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IMMUNOBIOLOGY
Surface expression patterns of negative regulatory molecules identify
determinants of virus-specific CD8⫹ T-cell exhaustion in HIV infection
Takuya Yamamoto,1 David A. Price,2,3 Joseph P. Casazza,1 Guido Ferrari,4 Martha Nason,5 Pratip K. Chattopadhyay,6
Mario Roederer,6 Emma Gostick,3 Peter D. Katsikis,7 Daniel C. Douek,2 Richard Haubrich,8 Constantinos Petrovas,1 and
Richard A. Koup1
1Immunology
Laboratory and 2Human Immunology Section, National Institutes of Health, Bethesda, MD; 3Department of Infection, Immunity and Biochemistry,
Cardiff University School of Medicine, Cardiff, United Kingdom; 4Department of Surgical Sciences, Duke University Medical Center, Durham, NC; 5Biostatistics
Research Branch, National Institute of Allergy and Infectious Diseases, and 6ImmunoTechnology Section, Vaccine Research Center, National Institutes of
Health, Bethesda, MD; 7Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA; and 8Division of Infectious
Diseases, Antiviral Research Center, University of California San Diego, San Diego, CA
A highly complex network of coinhibitory
and costimulatory receptors regulates the
outcome of virus-specific CD8ⴙ T-cell responses. Here, we report on the expression patterns of multiple inhibitory receptors on HIV-specific, cytomegalovirusspecific, and bulk CD8ⴙ T-cell memory
populations. In contrast to cytomegalovirus-specific CD8ⴙ T cells, the majority of
HIV-specific CD8ⴙ T cells exhibited an
immature phenotype and expressed Pro-
grammed Death-1, CD160 and 2B4 but not
lymphocyte activation gene-3. Notably,
before antiretroviral therapy, simultaneous expression of these negative regulators correlated strongly with both HIV
load and impaired cytokine production.
Suppression of HIV replication by antiretroviral therapy was associated with reduced surface expression of inhibitory
molecules on HIV-specific CD8ⴙ T cells.
Furthermore, in vitro manipulation of Pro-
grammed Death-1 and 2B4 inhibitory pathways increased the proliferative capacity
of HIV-specific CD8ⴙ T cells. Thus, multiple coinhibitory receptors can affect the
development of HIV-specific CD8ⴙ T-cell
responses and, by extension, represent
potential targets for new immune-based
interventions in HIV-infected persons.
(Blood. 2011;117(18):4805-4815)
Introduction
CD8⫹ T cells are a major component of the adaptive immune
response against viruses and cancers. Although they form a
heterogeneous population, they can be divided into distinct subsets
that define the major steps in a process of memory T-cell
differentiation.1,2 These multiple subsets display specific transcriptional programs and express distinct surface receptors and intracellular molecules, indicating quite different requirements for stimulation, survival, homing potential, and effector functions.3
In HIV infection, cellular immune responses fail to control the
virus, and the majority of HIV-infected persons progress to develop
AIDS.4 HIV-specific CD8⫹ T cells, which lack CD4⫹ T-cell help,
express an exhausted phenotype characterized by an impaired
ability to produce cytokines, and proliferate after in vitro stimulation.5 Furthermore, HIV-specific CD8⫹ T cells are sensitive to in
vitro cell death,6 which further compounds their exhausted phenotype. Therefore, therapeutic interventions that target the survival
and effector function of these cells could result in improved
immune control of HIV infection.
Some of the mechanisms that lead to T-cell exhaustion7-9 are
now being clarified. DNA microarray analyses of exhausted CD8⫹
T cells in murine models10 and humans11 suggest that T-cell
exhaustion is the result of both active transcriptional suppression
and defects in metabolism and cell signaling. Therefore, understanding how active inhibitory signals impact cellular immune responses
may lead to the development of novel immunotherapeutic strategies.
An initial series of studies12-14 demonstrated that dysfunctional
HIV-specific CD8⫹ T cells express high levels of Programmed
Death-1 (PD-1), a major marker of virus-specific CD8⫹ T-cell
exhaustion. Furthermore, a correlation between PD-1 expression
on the surface of HIV-specific CD8⫹ T cells and either viral load or
disease progression was observed.12,14 In addition, longitudinal
analysis of HIV-infected subjects before and after the initiation
of antiretroviral therapy (ART) showed that viral load reduction
led to decreased levels of PD-1 expression on HIV-specific
CD8⫹ T cells. Our group also demonstrated that PD-1–
expressing CD8⫹ T cells are more susceptible to both spontaneous and Fas-mediated apoptosis.13 Cross-linking of PD-1 with
an anti-PD-1 monoclonal antibody (mAb) preferentially triggered apoptosis in CD8⫹ T cells that expressed high levels of
PD-1. Conversely, blockade of the PD-1 pathway with an
anti-PD-L1 mAb enabled greater proliferation of HIV-specific
CD8⫹ T cells.13
More recently, Blackburn et al reported that CD8⫹ T-cell
responses during chronic viral infection in mice are regulated by
complex patterns of coexpressed inhibitory receptors.15 In this
latter study, several molecules that had previously been identified
by DNA microarray analysis10 were found to be highly expressed
on the surface of exhausted CD8⫹ T cells; these included PD-1,
CD160,16,17 2B4,18 and lymphocyte activation gene-3 (LAG-3).19,20
Furthermore, it appears that the greater the coexpression of these
Submitted November 3, 2010; accepted March 6, 2011. Prepublished online as
Blood First Edition paper, March 11, 2011; DOI 10.1182/blood-2010-11-317297.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
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BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
YAMAMOTO et al
inhibitory receptors, the greater the degree of exhaustion exhibited
by virus-specific CD8⫹ T cells in both mice and humans.21,22
In this study, we examined the simultaneous expression patterns
of PD-1, CD160, 2B4, and LAG-3 on CD8⫹ T-cell populations
with defined virus-derived antigen specificities. The expression of
inhibitory receptors varied with antigen specificity and T-cell
differentiation status in HIV-infected persons. Moreover, the simultaneous expression of these molecules correlated directly with HIV
load and inversely with the multiplicity of functional outputs
exhibited by HIV-specific CD8⫹ T cells reexposed to cognate
antigen. In addition, the proliferative capacity of HIV-specific
CD8⫹ T cells was restored by blocking both PD-1/PD-L1 and
2B4/CD48 interactions.
