of August 10, 2012.
This information is current as
Lymphocytes during Primary
Functional Exhaustion of CD4
Sandra Lecomte, Corinne Liesnard, Catherine Donner and
Pierre Antoine, Véronique Olislagers, Ariane Huygens,
published online 3 August 2012
is online at:
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The Journal of Immunology
at ULB - Bibliothèques on August 10, 2012
The Journal of Immunology
Functional Exhaustion of CD4+T Lymphocytes during
Primary Cytomegalovirus Infection
Pierre Antoine,* Ve ´ronique Olislagers,* Ariane Huygens,* Sandra Lecomte,*
Corinne Liesnard,†Catherine Donner,‡and Arnaud Marchant*
Human CMV establishes lifelong persistence after primary infection. Chronic CMV infection is associated with intermittent
viral reactivation inducing high frequencies of CD4+T lymphocytes with potent antiviral and helper properties. Primary
CMV infection is characterized by an intense viral replication lasting for several months. The impact of this prolonged exposure
to high Ag loads on the functionality of CD4+T cells remains incompletely understood. In pregnant women with primary CMV
infection, we observed that CMV-specific CD4+T lymphocytes had a decreased capacity to proliferate and to produce IL-2. Avery
large proportion of CMV-specific CD4+T cells had downregulated the expression of CD28, a costimulatory molecule centrally
involved in the production of IL-2. Unexpectedly, both CD282and CD28+CD4+T cells produced low levels of IL-2. This defective
production of IL-2 was part of a larger downregulation of cytokine production. Indeed, CMV-specific CD4+T cells produced
lower amounts of IFN-g and TNF-a and showed lower functional avidity during primary as compared with chronic infection.
Increased programmed death-1 expression was observed in CD28+CMV-specific CD4+T cells, and programmed death-1 inhi-
bition increased proliferative responses. These results indicate that primary CMV infection is associated with the exhaustion of
CMV-specific CD4+T cells displaying low functional avidity for viral Ags.
competent subjects but can lead to severe clinical consequences
in immunocompromised hosts and fetuses (1, 2). CD4+T lym-
phocytes play a central role in the control of CMV infection by
expressing antiviral functions and by promoting B and CD8+
T lymphocyte responses. CD4+T cell deficiency is correlated
with CMV reactivation in patients with HIV infection (3) and
with solid organ transplantation (4). Delayed appearance of
functional CMV-specific CD4+T lymphocytes is associated
with symptomatic infection in kidney transplanted patients with
primary infection (5). Following allogeneic stem cell trans-
plantation, an early CD4+T cell response to CMV is inversely
correlated with the risk of CMV viremia (6), and the survival of
adoptively transferred CMV-specific CD8+T cell clones is
correlated with the detection of an antiviral CD4+T cell response
During the chronic phase of CMVinfection, virus-specific CD4+
T cells undergo large oligoclonal expansions and include a vari-
able fraction of cells expressing a late differentiation phenotype
The Journal of Immunology, 2012, 189: 000–000.
umanCMVis a member of the herpesviridae family that
establishes lifelong persistence after primary infection.
CMV infection is usually asymptomatic in immuno-
characterized by the loss of expression of two costimulatory
receptors, CD27 and CD28 (9). This phenotype is typical of CMV-
specific cells (10) and is associated with a high capacity to pro-
duce effector cytokines (IFN-g and TNF-a) and with the acqui-
sition of MHC class II-restricted cytolytic activity (11).
The differentiation of CD4+T lymphocytes and their acquisition
of effector functions during primary CMV infection remains
poorly characterized. CMV-specific CD4+T cells producing ef-
fector cytokines can be detected rapidly during the course of
primary infection (12). In contrast, the proliferative capacity of
CD4+T cells is very low during the first months of infection when
viral excretion in urine and saliva can be detected (13, 14). This
suggests that active CMV replication interferes with the acquisi-
tion of at least some of CD4+T lymphocytes effector functions.
This interference could have significant clinical implications be-
cause low proliferative responses of CMV-specific T cells are
associated with in utero transmission of CMV following primary
infection in pregnancy (15–17) and with an increased risk of CMV
retinitis in HIV-infected patients (18). Previous studies suggest
that the low proliferative responses of CD4+T cells observed
during primary CMV infection is related to their differentiation
into effector T cells with a reduced capacity to produce IL-2 (19,
20). Optimal IL-2 production by T lymphocytes is dependent on
the signals provided by the costimulatory receptor CD28. Indeed,
CD282CD4+and CD282CD8+T cells produce lower levels of IL-
2 than their CD28+counterparts following polyclonal stimulation
(21). In addition, CD28 transduction of CD282CD8+T cells
restores their capacity to produce IL-2 (22). Taken together,
these observations suggest that active viral replication associated
with primary CMV infection selectively impairs the capacity of
CD4+T cells to produce IL-2 by promoting their differentiation
into CD282effector cells. The aim of our study is to test this
hypothesis and to comprehensively assess the functional capacity
of CD4+T cells and its link with cell differentiation in pregnant
women diagnosed with primary CMV infection and healthy adults
with chronic infection.
