Differential Association of Programmed Death-1 and CD57 with Ex Vivo Survival of CD8+ T Cells in HIV Infection

Article (PDF Available)inThe Journal of Immunology 183(2):1120-32 · July 2009with59 Reads
DOI: 10.4049/jimmunol.0900182 · Source: PubMed
Abstract
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 apoptosis, 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 alpha, high expression of CD95/Fas and high mitochondrial 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-1(L)CD57(H) phenotype exhibiting lower levels of cell death. The majority of HIV-specific CD8(+) T cells were found to express a PD-1(H)CD57(L) or PD-1(H)CD57(H) phenotype. 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.
Differential Association of Programmed Death-1 and CD57
with Ex Vivo Survival of CD8
T Cells in HIV Infection
1
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. Koup
2
*
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-1
L
CD57
H
phenotype exhibiting lower levels
of cell death. The majority of HIV-specific CD8
T cells were found to express a PD-1
H
CD57
L
or PD-1
H
CD57
H
phenotype. 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.
D
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-
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-
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.
1
The 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.
2
Address 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: petrovasc@mail.nih.gov and
rk@mail.nih.gov
3
Abbreviations 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
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900182
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-1
H/D
CD57
L
phenotype. CD8
T
cells expressing only CD57 are resistant to spontaneous and CD95/
Fas-induced apoptosis. PD-1
H
CD8
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
Donors
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).
Abs-fluorescent reagents
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, CD127-PE (Beckman
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.
Flow cytometry
Cells were analyzed with a modified LSRII flow cytometer (BD Immuno-
cytometry Systems) as previously described (6). Between 200,000 and 1
10
6
events 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-
phycocyanin, CD8-allophycocyanin, PD-1-SA-655, CD27-Cy5PE,
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% CO
2
. 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-
Cy5PE/CD45RO-TRPE/PD-1-SA-Cy7PE/CD127-PE/CCR7-Cascade blue
was used for further characterization of cells located in the CD27
H
CD45RO
H
compartment. CD70 expression was analyzed in
CD3/
CD28
stimulated PBMC from HIV
donors and sorted, live mDC (CD14
low
CD11c
high
) stimulated through TLR 7 and 8.
Apoptosis studies
PBMC (1–1.5 10
6
) 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
CaCl
2
was included.
Cytokine production
PBMC (2 10
6
) 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 10
6
cells 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
channels.
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 to3hat37°C with CH-11 Ab. Cells
1121The 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).
Statistical analysis
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).
Results
PD-1
H
CD27
L
CD45RO
H/D
CD8
T cells from HIV-infected
donors have the greatest sensitivity to ex vivo spontaneous and
CD95/Fas-induced apoptosis
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 (CD27
H
CD45RO
L
) 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, CD27
L
CD45RO
H
and
CD27
L
CD45RO
D
were the populations expressing the highest ap
-
optosis sensitivity (Fig. 1B). CD27
H
CD45RO
H
cells 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-1
high
phenotype in all memory
populations tested (Fig. 1C). A similar trend, although not statis-
tically significant, was observed between PD-1
D
and PD-1
L
cells
(Fig. 1C). In addition, CD8
T cells from HIV uninfected indi
-
viduals showed similar characteristics. PD-1
H
CD27
L
CD45RO
H
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-1
H
and PD-1
L
populations, respectively) and CD95/
Fas-induced apoptosis (5 1.9 vs 0.84 0.2 for PD-1
H
and
PD-1
L
populations, respectively). No correlation was found be
-
tween age, CD4 T cell counts, and the percentage of CD8
T cells
expressing a PD-1
H
phenotype in memory populations. A negative
correlation, however, was found between CD8 T cell counts and
this percentage in the CD27
L
CD45RO
D
population ( p 0.0023).
In contrast, a strong positive correlation was found between viral
load and the percentage of PD-1
H
CD8
T cells in the
CD27
H
CD45RO
H
( p 0.0007), CD27
L
CD45RO
H
( p 0.0001)
and CD27
L
CD45RO
D
( p 0.0029) populations. The percentage
of PD-1
H
CD27
L
CD45RO
H
CD8
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-1
H
cells, however, in the CD27
L
CD45RO
H
compartment 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.
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-1
H
as com
-
pared with PD-1
D
CD8
T cells in all memory compartments, with
the exception of CD27
L
CD45RO
L
cells (Fig. 2
A, upper left panel).