Methods
Study subjects and cell culture
HIV-1–infected antiretroviral-naive male and nonpregnant female subjects
with a minimum age of 13 years and plasma HIV RNA levels more than
2000 copies/mL were enrolled.23 The study protocol was approved by an
Institutional Review Board or ethics committee at each participating site.
All participants provided written informed consent, in accordance with the
Declaration of Helsinki. AIDS Clinical Trial Group A5142 was a phase 3,
randomized, multicenter, open-label trial.24 Longitudinal cryopreserved
peripheral blood mononuclear cell (PBMC) samples were collected from
HIV⫹ (n ⫽ 85) and HIV⫺ (n ⫽ 19) persons. All available subjects from
AIDS Clinical Trial Group A5142 who had adequate stored samples
from baseline and week 48 with more than 85% viability and HIV pVL
levels less than 50 copies after week 24 were studied. The median pre-ART
CD4⫹ T-cell count of the 85 HIV⫹ persons was 227.5 cells/␮L (range,
6-1052 cells/␮L), and the median pre-ART pVL was 38 905 RNA
copies/mL plasma (range, 1567-1 412 538 RNA copies/mL plasma). An
additional 11 HIV⫹ persons were recruited from Drexel University College
of Medicine, Philadelphia, PA; freshly isolated PBMCs from these persons
were used for functional analysis of inhibitory receptor blockade. A further
11 HIV⫹ persons (3 viremic and 8 aviremic) were recruited from Duke
University Medical Center (Durham, NC); PBMCs from these persons were
included in the combined intracellular cytokine and tetramer staining
experiments. PBMCs were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 U/mL
penicillin, and 100 ␮g/mL streptomycin (Invitrogen).
Antibodies
The following directly conjugated mAbs were used: (1) CD3Cy7allophycocyanin (APC), IL2-Cy5.5peridinin chlorophyll protein, interferon-␥ (IFN-␥)–APC and tumor necrosis factor-␣ (TNF-␣)–Cy7PE (BD
Biosciences); (2) CD45RO-TexasRedPE, 2B4-Cy5PE (clone C1.7),
CD160-PE (clone BY55), and CD27-Alexa700APC (Beckman Coulter);
(3) CD4-Qdot605 (Invitrogen); and (4) LAG-3-fluorescein isothiocyanate
(FITC; clone 17B4) and IFN-␥–Pacific Blue (eBioscience). The following
mAbs were conjugated in our laboratory: CCR7-Pacific Blue, CD14-Pacific
Blue, CD19-Pacific Blue, CD8-Qdot585, and CD8-Qdot705. Unconjugated
mAbs were obtained from BD Biosciences. Pacific Blue was obtained from
Invitrogen. Quantum dots were obtained from Invitrogen. The violet
(ViViD), Aqua amine reactive viability dyes were obtained from Invitrogen.
Biotinylated PD-1 mAb (clone; BAF 1086) was obtained from R&D
Systems, and conjugated streptavidin (Cy7PE or Qdot655) was obtained
from Invitrogen.
Polychromatic flow cytometry
Cells were analyzed with a modified LSRII flow cytometer (BD Immunocytometry Systems) as described previously.25 Briefly, 3 ⫻ 106 PBMCs
were incubated in 1 mL of medium containing 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 or cytomegalovirus (CMV) pp65 (2 ␮g/mL each
peptide, 5 ␮L/mL; National Institutes of Health AIDS Research and
Reference Reagent Program) for 6 hours. After washing, cells were surface
stained for PD-1, CD160, 2B4, LAG-3, CD4, CD8, CD27, CD45RO,
CCR7, and CD14/CD19; ViViD or Aqua was used to exclude dead cells
from the analysis. After permeabilization (Cytofix/Cytoperm kit; BD
Biosciences), cells were stained for CD3, IFN-␥, IL-2, and TNF-␣.
Between 300 000 and 2 ⫻ 106 events were collected in each case.
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 9.0.1 (TreeStar).
