pooled data showing the
percentage (%) of apoptotic memory CD8?T cells from HIV?donors (n ? 28) with respect to PD-1 and CD57 expression are shown in the lower panels.
Bars depict median values. p values were calculated using Mann-Whitney U test.
Differential association of PD-1 and CD57 with ex vivo spontaneous and CD95/Fas-induced apoptosis of CD8?T cells from HIV infected
1127The Journal of Immunology
cytokines. A, Representative flow cytometry plots depicting the expression of PD-1 and CD57 in Gag-specific CD8?T cells identified by IFN-??,
IFN-??TNF-??, or IFN-??IL-2?cytokine production (upper panel). Pooled data showing the phenotype of HIV- and CMV-specific CD8?T cells with
respect to expression of PD-1 and CD57 (lower panel). Bars depict median values. p values were calculated using Mann-Whitney U test. B, Functional
composition of the HIV- (n ? 8) and CMV-specific (n ? 8) CD8?T cell responses in relation to PD-1 expression. Each slice of the pie represents the
fraction of the total response that consists of CD8?T cells positive for a given number of functions (left panel). MFI values of PD-1 for every possible
combination of responses are shown on the x-axis (right panel). Boxes represent interquartile ranges; mean and SD lines are shown.
HIV-specific CD8?T cells express predominantly a PD-1HCD57Lphenotype that is not associated with their ex vivo ability to produce
1128 PROAPOPTOTIC STATUS OF PD-1HCD8?T CELLS
or CD95/actin and the relative distribution of these molecules was analyzed using the ImageStream Imaging Flow Cytometer. Histograms depict the values
of Bright Detailed Similarity R3 (an index of relative colocalization) for the pairs of molecules tested (left panel). Representative images showing the
relative distribution of these molecules in Jurkat cells for different values of BDS-R3 (right panel). B, PD-1-GFP transfected Jurkat cells were analyzed
under induction of CD95/Fas-capping. Histograms depict the BDS-R3 values for PD-1 and CD95/Fas localization in PD-1L, PD-1D, and PD-1Hpopulations
of total cells or cells in which CD95/Fas is capped (left panel). Representative images for different BDS-R3 values are shown (right panel). Draq 5 (a nuclear
staining) is shown in blue, PD-1-GFP in green and CD95/Fas-PE in red. C, Confocal images of Jurkat cells showing the localization of PD-1-GFP and
CD95/Fas under experimental conditions inducing CD95/Fas-capping.
Cotranslocation of PD-1 and CD95/Fas under ex vivo induction of CD95/Fas-capping. A, Jurkat cells were stained with either TCRab/CD3
1129 The Journal of Immunology
donors. A, Flow cytometry plots showing the expression of PD-1 (CD57) and CD95/Fas in “focused” sorted Vivid?CD3?CD8?CD27HT cells from an
HIV?donor analyzed by the “ImageStream Data Exploration and Analysis Software” (left panel). Histograms depict the BDS-R3 values for PD-1 (CD57)
vs CD95/Fas in total or CD95/Fas-capped cells and in relation to PD-1 (CD57) levels (right panel). Representative images for different BDS-R3 values
are also shown. B, Pooled data showing the percentages (%) of primary sorted Vivid?CD3?CD8?CD27LT cells expressing a phenotype characterized by
BDR-S3 values ?2. Group of cells expressing high PD-1, dim CD57, and high CD57 levels are shown. Bars depict means ? SD.
In contrast to PD-1, highly expressed CD57 is excluded from the area of CD95/Fas-capping in primary CD8?T cells from HIV-infected
1130PROAPOPTOTIC STATUS OF PD-1HCD8?T CELLS
(63, 64) have shown that TCR-induced apoptosis in HIV infection
is CD95/Fas-independent. Furthermore, upon TCR-stimulation, both
CD4?and CD8?T cells up-regulate PD-1 (65). Therefore de novo
synthesized PD-1 could also contribute to activation-induced cell
death. Our data also revealed that CD8?T cells can express both
PD-1 and CD57 and these cells are sensitive to apoptosis. The low
sensitivity to ex vivo apoptosis of CD57HCD8?T cells is in agree-
ment with recent reports of in vivo accumulation of this population in
HIV-infected donors (66). We have previously shown that HIV-spe-
cific CD8?T cells are characterized by increased sensitivity to CD95/
Fas-induced death even compared with other virus-specific CD8?T
cells, i.e., CMV-specific cells from the same HIV-infected donors
(2). The PD-1/CD57 profile of HIV- and CMV-specific CD8?T
cells described in this study, according to which the majority of
HIV-specific CD8?T cells are characterized by a PD-1Hand or
PD-1HCD57Hphenotype, is consistent with apoptosis sensitiv-
ity being predominantly mediated by PD-1.
CD95/Fas-capping is a very early and necessary molecular event
during the formation of DISC and the initiation of death signaling
(67). Recent studies have focused on the recruitment of other surface
receptors to the area of CD95/Fas-capping (50–52). Such comobili-
through physical interactions or by changing the dynamics of inter-
actions between CD95/Fas and other signaling molecules (68). Our
Fas capping in a large proportion of cells. In contrast, no such copo-
larization of CD95/Fas and CD57 was observed. How the cotranslo-
cation of PD-1 and CD95/Fas receptors could affect the initiation of
death signal(s) is currently under investigation.
In this study, we describe a population of CD8?T cells exhib-
iting a CD27HCD45ROHIL7R?HCCR7HPD-1LCD95LBcl-2H
Mitochondrial MassLphenotype. This population is characterized
by increased resistance to both spontaneous and CD95/Fas-in-
duced cell death. Central memory cells are long-lasting cells pre-
sumably resistant to cell death. We propose that the use of com-
bination of both differentiation and survival-related parameters
could potentially help to further identify and characterize central
memory cells. However, whether the above combination charac-
terizes “central memory” CD8?T cells or an early activated, less
matured population is under investigation.
We have recently proposed a model where HIV-specific CD8?
T cells could be eliminated upon repetitive encounter with FasL
expressed on HIV-infected cells (69). HIV-infected cells up-reg-
ulate FasL by a Nef-dependent mechanism (70, 71). HIV infection
could also potentially induce the expression of PD-L1. We hy-
pothesize that the orchestrated expression of both ligands in HIV-
infected cells could further contribute to the elimination of HIV-
specific CD8?T cells.
Our data point to a complex regulation of CD8?T cell survival
that is linked to their differentiation level and the ability of intra-
cellular pathways to transduce apoptotic signals. The low apoptotic
potential of less mature CD27HCD45ROHcells is consistent with
the hypothesis that expression of both PD-1 and CD95 are neces-
sary but not sufficient for apoptosis of CD8?T cells from HIV-
infected donors. Furthermore, the collaboration of two surface re-
ceptors (PD-1 and CD95/Fas) could be crucial in the process of
CD8?T cell exhaustion in chronic viral infections.
We thank Dr. Jeffrey I. Cohen (National Institute of Allergy and Infectious
Diseases) for providing access to the Image-Stream flow cytometer (Amnis).
The authors have no financial conflict of interest.
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1132PROAPOPTOTIC STATUS OF PD-1HCD8?T CELLS