The difference in Bcl-2 expression was most evident in the
CD27
L
CD45RO
H
population ( p 0.05 and p 0.027 for com
-
parison between PD-1
H
/PD-1
D
and PD-1
H
/PD-1
L
, respectively)
(Fig. 2A, upper left panel). Similarly, PD-1
H
cells were found to
express higher levels of CD95/Fas surface receptor than PD-1
D
cells, reaching statistical significance in CD27
H
CD45RO
H
( p
0.014 for PD-1
H
vs PD-1
D
) and CD27
L
CD45RO
H
cells ( p
0.0062 for PD-1
H
vs PD-1
D
) (Fig. 2A, upper right panel). Previ
-
ously, MitoTrackerGreen FM has been used as a marker of
“mitochondrial mass” (5, 43, 44). PD-1
H
cells showed increased
binding of MitoTracker in all memory CD8
T cell compartments
with the exception of CD27
L
CD45RO
L
cells (Fig. 2A, lower left
panel). This binding was significantly higher compared with 1)
PD-1
D
( p 0.03) and PD-1
L
( p 0.01) cells in the
CD27
H
CD45RO
H
compartment; 2) PD-1
L
( p 0.04) cells in the
CD27
L
CD45RO
H
compartment; and 3) PD-1
L
( p 0.03) cells in
CD27
L
CD45RO
D
memory 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 Mitotracker
H
4
The online version of this article contains supplementary material.
1122 PROAPOPTOTIC STATUS OF PD-1
H
CD8
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
CD27
H
CD45RO
H
compartment (Fig. 2A, lower right panel). This
expression was significantly reduced in PD-1
H
cells ( p 0.015 for
PD-1
H
vs PD-1
D
and 0.002 for PD-1
H
vs PD-1
L
, 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 CD27
H
CD45RO
H
cells
from HIV-infected donors were found to express a CCR7
H
phe
-
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-1
L
CD127
H
CCR7
H
phenotype (supplementary Fig.
1). This population exhibited a Bcl-2
H
CD95
L
Mitotracker
L
pheno
-
type and minimum levels of ex vivo spontaneous as well as
CD95/Fas-induced apoptosis (Fig. 1C, annexin V positivity for
PD-1
L
cells).
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 CD27
L
CD45RO
L
memory 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-
1
H
CD57
L
population exhibited the highest sensitivity to apo
-
ptosis for both types of treatments in all but the CD27
H
CD45RO
H
memory 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-1
L
CD57
H
cells was
significantly lower than that of CD8
PD-1
H
CD57
H
cells. Fur
-
thermore, spontaneous and CD95/Fas-induced apoptosis was
comparable within the CD8
PD-1
L
CD57
H
compartment 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-1
H
CD57
L
or PD-1
H
CD57
H
while PD-1
L
CD57
H
was 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-1
L
CD57
L
or PD-1
L
CD57
H
phe
-
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-1
L
CD57
L
or PD-1
H
CD57
L
phe
-
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
CD27
L
PD-1
L
cells 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 CD57
H
phenotype
(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.
Discussion
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
1123The Journal of Immunology
FIGURE 1. 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
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
1124 PROAPOPTOTIC STATUS OF PD-1
H
CD8
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 CD45RO
H
CD8
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 CD27
L
CD45RO
H
and CD27
L
CD45RO
D
compartments followed by lower levels in the highly
differentiated CD27
L
CD45RO
L
population. Although apoptosis in
the CD27
H
CD45RO
H
memory compartment was significantly
higher compared with the naive compartment (CD27
H
CD45RO
L
),
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 CD27
L
CD45RO
H
and CD27
L
CD45RO
D
cells.
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-1
H
compartment, 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-1
H
CD27
L
CD45RO
H
in 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-1
H
“ef
-
fector memory” cells upon HAART treatment needs further
investigation.
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 CD27
H
CD45RO
H
CD8
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 CD27
H
cells. Furthermore,
mitochondrial mass was higher in CD27
L
CD45RO
H
and
CD27
H
CD45RO
D
cells compared with CD27
H
CD45RO
H
cells
while IL-7R
was significantly higher on CD27
H
CD45RO
H
cells,
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-1
H
effector 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 CD27
H
CD45RO
H
cells) 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 CD27
L
CD45RO
L
population. We have previously shown
that CD57
H
CD8
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
FIGURE 2. CD8
PD-1
H
T cells are characterized by a proapoptotic phenotype. A, Pooled data showing the ex vivo expression of Bcl-2 (upper left),
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.
1126 PROAPOPTOTIC STATUS OF PD-1
H
CD8
T CELLS
FIGURE 3. Differential association of PD-1 and CD57 with ex vivo spontaneous and CD95/Fas-induced apoptosis of CD8
T cells from HIV infected
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.
1127The Journal of Immunology
FIGURE 4. HIV-specific CD8
T cells express predominantly a PD-1
H
CD57
L
phenotype that is not associated with their ex vivo ability to produce
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.
1128 PROAPOPTOTIC STATUS OF PD-1
H
CD8
T CELLS
FIGURE 5. Cotranslocation of PD-1 and CD95/Fas under ex vivo induction of CD95/Fas-capping. A, Jurkat cells were stained with either TCRab/CD3
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-1
L
, PD-1
D
, and PD-1
H
populations
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.