Forward scatter area versus forward scatter height was used to gate out cell
aggregates. CD14⫹, CD19⫹, and dead cells were removed from the analysis
to reduce background staining. The combinations ViViD/CD14–Pacific
Blue/CD19–Pacific Blue/CD3-Cy7APC/CD4-Qdot605/CD8-Qdot705/
CD27-Alexa700APC/CD45RO-TexasRedPE/PD-1-SA-Qdot655/2B4-Cy5PE/
CD160-PE/LAG-3-FITC, Aqua/CD3-Cy7APC/CD4-Qdot605/CD8-Qdot585/
CD27-Alexa700APC/CD45RO-TexasRedPE/CCR7–Pacific Blue/PD-1SA-Qdot655/2B4-Cy5PE/CD160-PE/LAG-3-FITC/TNF-␣–Cy7PE/IL2Cy5.5peridinin chlorophyll protein/IFN-␥–APC, and Aqua/CD3-Cy7APC/
CD4-Qdot605/CD8-Qdot585/CD27-Alexa700APC/CD45RO-TexasRedPE/
PD-1-SA-Qdot655/2B4-Cy5PE/CD160-PE/LAG-3-FITC/TNF-␣–Cy7PE/
IL2-Cy5.5peridinin chlorophyll protein/IFN-␥–Pacific Blue/tetramer-APC
were used for further characterization.
Peptide-MHC class I tetramers
Tetrameric peptide/human leukocyte antigen (HLA) class I tetramers
conjugated to APC were produced as described previously.26 The following
complexes were used in this study: HIV-1 Gag SLYNTVATL(SL9)/HLA
A*0201, CMV pp65 NLVPMVATV(NV9)/HLAA*0201, HIV-1 Nef TPQVPLRPM(TM9)/HLA B*0702, CMV pp65 TPRVTGGGAM(TM10)/HLA
B*0702, and HIV-1 Gag TSTLQEQIAW(TW10)/HLA B*5701.
Proliferation studies
Carboxyfluorescein succinimidyl ester dilution assays to examine CD8⫹
T-cell proliferative responses were performed as described previously,
using a final concentration of 0.2 ␮g/mL peptide with the costimulatory
mAbs CD28 and CD49d at 1 ␮g/mL each (BD Biosciences).25 Briefly,
freshly isolated PBMCs were incubated with or without the HIV-1 Gag
peptide pool containing isotype control antibody (IgG2b clone MPC.11;
10 ␮g/mL), purified anti–PD-L1 (10 ␮g/mL; eBioscience), purified antiCD48 (5 ␮g/mL; eBioscience), or purified anti–PD-L1 plus anti-CD48.
Cells were incubated for 6 days, stained with surface antibodies, and the
percentage of carboxyfluorescein succinimidyl esterlow CD8⫹ T cells was
analyzed by flow cytometry. For some experiments, the SL9, a gag-epitope
peptide, was used for stimulation. The production of IFN-␥ and TNF-␣ was
also analyzed after 6 days of culture in some experiments. In this case, cells
were restimulated on day 6 with the same peptide pool and cytokine
production was detected by intracellular staining.
Statistical analysis
Experimental variables were analyzed using the nonparametric MannWhitney U test, the Wilcoxon matched-pairs signed rank test, and the
Friedman test; correlations were performed using the nonparameric Spearman rank test. Bars represent median values. P values ⬍ .05 were
considered significant. The GraphPad Prism statistical analysis program
(Version 5.0c; GraphPad Software) was used throughout. Analysis and
graphical representation of cytokine production in relation to PD-1,
CD160, 2B4, and LAG-3 expression were conducted using the data analysis program Simplified Presentation of Incredibly Complex Evaluations
(Version 5.05013) provided by M. Roederer (National Institutes of Health,
Bethesda, MD).
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BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
Results
Expression patterns of inhibitory receptors vary across distinct
memory CD8ⴙ T-cell subsets
To characterize the role of inhibitory receptors in HIV⫹ persons, we
first analyzed the expression patterns of several costimulatory and
coinhibitory receptors on virus-specific CD8 T cells. Our preliminary studies showed negligible expression of Tim-3 and CTLA-4
on these cells (both surface and intracellular; data not shown). We
therefore focused our analyses in the expression of PD-1, CD160,
2B4, and LAG-3 on total and virus-specific CD8⫹ T cells from
infected donors before and after ART treatment (AIDS Clinical
Trial Group A5142 cohort).23,24 Representative gating schemes for
the flow cytometric analysis of individual surface molecule expression with respect to CD8⫹ T-cell maturation status are shown in
supplemental Figure 1 (available on the Blood Web site; see the
Supplemental Materials link at the top of the online article). The
surface markers CD27, CD45RO, and CCR7 were used to define
memory populations. Naive CD8⫹ T cells, the population with the
lowest levels of inhibitory receptors, were used as a reference to
define cells with high expression levels of the individual molecules
tested. As described previously, we found that PD-1 was expressed
primarily on central memory (CM), transitional memory (TM), and
effector memory (EM) CD8⫹ T cells27; CD160 was expressed
mainly on CM and TM CD8⫹ T cells (Figure 1). In contrast, the
expression of 2B4 on CD8⫹ T cells increased progressively with
maturation status; thus, the EM and effector (Eff) populations
expressed the highest levels of 2B4. Although the majority of
CD8⫹ T cells stimulated in vitro expressed high levels of LAG-3
(supplemental Figure 2), ex vivo measurements of this molecule
revealed elevated expression only within the CM population.
ART decreases inhibitory receptor expression on memory
CD8ⴙ T cells in HIVⴙ persons
In this cohort, plasma viral load (pVL) was consistently less than
50 copies/mL in all persons between week 24 and week 48 after the
initiation of ART. Reduced expression of all inhibitory molecules
(PD-1, CD160, 2B4, and LAG-3) within the total CD8⫹ T-cell pool
was observed in HIV⫹ persons on ART during this period
compared with baseline levels before treatment (P ⬍ .0001 in each
case for PD-1, CD160, 2B4, and LAG-3; Figure 1). Of note,
although ART reduced the elevated expression of CD160 on all
CD8⫹ T cells, this decrease was especially marked on naive
CD8⫹ T cells; thus, CD160 levels decreased to parallel those
observed on naive CD8⫹ T cells from HIV⫺ persons, suggesting
that CD160 may be an antigen-independent marker of general
activation. Expression levels of PD-1 on CD8⫹ T cells on CM,
TM, and EM compartments were reduced on ART, as was 2B4
expression on TM cells. Paradoxically, LAG-3 expression on
CD8⫹ T cells with a CM phenotype increased on ART.