*Institute for Medical Immunology, Universite ´ Libre de Bruxelles, 6041 Charleroi,
1070 Brussels, Belgium; and‡Gynecology Department, Ho ˆpital Erasme, Universite ´
Libre de Bruxelles, 1070 Brussels, Belgium
†Virology Department, Ho ˆpital Erasme, Universite ´ Libre de Bruxelles,
Received for publication April 22, 2011. Accepted for publication June 21, 2012.
P.A. is a research fellow and A.M. is a senior research assistant at the Fonds National
de la Recherche Scientifique (Belgium). The Institute for Medical Immunology is
cofunded by the government of the Walloon Region and by GlaxoSmithKline Bio-
Address correspondence and reprint requests to Dr. Arnaud Marchant, Institute for
Medical Immunology, Universite ´ Libre de Bruxelles, Rue A. Bolland 8, 6041 Char-
leroi, Belgium. E-mail address: email@example.com
Abbreviations used in this article: HCV, hepatitis C virus; MFI, mean fluorescence
intensity; PD-1, programmed death-1; TT, tetanus toxoid.
Published August 3, 2012, doi:10.4049/jimmunol.1101165
at ULB - Bibliothèques on August 10, 2012
Materials and Methods
This study was approved by the Ethics Committee of the Universite ´ Libre de
Bruxelles. Pregnant women referred with a diagnosis of primary CMV
infection to the fetal medicine outpatient clinic of the Ho ˆpital Erasme were
recruited. Diagnosis of primary infection was based on documented IgG
seroconversion or, in case of unknown status at the beginning of the
pregnancy, increased titers of CMV-specific IgM. A total of 41 patients
were recruited in the study. The median delay between the diagnosis of
primary infection and blood collection for this study was 32 d (range, 6–132
d). Twenty-two healthy subjects chronically infected with CMV were
recruited as controls. Following written informed consent, a 30 ml aliquot
of heparinized blood was collected. Viremia was assessed using a qualita-
tive in-house diagnostic PCR assay targeting the pp150 CMV gene. Fifty-
three percent of pregnant women were CMV PCR positive at the time of
analysis. CD4+T cell responses to CMV and control Ags were similar in
patients who were viremic or nonviremic at the time of analysis (data not
shown). In addition, samples from 21 pregnant women diagnosed with
primary CMV infection and participating in the ongoing GlaxoSmithKline
Biologicals-sponsored study (NCT01251744) were analyzed in agreement
with the study protocol and consent form, to complement the results.
PBMC were purified from fresh peripheral blood by gradient centrifugation
using Lymphoprep (Nycomed Pharma) and were cultured in RPMI 1640
medium containing 5% human serum, penicillin/streptomycin, and gluta-
mine in 96-well plates (200,000 cells/well). Cells were stimulated with
a lysate of CMV-infected fibrobasts (1 mg/ml) (Virusys), a pool of 15 aa
peptides overlapping by 11 and derived from the CMV pp65 tegument
protein (1.75 mg/ml) (BD Pharmingen), a pool of 20 aa peptides over-
lapping by 10 and derived from the CMV glycoprotein B (2 mg/ml), tet-
anus toxoid (TT) (1 mg/ml), or a preparation of split Jiangsu influenza
virus (1 mg/ml) (all provided by GlaxoSmithKline Biologicals) and cul-
tured for 6 or 7 d at 37˚C in the presence of 5% CO2. The role of IL-2 or
inhibitory receptors was explored by adding rIL-2 (5 U/ml) (R&D Sys-
tems), anti–programmed death-1 (PD-1) blocking Ab, anti-Tim3 blocking
Ab, or isotype control (5 mg/ml; all from BioLegend) to the culture me-
dium. Cells were pulsed with BrdU (BD Biosciences) for the last 18 h of
stimulation. Cells were stained according to the manufacturer’s protocol
with the following Abs: CD3 PerCP, CD4 Pacific Blue, CD8 PE-Cy7, Ki67
FITC, and BrdU allophycocyanin (all from BD Biosciences). Proliferating
cells were defined as Ki-67+and BrdU+. Data were obtained on a Cyan
ADP LX9 cytometer and analyzed using the Summit 4.3 software
(DakoCytomation) or FlowJo 9.0.1 software (Tree Star).