1129The Journal of Immunology
FIGURE 6. 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
donors. A, Flow cytometry plots showing the expression of PD-1 (CD57) and CD95/Fas in “focused” sorted Vivid
CD3
CD8
CD27
H
T 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
CD27
L
T 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.
1130 PROAPOPTOTIC STATUS OF PD-1
H
CD8
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 CD57
H
CD8
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-1
H
and or
PD-1
H
CD57
H
phenotype, 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-
zation could affect the sequestration of CD95/Fas and DISC formation
through physical interactions or by changing the dynamics of inter-
actions between CD95/Fas and other signaling molecules (68). Our
data show a copolarization between PD-1 and CD95/Fas upon ex vivo
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 CD27
H
CD45RO
H
IL7R
H
CCR7
H
PD-1
L
CD95
L
Bcl-2
H
Mitochondrial Mass
L
phenotype. 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 CD27
H
CD45RO
H
cells 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.
Acknowledgments
We thank Dr. Jeffrey I. Cohen (National Institute of Allergy and Infectious
Diseases) for providing access to the Image-Stream flow cytometer (Amnis).
Disclosures
The authors have no financial conflict of interest.
References
1. Pantaleo, G., and R. A. Koup. 2004. Correlates of immune protection in HIV-1
infection: what we know, what we don’t know, what we should know. Nat. Med.
10: 806 810.
2. Mueller, Y. M., S. C. De Rosa, J. A. Hutton, J. Witek, M. Roederer, J. D. Altman,
and P. D. Katsikis. 2001. Increased CD95/Fas-induced apoptosis of HIV-specific
CD8
T cells. Immunity 15: 871– 882.
3. Betts, M. R., M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles,
J. Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, et al.
2006. HIV nonprogressors preferentially maintain highly functional HIV-specific
CD8
T cells. Blood 107: 4781– 4789.
4. Petrovas, C., Y. M. Mueller, I. D. Dimitriou, P. M. Bojczuk, K. C. Mounzer,
J. Witek, J. D. Altman, and P. D. Katsikis. 2004. HIV-specific CD8
T cells
exhibit markedly reduced levels of Bcl-2 and Bcl-xL. J. Immunol. 172:
4444 4453.
5. Petrovas, C., Y. M. Mueller, I. D. Dimitriou, S. R. Altork, A. Banerjee, P. Sklar,
K. C. Mounzer, J. D. Altman, and P. D. Katsikis. 2007. Increased mitochondrial
mass characterizes the survival defect of HIV-specific CD8
T cells. Blood 109:
2505–2513.
6. Petrovas, C., J. P. Casazza, J. M. Brenchley, D. A. Price, E. Gostick,
W. C. Adams, M. L. Precopio, T. Schacker, M. Roederer, D. C. Douek, and
R. A. Koup. 2006. PD-1 is a regulator of virus-specific CD8
T cell survival in
HIV infection. J. Exp. Med. 203: 2281–2292.
7. Keir, M. E., M. J. Butte, G. J. Freeman, and A. H. Sharpe. 2008. PD-1 and its
ligands in tolerance and immunity. Annu. Rev. Immunol. 26: 677–704.
8. Duvall, M. G., M. L. Precopio, D. A. Ambrozak, A. Jaye, A. J. McMichael,
H. C. Whittle, M. Roederer, S. L. Rowland-Jones, and R. A. Koup. 2008. Poly-
functional T cell responses are a hallmark of HIV-2 infection. Eur. J. Immunol.
38: 350 –363.
9. Ishida, Y., Y. Agata, K. Shibahara, and T. Honjo. 1992. Induced expression of
PD-1, a novel member of the immunoglobulin gene superfamily, upon pro-
grammed cell death. EMBO J. 11: 3887–3895.
10. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe,
G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 T cells
during chronic viral infection. Nature 439: 682– 687.
11. Ha, S. J., S. N. Mueller, E. J. Wherry, D. L. Barber, R. D. Aubert, A. H. Sharpe,
G. J. Freeman, and R. Ahmed. 2008. Enhancing therapeutic vaccination by block-
ing PD-1-mediated inhibitory signals during chronic infection. J. Exp. Med. 205:
543–555.
12. Lukens, J. R., M. W. Cruise, M. G. Lassen, and Y. S. Hahn. 2008. Blockade of
PD-1/B7–H1 interaction restores effector CD8
T cell responses in a hepatitis C
virus core murine model. J. Immunol. 180: 4875– 4884.
13. Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S. Reddy,
E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, et al. 2006. PD-1 expres-
sion on HIV-specific T cells is associated with T-cell exhaustion and disease
progression. Nature 443: 350 –354.