HIV-specific CD8ⴙ T cells express increased levels of inhibitory
receptors in the absence of ART
Next, we investigated the expression levels of all 4 inhibitory
molecules on CD8⫹ T cells specific for HIV and CMV. Virusspecific CD8⫹ T cells were detected by intracellular staining for
IFN-␥ in PBMC preparations stimulated with overlapping peptide
pools spanning the HIV-1 Gag and CMV pp65 proteins (supplemental Figure 3). The expression profile of these inhibitory molecules
DETERMINANTS OF HIV-SPECIFIC CD8⫹ T-CELL EXHAUSTION
4807
on HIV-specific CD8⫹ T cells was similar regardless of the peptide
pool (Gag, Pol, Env, or Nef) used for stimulation (data not shown).
On this basis, the Gag peptide pool was selected for the detection of
HIV-specific CD8⫹ T cells.
Analysis of pre-ART samples showed that HIV-specific CD8⫹
T cells expressed significantly higher levels of PD-1 compared with
CMV-specific CD8⫹ T cells (mean fluorescence intensity [MFI],
P ⬍ .0001; percentage, P ⫽ .0014; Figure 2A), in line with previously reported data.13 After ART, PD-1 expression on HIV-specific
CD8⫹ T cells decreased (MFI, P ⬍ .0001; percentage, P ⫽ .0077).
Although the MFI of PD-1 on CMV-specific CD8⫹ T cells before
ART was higher compared with that in post-ART samples
(P ⫽ .0017), the percentage of PD-1⫹ CMV-specific CD8⫹ T cells
did not change significantly (P ⫽ .8142). CD160 expression was
substantially higher on HIV-specific compared with CMV-specific
CD8⫹ T cells in pre-ART samples (MFI, P ⬍ .0001; percentage,
P ⬍ .0001; Figure 2B). ART reduced the expression of CD160 on
both HIV-specific and CMV-specific CD8⫹ T cells (P ⬍ .0001 for
all comparisons; Figure 2B). The expression intensity (MFI) of
2B4 was also consistently higher on HIV-specific compared
CMV-specific CD8⫹ T cells (P ⫽ .0016 for pre-ART comparison;
P ⫽ .0002 for post-ART comparison; Figure 2C). However, the
percentage of 2B4⫹ cells was similar in the HIV-specific and
CMV-specific CD8⫹ T-cell populations (P ⫽ .0247 for pre-ART
comparison; P ⫽ .5490 for post-ART comparison). No differences
between HIV-specific and CMV-specific CD8⫹ T cells were
observed with respect to LAG-3 expression, either in pre- or
post-ART samples (Figure 2D).
HIV-specific and CMV-specific CD8ⴙ T cells display distinct
patterns of inhibitory receptor expression
In further experiments, we determined the simultaneous expression
of PD-1, CD160, 2B4, and LAG-3 on HIV-specific and CMVspecific CD8⫹ T cells (Figure 2E). Of the 16 potential permutational expression patterns of these 4 inhibitory molecules, 3 predominated. We therefore evaluated virus-specific CD8⫹ T cells that fell
within these 3 major populations: PD-1⫹CD160⫹2B4⫹LAG-3⫺,
PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD-1⫺CD160⫺2B4⫹LAG-3⫺. The
percentage of HIV-specific CD8⫹ T cells that expressed a
PD-1⫹CD160⫹2B4⫹LAG-3⫺ phenotype was significantly higher
compared with CMV-specific CD8⫹ T cells (P ⬍ .0001). After
ART, the percentage of both HIV-specific and CMV-specific CD8⫹
T cells bearing a PD-1⫹CD160⫹2B4⫹LAG-3⫺ expression pattern
decreased dramatically. The majority of CMV-specific CD8⫹
T cells were PD-1⫺CD160⫺2B4⫹LAG-3⫺; this phenotypic pattern
increased within both the HIV-specific and CMV-specific CD8⫹
T-cell populations after ART (Figure 2E).
Next, we assessed the maturation level of CD8⫹ T cells with
PD-1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and
PD-1⫺CD160⫺2B4⫹LAG-3⫺ expression patterns (Figure 3A-B).
The PD-1⫹CD160⫹2B4⫹LAG-3⫺ expression pattern was associated with a relatively immature differentiation status
(CCR7lowCD27hiCD45ROhi); the majority of HIV-specific CD8⫹
T cells accumulate in this phenotypic compartment.28,29 Single
expression of 2B4, however, characterized a highly differentiated
population (CCR7lowCD27lowCD45ROlow) both in HIV-specific
and CMV-specific CD8⫹ T-cell populations (P ⬍ .0001 in each
case; Figure 3C-D). Thus, our data suggest a relationship between
the expression of inhibitory receptors and the level of maturation.