Cytokine production and cell phenotype
Fresh PBMC were cultured in RPMI 1640 medium containing 10% FCS,
penicillin/streptomycin, and glutamine and were stimulated with a lysate of
CMV-infected fibroblasts (5 mg/ml), a pool of CMV pp65 overlapping
peptides (1.75 mg/ml), a pool of CMV gB overlapping peptides (2 mg/ml),
TT (10 mg/ml), or a preparation of split Jiangsu influenza virus (1 mg/ml)
for 18 h at 37˚C in the presence of 5% CO2. Brefeldin A (5 mg/ml) (Sigma-
Aldrich) was added to the medium for the last 16 h of stimulation. The role
of inhibitory receptors was assessed by adding anti–PD-1 blocking Ab,
Tim3 blocking Ab, or isotype control (5 mg/ml; all from BioLegend) to the
culture medium. Ag-specific CD4+T cells were identified on the basis of
the production of at least one among three cytokines (IFN-g, TNF-a, and
IL-2). The phenotype of Ag-specific cells was characterized using the
following Abs: CD3 FITC, CD4 allophycocyanin-H7 or V500 or V450,
CD8 PerCP, CD14 PE, CD16 PE, CD19 PE, CD56 PE, CD45RO PE-Cy7,
CD27 V450, CD28 ECD, MHC class II FITC, CD38 AF700, IFN-g PE-
Cy7 or AF700, TNF-a Pacific Blue or eFluor 450 or AF700 or allophy-
cocyanin, IL-2 allophycocyanin or PE, MIP-1b PE-Cy7, Bcl-2 FITC, Tim3
PE, and PD-1 allophycocyanin (all from BD Biosciences, except CD28
from Beckman Coulter; TNF-a Pacific Blue or eFluor 450 from eBio-
science; Tim3 and PD-1 from BioLegend). CD3, cytokines, and Bcl-2
were stained intracellularly after permeabilization with Cytofix/
Cytoperm (BD Biosciences) according to the manufacturer’s instruc-
tions. Data were obtained on a Cyan ADP LX9 cytometer (DakoCyto-
mation) and analyzed using the FlowJo 8.8.6, 8.8.7, or 9.0.1 software (Tree
Star). To provide consistent measurements of mean fluorescence intensities
(MFI) between experiments, flow cytometer settings were standardized
using SPHERO Rainbow Beads (BD Biosciences). The voltage of each
photomultiplier tube was set to detect the beads with the same median
of fluorescence before every data acquisition. A single batch SPHERO
Rainbow Beads was used for the duration of the study.
Fresh PBMC were cultured for 18 h at 37˚C with 5% CO2in RPMI 1640
medium containing 10% FCS, penicillin/streptomycin, and glutamine with
or without addition of anti-Fas agonist Ab (1 mg/ml) (Millipore). Cells
were then stained with the following Abs: CD3 Cascade Yellow, CD4
V450, CD8 PE-Cy7, CD28 allophycocyanin, AnnexinV FITC (all from
BD Biosciences, except CD3 from DakoCytomation; CD8 from Beckman
Coulter) and with 7-aminoactinomycin D (BD Biosciences), according to
the manufacturer’s instructions. Apoptotic cells were defined as Annex-
inV+and 7-aminoactinomycin D2. Data were obtained on a Cyan ADP
LX9 cytometer (DakoCytomation) and analyzed using the FlowJo 8.8.6 or
8.8.7 software (Tree Star).
Data are presented as individual results, medians and interquartile ranges or
means and SEs on the mean. Specific fluorescence intensity of Bcl-2 ex-
pression was calculated using the following formula: specific fluorescence
intensity = (MFI[Bcl-2] 2 MFI[fluorescence minus one])/MFI[fluorescence
minus one]. Multiple parameter comparisons of primary and chronic in-
fections were performed with the two-way ANOVA test. When significant
differences were observed, data obtained from primary and chronic infec-
tions were compared for each parameter using the Mann–Whitney U test.
Statistical significance was defined at p , 0.05. GraphPad Prism 5 was used
to perform the analyses.
Primary CMV infection induces high frequencies of
cytokine-producing CD4+T cells with limited proliferative
To assess the magnitude of the CD4+T lymphocyte response to
primary CMV infection, frequencies of cells producing at least
one of three cytokines (IFN-g, TNF-a, and IL-2) following short-
term stimulation with CMV or control Ags were measured (Fig.