14. Golden-Mason, L., B. Palmer, J. Klarquist, J. A. Mengshol, N. Castelblanco, and
H. R. Rosen. 2007. Upregulation of PD-1 expression on circulating and intrahe-
patic hepatitis C virus-specific CD8
T cells associated with reversible immune
dysfunction. J. Virol. 81: 9249 –9258.
15. Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B. Bessette,
M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, et al. 2006. Upregu-
lation of PD-1 expression on HIV-specific CD8
T cells leads to reversible
immune dysfunction. Nat. Med. 12: 1198 –1202.
16. D’Souza, M., A. P. Fontenot, D. G. Mack, C. Lozupone, S. Dillon, A. Meditz,
C. C. Wilson, E. Connick, and B. E. Palmer. 2007. Programmed death 1 expres-
sion on HIV-specific CD4
T cells is driven by viral replication and associated
with T cell dysfunction. J. Immunol. 179: 1979 –1987.
17. Elrefaei, M., C. A. Baker, N. G. Jones, D. R. Bangsberg, and H. Cao. 2008.
Presence of suppressor HIV-specific CD8
T cells is associated with increased
PD-1 expression on effector CD8
T cells. J. Immunol. 180: 7757–7763.
18. Dong, H., S. E. Strome, D. R. Salomao, H. Tamura, F. Hirano, D. B. Flies,
P. C. Roche, J. Lu, G. Zhu, K. Tamada, et al. 2002. Tumor-associated B7–H1
promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med.
8: 793– 800.
19. Muhlbauer, M., M. Fleck, C. Schutz, T. Weiss, M. Froh, C. Blank,
J. Scholmerich, and C. Hellerbrand. 2006. PD-L1 is induced in hepatocytes by
viral infection and by interferon-
and -
and mediates T cell apoptosis. J. Hepa-
tol. 45: 520 –528.
20. Petrovas, C., D. A. Price, J. Mattapallil, D. R. Ambrozak, C. Geldmacher,
V. Cecchinato, M. Vaccari, E. Tryniszewska, E. Gostick, M. Roederer, et al.
2007. SIV-specific CD8
T cells express high levels of PD1 and cytokines but
have impaired proliferative capacity in acute and chronic SIVmac251 infection.
Blood 110: 928 –936.
21. Zhang, Z., J. Y. Zhang, E. J. Wherry, B. Jin, B. Xu, Z. S. Zou, S. Y. Zhang,
B. S. Li, H. F. Wang, H. Wu, et al. 2008. Dynamic programmed death 1 expres-
sion by virus-specific CD8 T cells correlates with the outcome of acute hepatitis
B. Gastroenterology 134: 1938 –1949.
22. Marrack, P., and J. Kappler. 2004. Control of T cell viability. Annu. Rev. Immu-
nol. 22: 765–787.
23. Chemnitz, J. M., R. V. Parry, K. E. Nichols, C. H. June, and J. L. Riley. 2004.
SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of
programmed death 1 upon primary human T cell stimulation, but only receptor
ligation prevents T cell activation. J. Immunol. 173: 945–954.
24. Parry, R. V., J. M. Chemnitz, K. A. Frauwirth, A. R. Lanfranco, I. Braunstein,
S. V. Kobayashi, P. S. Linsley, C. B. Thompson, and J. L. Riley. 2005. CTLA-4
1131The Journal of Immunology
and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol. Cell.
Biol. 25: 9543–9553.
25. Frauwirth, K. A., and C. B. Thompson. 2004. Regulation of T lymphocyte me-
tabolism. J. Immunol. 172: 4661– 4665.
26. Kane, L. P., and A. Weiss. 2003. The PI-3 kinase/Akt pathway and T cell acti-
vation: pleiotropic pathways downstream of PIP3. Immunol. Rev. 192: 7–20.
27. Brenchley, J. M., N. J. Karandikar, M. R. Betts, D. R. Ambrozak, B. J. Hill,
L. E. Crotty, J. P. Casazza, J. Kuruppu, S. A. Migueles, M. Connors, et al. 2003.
Expression of CD57 defines replicative senescence and antigen-induced apoptotic
death of CD8
T cells. Blood 101: 2711–2720.
28. Papagno, L., C. A. Spina, A. Marchant, M. Salio, N. Rufer, S. Little, T. Dong,
G. Chesney, A. Waters, P. Easterbrook, et al. 2004. Immune activation and CD8
T-cell differentiation towards senescence in HIV-1 infection. PLoS Biol. 2: E20.
29. Le Priol, Y., D. Puthier, C. Lecureuil, C. Combadiere, P. Debre, C. Nguyen, and
B. Combadiere. 2006. High cytotoxic and specific migratory potencies of senes-
cent CD8
CD57
cells in HIV-infected and uninfected individuals. J. Immunol.