However, it is not clear whether these molecules play an active role
in regulating the differentiation process of virus-specific CD8⫹
T cells. Next, the association between the simultaneous expression
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YAMAMOTO et al
BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
Figure 1. Expression patterns of inhibitory receptors vary across distinct memory CD8ⴙ T-cell subsets. Pooled data showing the percentage of CD8⫹ T cells expressing
PD-1, CD160, 2B4, and LAG-3 within the total CD8⫹ T-cell pool from healthy donors (n ⫽ 19) and HIV⫹ persons before and after ART (n ⫽ 85). PD-1⫹, CD160⫹, 2B4⫹, and
LAG-3⫹ expression in naive and memory CD8⫹ T-cell subsets from healthy donors and HIV⫹ persons before and after ART is also shown. Horizontal lines indicate median
values. The P values were calculated using a combination of a paired test for the pre- and post-initiation of therapy measurements, and Mann-Whitney unpaired test to compare
the HIV-negative group with HIV⫹ pre-ART of measures. *P ⬍ .01, **P ⬍ .001, ***P ⬍ .0001.
of PD-1, CD160, and 2B4 and activation level of HIV-specific
CD8⫹ T cells was investigated. A significant correlation was found
between this “coinhibitory pattern” and the expression of CD38
(P ⫽ .03). No correlation was found between this pattern and
HLADR or CD57, a marker of senescence.
The frequency of hyperexhausted HIV-specific CD8ⴙ T cells
correlates with viremia
Previously published data have shown a correlation between
pVL and PD-1 expression on HIV-specific CD8⫹ T cells.12,14
Therefore, we analyzed the relationship between pVL and the
expression of inhibitory molecules in HIV⫹ persons before ART.
No significant correlation was detected between pVL and single
expression of any of the 4 inhibitory receptors on HIV-specific
CD8⫹ T cells (Figure 4A). However, a significant correlation was
found between pVL and the simultaneous expression of PD-1,
CD160, and 2B4 on HIV-specific CD8⫹ T cells (P ⫽ .0034). No
correlation was observed for the other 2 major populations,
PD-1⫹CD160⫺2B4⫹LAG-3⫺ and PD-1⫺CD160⫺2B4⫹LAG-3⫺
(Figure 4B). These data imply that the simultaneous expression of
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BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
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Figure 2. HIV-specific CD8ⴙ T cells express high levels of inhibitory molecules before ART. (A-D) Comparisons of MFI and percentage of PD-1, CD160, 2B4, and LAG-3
expression on HIV-specific and CMV-specific CD8⫹ T cells in matched samples from 85 HIV⫹ persons. HIV-specific (n ⫽ 52, blue circles) and CMV-specific (n ⫽ 60, dark gray
circles) CD8⫹ T cells before ART; HIV-specific (n ⫽ 59, red circles) and CMV-specific (n ⫽ 62, light gray circles) CD8⫹ T cells after ART. Each dot represents one person at one
time point. (E) Percentage expression of inhibitory molecules on HIV-specific CD8⫹ T cells before ART (n ⫽ 52, blue bars) and after ART (n ⫽ 59, red bars), and on
CMV-specific CD8⫹ T cells before ART (n ⫽ 60, dark gray bars) and after ART (n ⫽ 62, light gray bars) are shown for each of the 16 phenotypic subsets based on the
expression profiles of PD-1, CD160, 2B4, and LAG-3. *P ⬍ .01, **P ⬍ .001, ***P ⬍ .0001.
PD-1, CD160, and 2B4 may better characterize the exhausted
phenotype of HIV-specific CD8⫹ T cells under conditions of
chronic virus-specific stimulation.
Simultaneous expression of inhibitory receptors correlates
inversely with polyfunctionality
Next, we investigated the capacity of virus-specific CD8⫹ T cells to
produce cytokines with respect to the expression of inhibitory
molecules. Cells were stimulated with cognate peptides, and total
antigen-specific CD8⫹ T-cell populations (tetramer⫹cytokine⫹,
tetramer⫹cytokine⫺, and tetramer⫺cytokine⫹) were analyzed for
inhibitory molecule expression patterns (Figure 5A). The frequencies of nonfunctional HIV-specific CD8⫹ T cells within the
PD-1⫹CD160⫹2B4⫹LAG-3⫺ and PD-1⫹CD160⫺2B4⫹LAG-3⫺
compartments were significantly higher compared with the corresponding frequencies in the PD-1⫺CD160⫺2B4⫹LAG-3⫺ compartment (P ⫽ .0156 and P ⫽ .0469, respectively; Figure 5A). The
frequencies of nonfunctional CMV-specific CD8⫹ T cells within
the PD-1⫹CD160⫹2B4⫹LAG-3⫺ and PD-1⫹CD160⫺2B4⫹LAG-3⫺
compartments were also higher compared with the corresponding
frequencies in the PD-1⫺CD160⫺2B4⫹LAG-3⫺ compartment
(P ⫽ .0137 and P ⫽ .002, respectively; Figure 5A).