1A). During primary infection, median frequencies of cytokine-
CD4+T cells during primary and chronic infection. (A) Frequencies of
total cytokine-producing CD4+T cells (producing at least one cytokine
among IFN-g, TNF-a, and IL-2) were measured among CD4+T cells after
overnight stimulation with whole CMV lysate, pp65 peptide pool, gB
peptide pool, TT, and influenza split virus. (B) CD4+T lymphocytes
proliferative responses to CMV and third-party Ags were measured using
the BrdU incorporation assay. Figures show medians 6 interquartile
ranges of 6–21 subjects depending on the Ag tested and readout. *p ,
0.05, ***p , 0.001.
Frequencies and proliferative responses of CMV-specific
2EXHAUSTION OF CD4+T LYMPHOCYTES IN PRIMARY CMV INFECTION
at ULB - Bibliothèques on August 10, 2012
producing cells ranged between 0.11 and 0.45% of CD4+T cells,
depending on the Ag tested. Similar frequencies of CMV-specific
cells were detected in chronically infected subjects. Also, subjects
with primary or chronic CMV infection had similar frequencies of
CD4+T cells producing cytokines in response to TTand influenza
Ags. Proliferative responses to CMV and control Ags were mea-
sured using the BrdU incorporation assay (Fig. 1B). As previously
reported (13, 14), proliferative responses of CD4+T cells to CMV
Ags were significantly lower during primary as compared with
chronic infection. In contrast, subjects with primary or chronic
CMV infection had similar proliferative responses to control Ags.
Taken together, these data indicate that primary CMV infection
induces the expansion of high frequencies of cytokine-producing
CD4+T cells with a limited proliferative capacity. The defective
proliferation appears to selectively affect CMV-specific cells.
Activation of CMV-specific CD4+T lymphocytes during
During primary CMVinfection, active viral replication is expected
to induce more intense T cell activation as compared with chronic
infection (23). The state of activation of CMV-specific CD4+
T cells was assessed by measuring the expression of the membrane
molecules CD38 and MHC class II as well as the intracellular
content of the antiapoptotic molecule Bcl-2 following short-term
stimulation with CMV or control Ags and gating on cytokine-
positive cells (Fig. 2). During primary infection, CMV-specific
CD4+T cells expressed high levels of CD38, whereas low lev-
els of expression were detected in chronically infected subjects
(Fig. 2A). Similar results were obtained with HLA class II ex-
pression, although significant differences where only observed for
pp65-specific cells. T cell activation during primary viral infec-
tions is associated with reduced expression of the anti-apoptotic
Bcl-2 protein and with an increased susceptibility to apoptosis
(24). In subjects with primary infection, CMV-specific CD4+
T cells expressed low levels of Bcl-2 as compared with chroni-
cally infected subjects (Fig. 2B, 2C). In contrast, similar levels of
Bcl-2 were detected in TT and influenza-specific cells from sub-
jects with primary or chronic infection. The biological conse-
quence of Bcl-2 downregulation was assessed by measuring the
susceptibility of CD4+T cells to spontaneous or Fas agonist-
induced apoptosis (Fig. 2D). In these experiments, CD28 nega-
tivity was used as a marker of CMV-specific cells to avoid Ag
stimulation (10). Susceptibility of CD282CD4+T cells to spon-
taneous apoptosis following overnight incubation was signifi-
cantly higher during primary as compared with chronic infection.
Incubation with anti-Fas agonist Abs further increased the apo-
ptosis of CD282CD4+T cells in subjects with primary infection
but not in chronically infected subjects. Taken together, these data
indicate that during primary infection CMV-specific CD4+T cells
express a phenotype of activation that is more marked than that of
cells of other specificities or CMV-specific cells during chronic
infection. This state of activation further validates the diagnosis of
primary CMV infection.
Defective production of IL-2 by CMV-specific CD4+T cells
during primary infection
To identify the mechanisms underlying the defective proliferative
responses of CD4+T cells associated with primary CMVinfection,
we measured the capacity of CMV-specific cells to produce IL-2.
The proportions of cells producing IL-2 among the total pop-
ulation of cytokine-producing CD4+T cells was assessed fol-
lowing stimulation with CMV or control Ags (Fig. 3A, 3B).