177: 5145–5154.
30. Dewson, G., G. M. Cohen, and A. J. Wardlaw. 2001. Interleukin-5 inhibits trans-
location of Bax to the mitochondria, cytochrome c release, and activation of
caspases in human eosinophils. Blood 98: 2239 –2247.
31. Uehara, T., T. Miyawaki, K. Ohta, Y. Tamaru, T. Yokoi, S. Nakamura, and
N. Taniguchi. 1992. Apoptotic cell death of primed CD45RO
T lymphocytes in
Epstein-Barr virus-induced infectious mononucleosis. Blood 80: 452– 458.
32. Chong, L. K., R. J. Aicheler, S. Llewellyn-Lacey, P. Tomasec, P. Brennan, and
E. C. Wang. 2008. Proliferation and interleukin 5 production by CD8
hi
CD57
T
cells. Eur. J. Immunol. 38: 995–1000.
33. Kozak, M. 1987. At least six nucleotides preceding the AUG initiator codon
enhance translation in mammalian cells. J. Mol. Biol. 196: 947–950.
34. Stauber, R. H., K. Horie, P. Carney, E. A. Hudson, N. I. Tarasova,
G. A. Gaitanaris, and G. N. Pavlakis. 1998. Development and applications of
enhanced green fluorescent protein mutants. BioTechniques 24: 462–466,
468 471.
35. Rosati, M., A. von Gegerfelt, P. Roth, C. Alicea, A. Valentin, M. Robert-Guroff,
D. Venzon, D. C. Montefiori, P. Markham, B. K. Felber, and G. N. Pavlakis.
2005. DNA vaccines expressing different forms of simian immunodeficiency vi-
rus antigens decrease viremia upon SIVmac251 challenge. J. Virol. 79:
8480 8492.
36. Cremesti, A., F. Paris, H. Grassme, N. Holler, J. Tschopp, Z. Fuks, E. Gulbins,
and R. Kolesnick. 2001. Ceramide enables fas to cap and kill. J. Biol. Chem. 276:
23954 –23961.
37. George, T. C., S. L. Fanning, P. Fitzgeral-Bocarsly, R. B. Medeiros, S. Highfill,
Y. Shimizu, B. E. Hall, K. Frost, D. Basiji, W. E. Ortyn, et al. 2006. Quantitative
measurement of nuclear translocation events using similarity analysis of multi-
spectral cellular images obtained in flow. J. Immunol. Methods 311: 117–129.
38. Precopio, M. L., M. R. Betts, J. Parrino, D. A. Price, E. Gostick, D. R. Ambrozak,
T. E. Asher, D. C. Douek, A. Harari, G. Pantaleo, et al. 2007. Immunization with
vaccinia virus induces polyfunctional and phenotypically distinctive CD8
T cell
responses. J. Exp. Med. 204: 1405–1416.
39. Arnoult, D., F. Petit, J. D. Lelievre, D. Lecossier, A. Hance, V. Monceaux,
B. Hurtrel, R. Ho Tsong Fang, J. C. Ameisen, and J. Estaquier. 2003. Caspase-
dependent and -independent T-cell death pathways in pathogenic simian immu-
nodeficiency virus infection: relationship to disease progression. Cell. Death Dif-
fer. 10: 1240 –1252.
40. de Oliveira Pinto, L. M., S. Garcia, H. Lecoeur, C. Rapp, and M. L. Gougeon.
2002. Increased sensitivity of T lymphocytes to tumor necrosis factor receptor 1
(TNFR1)- and TNFR2-mediated apoptosis in HIV infection: relation to expres-
sion of Bcl-2 and active caspase-8 and caspase-3. Blood 99: 1666 –1675.
41. Genesca, M., T. Rourke, J. Li, K. Bost, B. Chohan, M. B. McChesney, and
C. J. Miller. 2007. Live attenuated lentivirus infection elicits polyfunctional sim-
ian immunodeficiency virus Gag-specific CD8
T cells with reduced apoptotic
susceptibility in rhesus macaques that control virus replication after challenge
with pathogenic SIVmac239. J. Immunol. 179: 4732– 4740.
42. Katsikis, P. D., M. E. Garcia-Ojeda, J. F. Torres-Roca, I. M. Tijoe, C. A. Smith,
L. A. Herzenberg, and L. A. Herzenberg. 1997. Interleukin-1
converting en-
zyme-like protease involvement in Fas-induced and activation-induced peripheral
blood T cell apoptosis in HIV infection. TNF-related apoptosis-inducing ligand
can mediate activation-induced T cell death in HIV infection. J. Exp. Med. 186:
1365–1372.