Previous studies30 have revealed the importance of T-cell
polyfunctionality, defined by the multiplicity of antigen-specific
cytokine production and other effector functions, in HIV pathogenesis. Such functional profiling has been used as an indicator of
T-cell quality, and an inverse correlation has been found between
pVL and the polyfunctionality of HIV-specific CD8⫹ T cells in
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YAMAMOTO et al
BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
Figure 3. Virus-specific CD8ⴙ T cells become less exhausted with increasing maturity. (A-B) The surface phenotype of HIV-specific and CMV-specific CD8⫹ T cells is
shown for representative exhausted populations (PD-1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD-1⫺CD160⫺2B4⫹LAG-3⫺) from a single HIV⫹ person by
overlaying responding cells (red contour plots) on density plots of the corresponding total CD8⫹ T-cell populations for CD27 and CD45RO expression. (C-D) Maturation
decreases inhibitory molecule expression on PD-1⫹CD160⫹2B4⫹LAG-3⫺ (dark purple), PD-1⫹CD160⫺2B4⫹LAG-3⫺ (purple), and PD-1⫺CD160⫺2B4⫹LAG-3⫺ (light purple)
HIV-specific CD8⫹ T cells, and PD-1⫹CD160⫹2B4⫹LAG-3⫺ (dark gray), PD-1⫹CD160⫺2B4⫹LAG-3⫺ (gray), and PD-1⫺CD160⫺2B4⫹LAG-3⫺ (light gray) CMV-specific CD8⫹
T cells, in HIV⫹ persons before ART (n ⫽ 47 for HIV-specific CD8⫹ T cells; n ⫽ 60 for CMV-specific CD8⫹ T cells). The phenotypic complexity of virus-specific CD8⫹ T cells was
assessed by analyzing the individual phenotypic patterns. The y-axis displays the percentage of each phenotypic pattern, the composition of which is denoted on the x-axis with
⫹ for the presence of CD27, CD45RO, and CCR7 on PD-1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD-1⫺CD160⫺2B4⫹LAG-3⫺ virus-specific CD8⫹ T cells.
The proportion of total memory CD8⫹ T cells accounted for by each phenotypic pattern, together with the median and interquartile ranges, are shown. The phenotypic patterns
are grouped and color-coded according to phenotype and summarized in pie chart form; each pie slice represents the mean proportion of total memory CD8⫹ T cells for each
inhibitory receptor expression pattern. Red represents CM; blue, TM; green, EM; and yellow, Eff.
HIV-infected persons.5 Therefore, we analyzed the polyfunctionality of HIV-specific and CMV-specific CD8⫹ T cells with PD1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD1 ⫺ CD160 ⫺ 2B4 ⫹ LAG-3 ⫺ patterns of inhibitory receptor
expression (Figure 5B). HIV-specific CD8⫹ T cells with a
PD-1⫹CD160⫹2B4⫹LAG-3⫺ phenotype were less polyfunctional compared with HIV-specific CD8⫹ T cells with a PD1⫺CD160⫺2B4⫹LAG-3⫺ phenotype (n ⫽ 47, P ⫽ .002). Similar findings applied to CMV-specific CD8⫹ T cells (n ⫽ 60,
P ⫽ .016). Moreover, there was a significant inverse correlation
between the percentage of PD-1⫹CD160⫹2B4⫹LAG-3⫺ cells
within the HIV-specific CD8⫹ T-cell population and the percentage of polyfunctional HIV-specific CD8⫹ T cells (P ⫽ .0027;
Figure 5C). This was not the case, however, for the PD-
1⫹CD160⫺2B4⫹LAG-3⫺ and PD-1⫺CD160⫺2B4⫹LAG-3⫺ HIVspecific CD8⫹ T-cell populations (Figure 5C). Thus, the simultaneous expression of several inhibitory molecules correlates
inversely with the capacity of CD8⫹ T cells to produce
cytokines.
Blockade of both PD-1 and 2B4 engagement restores
HIV-specific CD8ⴙ T-cell proliferation
The proliferative potential of AIDS virus-specific CD8⫹ T cells can
be restored by manipulating the interaction between PD-1 and its
ligand, PD-L1, both in vitro12-14 and in vivo.31 To extend these
observations, we investigated the effects of additional inhibitory
pathway manipulations on the proliferation profile of virus-specific
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BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
DETERMINANTS OF HIV-SPECIFIC CD8⫹ T-CELL EXHAUSTION
4811
Figure 4. Hyperexhausted HIV-specific CD8ⴙ T cells correlate with viremia. (A) Percentage of single inhibitory molecule expression on HIV-specific CD8⫹ T cells as a
function of pVL (n ⫽ 52). (B) Percentage of PD-1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD-1⫺CD160⫺2B4⫹LAG-3⫺ expression profiles on HIV-specific
CD8⫹ T cells as a function of pVL (n ⫽ 47). Lines indicate correlations detected by Prism software Version 5.0c.
CD8⫹ T cells in vitro (Figure 6). Our preliminary studies
showed a similar positive effect of blocking PD-1/PD-L1 in
proliferation of virus-specific CD8 T cells in the absence or
presence of anti-CD28/49d costimulation. The addition of
anti-CD28/49d, however, resulted in higher proliferation rates
(data not shown). Simultaneous blockade of the PD-1/PD-L1
and 2B4/CD48 interactions exhibited a superior effect in terms
of restoring the proliferation of HIV-specific CD8⫹ T cells
compared with PD-1/PD-L1 blockade alone (n ⫽ 11, P ⬍ .05;
Figure 6B) when cells were stimulated with viral peptide pools.