During primary infection, CMV-specific CD4+T cells included
low frequencies of cells producing IL-2 as compared with chronic
infection. In contrast, similar frequencies of TT and influenza-
specific cells producing IL-2 were detected in the two study
groups. The effect of addition of exogenous IL-2 on the prolif-
erative responses of CD4+T cells to CMVAgs was then assessed
(Fig. 3C). In subjects with primary CMVinfection, addition of low
doses of IL-2 significantly increased the proliferation of CMV-
specific CD4+T cells, whereas no significant effect was ob-
served in chronically infected subjects. Proliferative responses to
CMV lysate and to pp65 peptide pool in the presence of IL-2 were
comparable in subjects with primary and chronic infection,
whereas the responses to the gB peptide pool remained lower in
primary as compared with chronic infection. These results show
that, during primary infection, the defective proliferative response
of CMV-specific CD4+T lymphocytes is associated with a de-
creased capacity to produce IL-2 and that addition of IL-2 alone
can restore cell proliferation to levels similar to that observed
during chronic infection.
Role of CD28 downregulation in the defective production of
IL-2 during primary CMV infection
The capacity of T lymphocytes to produce IL-2 depends on their
expression of the costimulatory molecule CD28 (25). To gain
insight into the mechanisms involved in the downregulation of IL-
infection. (A) Expression of CD38 (left panel) and MHC class II (right
panel) was measured at the surface of CMV-specific CD4+T lymphocytes
producing at least one cytokine after overnight Ag stimulation (medians 6
interquartile ranges of five to six subjects per Ag tested). (B) Represen-
tative dot plots of Bcl-2 expression by Ag-specific CD4+T cells. For this
two-parameter representation, dominant cytokines were selected (IFN-g for
CMV-specific cells and IL-2 for TT and influenza-specific cells). (C)
Quantification of Bcl-2 expression by CD4+T cells producing at least one
cytokine in response to stimulation with CMVor control Ags in subjects with
primary (closed symbols) or chronic infection (open symbols). (D) Apoptosis
of CD282CD4+T cells was measured after overnight incubation in the
absence and presence of Fas agonist Abs in subjects with primary (closed
symbols) or chronic infection (open symbols). *p , 0.05, ***p , 0.001.
Activation of CMV-specific CD4+T cells during primary
The Journal of Immunology3
at ULB - Bibliothèques on August 10, 2012
2 production by CD4+T cells during primary CMV infection, the
differentiation phenotype of CMV-specific cells was assessed (Fig.
4A). During primary infection, CMV-specific CD4+T cells in-
cluded very large proportions of CD28-cells. These proportions
were significantly higher than those observed during chronic in-
fection. In contrast, low and similar frequencies of CD282cells
were detected among TT and influenza-specific cells during pri-
mary and chronic infection. To evaluate the role of CD28 down-
regulation in the defective production of IL-2, the proportion of
IL-2–producing cells was assessed within the CD282and CD28+
subsets of CMV-specific CD4+T cells (Fig. 4B). As expected,
very low frequencies of IL-2–producing cells were detected within
the CD282subset of CMV-specific cells during both primary and
chronic infection. Interestingly, CD28+CMV-specific CD4+
T cells were markedly less able to produce IL-2 during primary as
compared with chronic infection. Taken together, these results
indicate that the defective production of IL-2 by CD4+T cells
during primary CMV infection involves both the rapid differen-
tiation of high frequencies of CD28-cells as well as a decreased
capacity of CD28+cells to produce this cytokine.
Primary CMV infection is associated with a defective per cell
production of IFN-g and TNF-a by CD4+T cells
The observation that the defective IL-2 production affected CD28+
T lymphocytes suggested that the functional impairment of CMV-
specific CD4+T cells may not be restricted to IL-2. Because the
proportion of CD4+T cells producing either IFN-g or TNF-a was
similar in subjects with primary or chronic infection, the amount
of cytokines synthesized on a per cell basis was measured (Fig. 5).
During primary infection, CMV-specific CD4+T cells produced
significantly lower amounts of IFN-g and TNF-a, as indicated by
lower MFI, as compared with chronic infection (Fig. 5A). This
difference was specific to CMV as TT and influenza-specific cells
expressed similar IFN-g, TNF-a, and IL-2 MFI in the two study
groups (Fig. 5A, 5B). The expression of CD28 by CMV-specific
CD4+T cells did not influence the amount of cytokines produced
as both CD282and CD28+cells expressed lower IFN-g and TNF-
a MFI in subjects with primary infection as compared with
chronic infection (Fig. 5C). To further characterize the capacity of
CMV-specific CD4+T cells to produce effector cytokines during
primary infection, their functional avidity was assessed by mea-
suring the impact of Ag titration on cytokine responses (Fig. 6).