43. Fu, X., S. Wan, Y. L. Lyu, L. F. Liu, and H. Qi. 2008. Etoposide induces ATM-
dependent mitochondrial biogenesis through AMPK activation. PLoS. ONE 3:
e2009.
44. Pendergrass, W., N. Wolf, and M. Poot. 2004. Efficacy of MitoTracker Green and
CMXrosamine to measure changes in mitochondrial membrane potentials in liv-
ing cells and tissues. Cytometry A 61: 162–169.
45. Surh, C. D., O. Boyman, J. F. Purton, and J. Sprent. 2006. Homeostasis of mem-
ory T cells. Immunol. Rev. 211: 154 –163.
46. Ibegbu, C. C., Y. X. Xu, W. Harris, D. Maggio, J. D. Miller, and A. P. Kourtis.
2005. Expression of killer cell lectin-like receptor G
1
on antigen-specific human
CD8
T lymphocytes during active, latent, and resolved infection and its relation
with CD57. J. Immunol. 174: 6088 6094.
47. Merino, J., M. A. Martinez-Gonzalez, M. Rubio, S. Inoges, A. Sanchez-Ibarrola,
and M. L. Subira. 1998. Progressive decrease of CD8
high
CD28
CD57
cells
with ageing. Clin. Exp. Immunol. 112: 48 –51.
48. Algeciras-Schimnich, A., L. Shen, B. C. Barnhart, A. E. Murmann,
J. K. Burkhardt, and M. E. Peter. 2002. Molecular ordering of the initial signaling
events of CD95. Mol. Cell. Biol. 22: 207–220.
49. Gajate, C., and F. Mollinedo. 2001. The antitumor ether lipid ET-18-OCH(3)
induces apoptosis through translocation and capping of Fas/CD95 into membrane
rafts in human leukemic cells. Blood 98: 3860 –3863.
50. Moretti, S., A. Procopio, R. Lazzarini, M. R. Rippo, R. Testa, M. Marra,
L. Tamagnone, and A. Catalano. 2008. Semaphorin3A signaling controls Fas
(CD95)-mediated apoptosis by promoting Fas translocation into lipid rafts. Blood
111: 2290 –2299.
51. Mielgo, A., V. Brondani, L. Landmann, A. Glaser-Ruhm, P. Erb, D. Stupack, and
U. Gunthert. 2007. The CD44 standard/ezrin complex regulates Fas-mediated
apoptosis in Jurkat cells. Apoptosis 12: 2051–2061.
52. Giraud, S., C. Lautrette, B. Bessette, C. Decourt, M. Mathonnet, and
M. O. Jauberteau. 2005. Modulation of Fas-induced apoptosis by p75 neurotro-
phin receptor in a human neuroblastoma cell line. Apoptosis 10: 1271–1283.
53. Gougeon, M. L., H. Lecoeur, A. Dulioust, M. G. Enouf, M. Crouvoiser,
C. Goujard, T. Debord, and L. Montagnier. 1996. Programmed cell death in
peripheral lymphocytes from HIV-infected persons: increased susceptibility to
apoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and with
disease progression. J. Immunol. 156: 3509 –3520.
54. McCloskey, T. W., S. Bakshi, S. Than, P. Arman, and S. Pahwa. 1998. Immu-
nophenotypic analysis of peripheral blood mononuclear cells undergoing in vitro
apoptosis after isolation from human immunodeficiency virus-infected children.
Blood 92: 4230 4237.
55. Blattman, J. N., E. J. Wherry, S. J. Ha, R. G. van der Most, and R. Ahmed. 2009.
Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during
chronic infection. J. Virol. 83: 4386 4394.
56. Kinter, A. L., E. J. Godbout, J. P. McNally, I. Sereti, G. A. Roby, M. A. O’Shea,
and A. S. Fauci. 2008. The common
-chain cytokines IL-2, IL-7, IL-15, and
IL-21 induce the expression of programmed death-1 and its ligands. J. Immunol.
181:6738 6746.
57. Dolfi, D. V., A. C. Boesteanu, C. Petrovas, D. Xia, E. A. Butz, and P. D. Katsikis.
2008. Late signals from CD27 prevent Fas-dependent apoptosis of primary CD8
T cells. J. Immunol. 180: 2912–2921.
58. Ramaswamy, M., C. Dumont, A. C. Cruz, J. R. Muppidi, T. S. Gomez,
D. D. Billadeau, V. L. Tybulewicz, and R. M. Siegel. 2007. Cutting edge: Rac
GTPases sensitize activated T cells to die via Fas. J. Immunol. 179: 6384 6388.
59. Scaffidi, C., S. Fulda, A. Srinivasan, C. Friesen, F. Li, K. J. Tomaselli,
K. M. Debatin, P. H. Krammer, and M. E. Peter. 1998. Two CD95 (APO-1/Fas)
signaling pathways. EMBO J. 17: 1675–1687.