Use of an epitope-specific peptide showed a similar induction of
proliferation (data not shown). Similarly, the percentage of
CD8⫹ T cells capable of producing IFN-␥ and TNF-␣ was
increased in the presence of the blocking antibodies (data not
shown). Identical manipulations had no effect on the proliferation of HIV-specific CD8⫹ T cells in the absence of T-cell
receptor stimulation. These data are in line with recently
published work in the murine lymphocytic choriomeningitis
virus model.15
Discussion
Recently published data suggest that the manipulation of T cell
costimulatory pathways may represent a novel approach to
enhance and restore virus-specific CD8⫹ T-cell responses,
especially in the context of persistent infections, such as
HIV.7,15,32,33 In this study, we describe the selective increase and
simultaneous expression of several coinhibitory receptors on
HIV-specific CD8⫹ T cells. These observations are consistent
with recent reports of an active role for multiple coinhibitory
receptors in the regulation of virus-specific CD8⫹ T cells.15,34
Furthermore, the pattern of inhibitory receptor expression was
found to correlate with the level of HIV-specific CD8⫹ T-cell
exhaustion; again, this is consistent with previous studies in
other systems.15 However, it remains unclear whether this
simultaneous expression of inhibitory molecules represents a
biologic need for tight regulation of virus-specific CD8⫹ T-cell
responses.
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4812
YAMAMOTO et al
BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
Figure 5. Inhibitory marker coexpression correlates inversely with polyfunctionality. (A) 10 HIV⫹ subjects from Duke University were studied. Representative plots of
intracellular cytokine staining combined with SL9/HLAA*0201, NV9/HLAA*0201, TM9/HLA B*0702, TM10/HLA B*0702, or TW10/HLA B*5701 tetramer staining of CD8⫹ T cells
are shown. Cells were independently stimulated with individual epitopic peptides for 6 hours. Pooled data lower panels represent the percentage of nonfunctional CD8⫹ T cells
with PD-1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD-1⫺CD160⫺2B4⫹LAG-3⫺ expression profiles within tetramer⫹ or any cytokine⫹ populations
(HIV-specific responses, n ⫽ 7; CMV-specific responses, n ⫽ 10). (B) The fraction of the total response composing cells expressing each of the 7 possible combinations of
IFN-␥, IL-2, and TNF-␣ production within the PD-1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD-1⫺CD160⫺2B4⫹LAG-3⫺ populations. Cells were stimulated
with the HIV-1 Gag peptide pool (n ⫽ 47) or the CMV pp65 peptide pool (n ⫽ 60), respectively. (C) Comparisons between polyfunctionality and the percentage of
PD-1⫹CD160⫹2B4⫹LAG-3⫺, PD-1⫹CD160⫺2B4⫹LAG-3⫺, and PD-1⫺CD160⫺2B4⫹LAG-3⫺ expression profiles on HIV-specific CD8⫹ T cells as a function of pVL (n ⫽ 47).
Lines indicate correlations detected by Prism software Version 5.0c.
In more detailed analyses, we observed a correlation between
cellular differentiation and the expression of PD-1 and CD160;
specifically, these inhibitory receptors were expressed predominantly in the CM, TM, and EM compartments. In contrast, the
pattern of 2B4 expression was consistently high in all bulk memory
populations in samples from HIV⫹ donors and exhibited a trend for
greater expression with greater degrees of differentiation. Previous
studies have shown that 2B4 can elicit both “stimulatory” and
“inhibitory” signals depending on the cell type, the level of its
expression, and the abundance of adaptor molecules.18,35 Whether
the expression of 2B4 in different memory compartments is
associated with a positive or negative signaling for T-cell activation
is not clear and should be addressed in future studies. It is possible
that these coexpression patterns define signals that are necessary
during CD8⫹ T maturation, especially within the less differentiated
memory compartments (CM, TM, and EM), with each receptor
mediating a distinct and determinative cytosolic signal during the
maturation process. However, further work is necessary to clarify
the function of these individual inhibitory receptors in different
memory CD8⫹ T-cell populations.
The inhibitory receptors examined in this study were expressed
to a greater degree on CD8⫹ T cells from HIV⫹ persons compared
with those from HIV⫺ persons. HIV-specific T-cell receptormediated stimulation, general immune activation, and direct functional effects of viral particles/proteins could be among the
mechanisms that lead to this pervasive expression pattern.4,36-38
Treatment with ART reduced inhibitory receptor expression, in
some cases even to the levels observed on CD8⫹ T cells from HIV⫺
persons; this is consistent with previously observed effects of ART
on CD8⫹ T-cell functionality.39 It is notable that CD160 expression
was significantly higher on naive CD8⫹ T cells from HIV⫹ persons
compared with the corresponding cells from HIV⫺ persons and that
this was reversed by ART. This particular expression pattern could
potentially be attributable to the suppression of the general immune
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BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
DETERMINANTS OF HIV-SPECIFIC CD8⫹ T-CELL EXHAUSTION
4813
Figure 6. Blockade of both PD-1 and 2B4 engagement restores HIV-specific CD8ⴙ T-cell proliferation. (A) Representative data showing expansion of proliferating CD8⫹
T cells (carboxyfluorescein succinimidyl ester [CFSE] low) in response to stimulation with HIV-1 Gag peptides in the presence or absence of anti-PD-L1 and/or anti-CD48
antibody blockade. (B) Summary data for expansion of HIV-specific CD8⫹ T cells under conditions of inhibitory pathway blockade (n ⫽ 11). Statistical comparisons were
conducted using the Friedman test.
activation after ART. We did observe an up-regulation of LAG-3
only on bulk CM CD8⫹ T cells and not on virus-specific CD8⫹
T cells. We assume that this change is not related to antigen-specific
T-cell receptor stimulation and could be a bystander effect of
altered CD8 T-cell dynamics because of highly active antiretroviral
therapy.
No correlation was found between CD4 counts and simultaneous expression of PD-1, CD160, 2B4 either before or after ART,
indicating that this pattern is not related to CD4 reconstitution.