Percentage of cytokine-producing CD4+T cells following stimu-
lation with optimal concentrations of pp65 and gB peptide pools
was defined as 100%. Ag titration resulted in a significantly
sharper decline of cytokine responses, indicating lower functional
avidity of CD4+T cells, in subjects with primary infection as
compared with subjects with chronic infection. Similar results
were obtained with total cytokine or with IFN- g responses fol-
lowing stimulation with either gB or pp65 peptide pools. Taken
together, these data indicate that during primary CMV infection
CD4+T cells are impaired in their capacity to produce multiple
cytokines and have a lower functional avidity as compared with
CD28+CMV-specific CD4+T cells express increased levels of
PD-1 during primary infection
The reduced capacity of CMV-specific CD4+T cells to proliferate
and to produce cytokines during primary infection suggests a state
of functional exhaustion. Functional exhaustion of T cells is ob-
served in patients with chronic hepatitis or HIV infection and is
associated with increased expression of inhibitory receptors, in-
CMV-specific CD4+T lymphocytes during primary CMV. (A) The pro-
portion of IL-2–producing cells among total cytokine-producing CD4+
T cells was measured after overnight stimulation with CMVor control Ags
(medians 6 interquartile ranges of 4–13 subjects). (B) Representative dot
plots of IFN-g and IL-2 production during primary (top panel) or chronic
infection (bottom panel). (C) Influence of exogenous IL-2 on proliferative
responses of CD4+T cells to CMV Ags was studied using the BrdU in-
corporation assay (medians 6 interquartile ranges of 6–20 subjects). *p ,
0.05, ***p , 0.001.
Defective production of IL-2 and proliferative responses of
tion of IL-2 during primary infection. (A) The proportion of CD282cells
was measured among CD4+T lymphocytes producing at least one cytokine
in response to CMVor control Ags (medians 6 interquartile ranges of 4–
13 subjects). (B) The proportion of cells producing IL-2 was measured
among CD282and CD28+CD4+T cells producing at least one cytokine
(graphs show individual results and medians). **p , 0.01, ***p , 0.001.
Differentiation of CMV-specific CD4+T cells and produc-
4 EXHAUSTION OF CD4+T LYMPHOCYTES IN PRIMARY CMV INFECTION
at ULB - Bibliothèques on August 10, 2012
cluding PD-1 and Tim-3 (26). To gain insight into the mechanisms
regulating the function of CMV-specific CD4+T cells, the ex-
pression of PD-1 and Tim-3 was measured at the level of CMV
and TT-specific CD4+T during primary and chronic infection.
Primary infection was associated with increased expression of PD-
1 by CMV-specific CD4+T cells (Fig. 7A). This increased ex-
pression was observed only in CD28+cells, whereas CMV-specific
CD282CD4+T cells expressed similar levels of PD-1 during
primary and chronic infection. As shown in Fig. 7A, TT-specific
CD4+T cells expressed low and similar levels of PD-1 during
primary and chronic infection. In contrast to PD-1, low and similar
levels of Tim-3 were observed in CMVand TT-specific cells in the
two study groups (Fig. 7B). Addition of anti–PD-1 blocking Ab
increased the proliferative responses of CMV-specific CD4+
T cells during primary infection but had no impact on the
responses measured during chronic infection (Fig. 7C). In con-
trast, addition of anti–Tim-3 blocking Ab did not significantly
influence proliferative responses to CMV Ags in primary or
chronic infection. PD-1 or Tim-3 inhibition did not influence the
proliferative responses of TT-specific or influenza-specific CD4+
T cells (data not shown). Analyses of cytokine production (per-
centage of positive cells and MFI) by total, CD28+, or CD282
CMV-specific CD4+T cells did not reveal any impact of PD-1 or
Tim-3 inhibition (data not shown). These results indicate that the
defective proliferative responses associated with primary CMV
infection involve the upregulation of PD-1 expression by a subset
of CMV-specific CD4+T cells.
This study shows for the first time, to our knowledge, that the
functional capacity of CMV-specific CD4+T lymphocytes is se-
lectively impaired during primary infection. In subjects with pri-
mary CMV infection, CMV-specific CD4+T cells have a reduced
capacity to produce IFN-g, TNF-a, and IL-2 as compared with
subjects with chronic infection, whereas the function of CD4+
T cells of other specificities is preserved. During primary infec-
tion, CMV-specific CD4+T cells expressed an activation pheno-
type indicating recent antigenic exposure. We observed no
correlation between CD4+T cell response to CMVor control Ags
and the detection of viremia at the time of analysis (data not
shown). Excretion of CMV in urine and saliva lasts for several
months following primary infection, whereas viremia is more
transient (14, 27). Our results, therefore, suggest that the pro-
longed replication of CMVin tissues is an important source of Ags
stimulating CD4+T cells.