60. Petrovas, C., Y. M. Mueller, G. Yang, S. R. Altork, J. M. Jacobson, P. G. Pitsakis,
K. C. Mounzer, J. D. Altman, and P. D. Katsikis. 2007. Actin integrity is indis-
pensable for CD95/Fas-induced apoptosis of HIV-specific CD8
T cells. Apo
-
ptosis 12: 2175–2186.
61. Hughes, P. D., G. T. Belz, K. A. Fortner, R. C. Budd, A. Strasser, and P. Bouillet.
2008. Apoptosis regulators Fas and Bim cooperate in shutdown of chronic im-
mune responses and prevention of autoimmunity. Immunity 28: 197–205.
62. Weant, A. E., R. D. Michalek, I. U. Khan, B. C. Holbrook, M. C. Willingham,
and J. M. Grayson. 2008. Apoptosis regulators Bim and Fas function concurrently
to control autoimmunity and CD8
T cell contraction. Immunity 28: 218 –230.
63. Estaquier, J., M. Tanaka, T. Suda, S. Nagata, P. Golstein, and J. C. Ameisen.
1996. Fas-mediated apoptosis of CD4
and CD8
T cells from human immu
-
nodeficiency virus-infected persons: differential in vitro preventive effect of cy-
tokines and protease antagonists. Blood 87: 4959 4966.
64. Katsikis, P. D., M. E. Garcia-Ojeda, E. S. Wunderlich, C. A. Smith, H. Yagita,
K. Okumura, N. Kayagaki, M. Alderson, L. A. Herzenberg, and L. A. Herzen-
berg. 1996. Activation-induced peripheral blood T cell apoptosis is Fas indepen-
dent in HIV-infected individuals. Int. Immunol. 8: 1311–1317.
65. Cai, G., A. Karni, E. M. Oliveira, H. L. Weiner, D. A. Hafler, and G. J. Freeman.
2004. PD-1 ligands, negative regulators for activation of naive, memory, and
recently activated human CD4
T cells. Cell Immunol. 230: 89 –98.
66. Ladell, K., M. K. Hellerstein, D. Cesar, R. Busch, D. Boban, and J. M. McCune.
2008. Central memory CD8
T cells appear to have a shorter lifespan and re
-
duced abundance as a function of HIV disease progression. J. Immunol. 180:
7907–7918.
67. Lee, K. H., C. Feig, V. Tchikov, R. Schickel, C. Hallas, S. Schutze, M. E. Peter,
and A. C. Chan. 2006. The role of receptor internalization in CD95 signaling.
EMBO J. 25: 1009 –1023.
68. Koncz, G., K. Kerekes, K. Chakrabandhu, and A. O. Hueber. 2008. Regulating
Vav1 phosphorylation by the SHP-1 tyrosine phosphatase is a fine-tuning mech-
anism for the negative regulation of DISC formation and Fas-mediated cell death
signaling. Cell. Death Differ. 15: 494 –503.
69. Petrovas, C., Y. M. Mueller, and P. D. Katsikis. 2005. Apoptosis of HIV-specific
CD8
T cells: an HIV evasion strategy. Cell. Death Differ. 12 Suppl. 1: 859870.
70. Xu, X. N., B. Laffert, G. R. Screaton, M. Kraft, D. Wolf, W. Kolanus,
J. Mongkolsapay, A. J. McMichael, and A. S. Baur. 1999. Induction of Fas ligand
expression by HIV involves the interaction of Nef with the T cell receptor zeta
chain. J. Exp. Med. 189: 1489 –1496.
71. Xu, X. N., G. R. Screaton, F. M. Gotch, T. Dong, R. Tan, N. Almond, B. Walker,
R. Stebbings, K. Kent, S. Nagata, J. E. Stott, and A. J. McMichael. 1997. Evasion
of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas
ligand (CD95L) expression on simian immunodeficiency virus-infected cells.