Previous studies have demonstrated a correlation between pVL and
PD-1 expression on HIV-specific CD8⫹ T cells.12,14 In agreement
with our previous data,13 we were not able to confirm such a
relationship in this study. A strong correlation was observed,
however, between pVL and the simultaneous expression of PD-1,
CD160, and 2B4 on HIV-specific CD8⫹ T cells (Figure 4). This
association indicates that the simultaneous expression of these
inhibitory markers could serve as a more accurate phenotypic
composite to demarcate exhausted HIV-specific CD8⫹ T cells.
Furthermore, the reduced expression of these markers after ART
was found to be associated with undetectable viral loads, indicating
that this expression pattern could also be used to predict viral
suppression. More experiments, however, are needed to formally
determine whether the expression patterns of these markers can act
as a surrogate for full virus suppression.
The importance of the PD-1/PD-L1 pathway in the functional
exhaustion of CD8⫹ T cells in HIV⫹ persons has been reported by
several groups.12-14 However, our previous work revealed no
association between PD-1 expression and the ex vivo production of
cytokines by virus-specific CD8⫹ T cells.13 Here we report that the
simultaneous expression of several inhibitory receptors was correlated with the ex vivo capacity of both HIV-specific and CMVspecific CD8⫹ T cells to produce multiple cytokines (Figure 5).
Thus, the relative expression of these inhibitory receptors in
combination on HIV-specific CD8⫹ T cells could potentially
explain contradictory data in the literature12-14 regarding the role of
“exhaustion” molecules in cytokine production by virus-specific
CD8⫹ T cells. Our data also demonstrate that simultaneous
coexpression of 3 markers (PD-1, CD160, and 2B4) better defines
cytokine-exhausted CD8⫹ T cells than do any one of those markers
individually.
Manipulation of costimulatory molecules has been shown to
alter the proliferation/survival capacity of virus-specific CD8⫹
T cells both in vitro12-14 and in vivo.31 Simultaneous blockade of the
PD-1/PD-L1 and 2B4/CD48 interactions in the current study
revealed a synergistic effect on the proliferation of HIV-specific
CD8⫹ T cells, thereby indicating that these receptors can function
independently, at least in vitro (Figure 6). Such functional independence could be critical in vivo, where inhibitory receptor expression likely varies on different cell populations and at different
anatomic sites, and where the expression kinetics and distribution
of their respective cognate ligands may also differ.
What are the clinical implications of these findings? Although
our data indicate that manipulation of multiple negative regulators
of T-cell function may be more effective at restoring T-cell function
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4814
BLOOD, 5 MAY 2011 䡠 VOLUME 117, NUMBER 18
YAMAMOTO et al
than approaches targeting individual factors, one must remember
that T cells express these negative regulators for a reason, and
relieving them from the natural inhibitory effects of these molecules could have detrimental effects on the host. Experimental
approaches targeting these negative regulators should first be tested
in animal models specifically looking for evidence of unrestrained
immune activation or autoimmune phenomena. In addition, shortterm, rather than long-term, manipulation of these negative regulators (such as acute therapy to help clear infection or as adjuvants in
vaccination) would seem to offer a safer environment in which to
test such therapy.
Overall, our data revealed a highly complex expression pattern
of inhibitory receptors on HIV-specific CD8⫹ T cells. Specific
combinations of inhibitory receptor expression enable a better
definition of exhausted HIV-specific CD8⫹ T-cell populations and
provide information that relates to the immune control of viremia
and disease status. Furthermore, these inhibitory receptor-mediated
pathways represent potential targets for novel immune therapies in
HIV-infected persons.
Acknowledgments
The authors thank the study subjects for their participation as well
as D. Ambrozak, T. Prout, A. Heisler, B. J. Hill, M. F. Quigley, and
J. R. Almeida for technical assistance and thoughtful discussions.
This work was supported by the Intramural Research Program
of the Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, National Institutes of Health (K24 AI64086;
R.H.), the University of California San Diego Center for AIDS
Research (AI36214; R.H.), and the San Diego AIDS Clinical Trial
Group (CTU AI69432; R.H.). D.A.P. is a Medical Research
Council (United Kingdom) Senior Clinical Fellow.
Authorship
Contribution: T.Y., C.P., and R.A.K. provided primary conception,
execution, and data analysis; D.A.P., J.P.C., G.F., P.D.K., D.C.D.,
and R.H. contributed to primary conception, interim discussions,
and manuscript preparation; M.N. performed the statistical analysis; P.K.C. and M.R. provided expertise in flow cytometry and
panel development and supplied in-house conjugated antibodies;
E.G. provided the tetramers; T.Y. and C.P. performed experiments
and data acquisition; T.Y., D.A.P., C.P., and R.A.K. wrote the
paper; and all the coauthors assisted in manuscript preparation.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Richard A. Koup, Immunology Laboratory,
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Dr, MSC
3022, Bldg 40, Rm 3502, Bethesda, MD 20892; e-mail:
rkoup@mail.nih.gov.
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2011 117: 4805-4815
doi:10.1182/blood-2010-11-317297 originally published
online March 11, 2011
Surface expression patterns of negative regulatory molecules identify
determinants of virus-specific CD8 + T-cell exhaustion in HIV infection
Takuya Yamamoto, David A. Price, Joseph P. Casazza, Guido Ferrari, Martha Nason, Pratip K.
Chattopadhyay, Mario Roederer, Emma Gostick, Peter D. Katsikis, Daniel C. Douek, Richard
Haubrich, Constantinos Petrovas and Richard A. Koup
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