The reduced capacity of CMV-specific CD4+T cells to produce
IL-2 was associated with defective proliferative responses, and
addition of exogenous IL-2 restored proliferative responses to the
levels observed during chronic infection. These results are in line
with the low frequencies of IL-2–producing CD4+T cells ob-
and TNF-a by CMV-specific CD4+T cells
during primary infection. (A and B) Per cell
production of individual cytokines by CD4+
T cells was measured following stimulation
with CMV or control Ags and is expressed
as MFI (medians 6 interquartile ranges of
4–10 subjects). (C) Per cell production of
cytokines by CMV-specific CD28+and
CD282CD4+T cells was measured as in (A)
and (B) and is expressed as individual
results and medians. *p , 0.05, **p , 0.01,
***p , 0.001.
Defective production IFN-g
(left panel) or producing IFN-g (right panel) in response to serial dilutions of optimal concentrations of pp65 (A) or gB (B) peptide pools were measured.
Results are expressed as mean percentage (6SE on the mean) of the responses to optimal peptide pool concentrations. Figures show five to nine subjects
depending on the Ag tested and the readout.
Reduced functional avidity of CMV-specific CD4+during primary infection. Frequencies of CD4+T cells producing one of three cytokines
The Journal of Immunology5
at ULB - Bibliothèques on August 10, 2012
served by Harari et al. (20) in a restricted series of HIV-infected
patients with primary CMV infection and with the progressive
increase in frequencies of IL-2–producing CD4+T lymphocytes
observed by Lilleri et al. (19) in HIV-seronegative subjects with
primary CMV infection. Limited production of IL-2 by CD4+
T cells and low proliferative responses have been observed in
other chronic viral infection including HIV, hepatitis C virus
(HCV), and EBV (28–31). In HIV and HCV infections, the de-
fective production of IL-2 is associated with active viral replica-
tion (28, 32, 33). In HIV-infected patients, anti-viral therapy
improves the capacity of CD4+T cells to produce IL-2, suggesting
that high viral loads and intense T cell stimulation directly
downregulates IL-2 production (34, 35).
The defective production of IL-2 was associated with an
advanced stage of differentiation of CMV-specific CD4+T lym-
phocytes. Indeed, higher frequencies of CMV-specific cells lack-
ing the expression of the CD28 molecule were detected during
primary as compared with chronic infection. The progressive ac-
cumulation of CD282CD8+T lymphocytes with age in CMV-
seropositive subjects has led to the suggestion that CD282CMV-
specific T cells accumulate slowly over time because of repetitive
antigenic stimulation (36–38). Our results indicate that the differ-
entiation of CMV-specific CD4+T cells occurs early during the
course of primary infection. Because optimal production of IL-2 by
T lymphocytes is dependent on the signals provided by the co-
stimulatory receptor CD28 (25), the higher proportions of CD282
cells among CMV-specific CD4+T lymphocytes during primary
infection could have explained their defective production of IL-2.
As expected, we observed that CD282CMV-specific CD4+T cells
produce very low levels of IL-2 during both primary and chronic
infection. Unexpectedly, the capacity of CMV-specific CD28+cells
to produce IL-2 was significantly lower during primary as com-
pared with chronic infection. These results indicate that the de-
fective production of IL-2 by CMV-specific CD4+T cells during
primary infection is only partly related to their advanced differ-
The fact that the defectivecapacity to produce IL-2 affected both
CD282and CD28+CD4+T cells suggested broader functional
alterations of CD4+T cells. In agreement with this hypothesis, we
observed that although the frequencies of CMV-specific CD4+
T cells producing IFN-g and TNF-a were comparable during
primary and chronic infection, the per cell production of these
cytokines was significantly lower during primary infection. This
defective production of effector cytokines was independent on the
expression of CD28 by CD4+T cells. Because CMV-specific
CD4+T cells were detected following in vitro Ag stimulation,
cells unable to produce the measured cytokines would have been
excluded from our analyses, and functional exhaustion, therefore,
may have been underestimated. Functional analyses of CMV-
specific CD4+T cells detected by MHC class II tetramer stain-
ing should provide a more sensitive evaluation of their functional
An impairment of the production of multiple cytokines is
characteristic of the T cell exhaustion phenotype that has been
described in several models of chronic viral infections (39).