J. Exp. Med. 186: 7–16.
1132 PROAPOPTOTIC STATUS OF PD-1
H
CD8
T CELLS
    • "It is possible that repetitive infection with influenza virus or chronic stimulation of T cells responding to CMV or HIV could result in distinct patterns of senescence and/or exhaustion [7, 15]. HIV or CMV infection can lead to premature " aging " of the immune system , resulting in increased senescent and/or exhausted CD8 T cells and decreased function [30, 32, 49, 58]. This decrease in function is thought to be a result of chronic antigenic stimulation and agrees with mouse models of chronic infection [46]. "
    [Show abstract] [Hide abstract] ABSTRACT: Aged individuals have increased morbidity and mortality following influenza and other viral infections, despite previous exposure or vaccination. Mouse and human studies suggest increased senescence and/or exhaustion of influenza virus-specific CD8 T cells with advanced age. However, neither the relationship between senescence and exhaustion nor the underlying transcriptional pathways leading to decreased function of influenza virus-specific cellular immunity in elderly humans are well-defined. Here, we demonstrate that increased percentages of CD8 T cells from aged individuals express CD57 and KLRG1, along with PD-1 and other inhibitory receptors, markers of senescence, or exhaustion, respectively. Expression of T-box transcription factors, T-bet and Eomes, were also increased in CD8 T cells from aged subjects and correlated closely with expression of CD57 and KLRG1. Influenza virus-specific CD8 T cells from aged individuals exhibited decreased functionality with corresponding increases in CD57, KLRG1, and T-bet, a molecular regulator of terminal differentiation. However, in contrast to total CD8 T cells, influenza virus-specific CD8 T cells had altered expression of inhibitory receptors, including lower PD-1, in aged compared with young subjects. Thus, our data suggest a prominent role for senescence and/or terminal differentiation for influenza virus-specific CD8 T cells in elderly subjects.
    Full-text · Article · Feb 2013
    • "Although CD57? cells have been described as senescent cells, the fact that some CD57? cells could still proliferate (Chong et al. 2008), contrary to lack of proliferation ability that has been widely described as a major characteristic of senescent cells indicated the possible presence of a more definite subpopulation within the CD57? cells that might be more closely associated with senescence. A recent study has shown variations in the response to apoptosis of different subpopulations of CD57? cells based on their expression of PD-1 (Petrovas et al. 2009). Our present study provides more evidence in support of variations in the behaviour of subpopulations of CD57? cells. "
    [Show abstract] [Hide abstract] ABSTRACT: CD28-, CD57+ and KLRG1+ are cell surface markers that have been used to describe senescent T-lymphocytes in humans. However, the relationship among these phenotypes during aging, and their relationship with the concept of in vitro cellular aging have not been well established. Using five-colour flow cytometry, we analyzed peripheral blood T-lymphocytes for their expression of CD28, CD57 and KLRG1 in 11 young (Y) and 11 old (O) apparently healthy human subjects. The proportions of CD28- and CD57+ cells were significantly higher among the T-cell populations of O compared to Y subjects; the proportion of KLRG1+ cells was significantly higher only among CD8+ cells. Populations that were more frequent in the elderly participants were characterised as CD28+ CD57+, CD28- CD57+ or CD28- CD57-. The expression of p16 and p21, considered as markers for in vitro senescence, was higher in CD28+ CD57+ cells than in other subpopulations in both age groups. The expression of p21 was age-related, which was not the case for p16. Thus, although both p16 and p21 are involved in T-cell senescence, they appear to behave differently. CMV infection and shifts in subpopulations are unlikely as explanations of the observed differences. Their higher levels of p16 and p21 expression, coupled with their higher prevalence in the elderly participants make CD28+ CD57+ cells the subpopulation of T-cells most closely corresponding to the concept of senescent cells.
    Full-text · Article · Nov 2011
    • "Another possible explanation is a higher CD8 naive T cells apoptotic death. Unfortunately, as a limitation of the study, we do not dispose of apoptotic rates or PD-1 expression (Petrovas et al. 2009) from CD4 and/or CD8 T cells to clarify this hypothesis and further research is necessary to really clarify this point. Some authors point out that using CD45RA "
    [Show abstract] [Hide abstract] ABSTRACT: Immunosenescence is characterized by phenotypic and functional changes of effector memory T cells. In spite of the well-described senescent defects of these experienced T cells, immune responses to new pathogens are also deeply affected in elderly humans, suggesting that naive T cells could also show age-related defects. It has been reported in both, animal models and humans, alterations of the naive T cell turnover associated to advanced age or low thymic function. However, as far as we know, homeostatic mechanisms involved in the deregulation of naive T cell peripheral dynamics and their consequences are still not well understood. Thus, the aim of our study was to analyze homeostatic parameters of peripheral naive T cells and their relationship with thymic function in young and elderly humans. Our results show that lower naive T cell numbers were associated with a lower thymic function and higher activation and proliferating naive T cell levels. We then analyzed sjTREC numbers and relative telomere length from sorted naive T cells. Our results show that the aberrant activation and proliferation status was related to lower sjTREC numbers (a peripheral proliferation marker) and both, higher CD57 expression levels and shortened telomeres (replicative senescence-related markers). Elderly individuals show a greater contraction of the CD8 naive T cell numbers and all homeostatic alterations were more severe in this compartment. In addition, we found that low functional thymus show a CD4-biased thymocyte production. Taken together, our results suggest a homeostatic deregulation, affecting mostly the naive CD8 T cell subset, leading to the accumulation of age-associated defects in, otherwise, phenotypically naive T cells.
    Full-text · Article · Jun 2011
Show more