Functional exhaustion of T cells is induced by prolonged exposure
to high Ag loads and is characterized by the partial or complete
loss of capacity to produce effector cytokines. Functionally
exhausted CD8+T cells have been characterized in details in
several animal models and human diseases. Much less is known
about this process in CD4+T lymphocytes. Exhaustion of CD4+
T cells has been observed in the early phase of lymphocytic
choriomeningitis virus infection in mice and in patients infected
with HIVor HCV (40–42). T lymphocyte exhaustion can involve
multiple mechanisms including an increased expression of inhib-
itory receptors, such as PD-1 and Tim-3, modulating TCR sig-
naling (26, 43). We observed that CMV-specific CD4+T cells
expressed increased levels of PD-1 and similar and low levels of
Tim-3 during primary as compared with chronic infection. Sur-
prisingly, the increased expression of PD-1 was observed on
CD28+and not CD282CD4+T cells. This suggests that the signals
triggering PD-1 upregulation in vivo involved CD28 costimula-
tion. Increased expression of PD-1 by CD28+CD4 T cells has also
been observed in HIV-infected patients (44). Inhibition of PD-1
increased the proliferative responses of CMV-specific CD4+
T cells during primary infection. These results are in line with
those reported by Sester et al. (45) showing increased proliferation
of CMV-specific CD4+T cells following programmed death
ligands 1 and 2 blockade in transplanted patients with CMV vi-
during primary infection. The expression of PD-1 (A) and Tim-3 (B) was
measured on total, CD28+, and CD282CD4+T cells producing at least one
cytokine in response to stimulation with CMV-infected fibroblasts lysate
(left panel) or TT (right panel) in subjects with primary (closed symbols)
or chronic CMV infection (open symbols) and is expressed as MFI. (C)
The influence of anti–PD-1 and anti–Tim-3 blocking Abs on the prolif-
erative responses of CD4+T cells to CMVAgs was studied using the BrdU
incorporation assay (medians 6 interquartile ranges of six to seven sub-
jects). *p , 0.05.
CD28+CMV-specific CD4+express increased level of PD-1
6 EXHAUSTION OF CD4+T LYMPHOCYTES IN PRIMARY CMV INFECTION
at ULB - Bibliothèques on August 10, 2012
remia. Our results indicated that PD-1 inhibition did not restore
proliferative responses to the levels observed during chronic in-
fection, suggesting that PD-1 upregulation is only one of the
factors controlling CMV-specific CD4+T cell proliferation. Also,
our results suggest that the upregulation of PD-1 is not central to
the control of cytokine production by CMV specific CD4+T cells.
Indeed, decreased cytokine responses were observed in both
CD28+and CD282subsets, whereas PD-1 upregulation was
specifically observed in CD28+cells. In addition, PD-1 inhibition
did not influence the production of cytokines following short-term
cell stimulation of CMV-specific CD4+T cells. These results are
in line those obtained by Serriari et al. (23) in transplanted patients
with primary CMV infection and showing that PD-1 controls the
proliferation of CMV-specific CD8+T cells but not their capacity
to produce effector cytokines. Taken together, these observations
indicate that additional mechanisms to PD-1 upregulation and
CD28 downregulation control the functions of CMV-specific
T cells during primary infection.
The functional exhaustion of CMV-specific CD4+T cells dur-
ing primary infection was associated with a decreased functional
avidity of these cells. Indeed, Ag dilution experiments revealed
that CD4+T cells were more sensitive to peptide titration during
primary as compared with chronic infection. A similar association
between functional avidity and production of cytokines was ob-
served in CD8+T cells from HIV-infected patients (46). CD8+
T cells with high functional avidity are detected in patients with
slow HIV disease progression and exhibit increased poly-
functionality and clonal turnover, whereas low-avidity CD8+
T cells have defective cytokine production and proliferative ca-
pacity. The cellular mechanisms underlying these differences of
functional avidity have not been fully characterized. Nonstructural
mechanisms including inhibitory receptors, regulation of TCR
signaling, transcriptional control of cytokine gene expression, or
posttranscriptional control of cytokine synthesis associated with
cellular stress could be involved (47, 48). The structural charac-
teristics of the TCR could also play an important role as low-
affinity interactions with the cognate MHC–peptide complexes
would result in low functional avidity and low-intensity signaling.
CD4+T cells from HIV controllers were recently shown to express
higher affinity TCRs than viremic or treated patients, suggesting
that immune control of HIV replication may be favored by high-
affinity CD4+T cells (49). Further studies are needed to charac-
terize the role of nonstructural and TCR-related mechanisms in
the functional exhaustion of CD4+T lymphocytes during primary
The mechanisms underlying the emergence of CD4+T cells
with higher functional capacity and avidity during the chronic
phase of CMVinfection remain to be elucidated. As viral replication
decreases and Ag load is reduced, cells could recover from func-
tional exhaustion and produce larger amounts of effector cytokines.
Alternatively, new clones may emerge and acquire higher functional
capacities when Ag load is reduced. The second hypothesis is
supported by the observation that CMV-specific CD4+T cells
undergo intense clonal turnover between the primary and the
chronic phase of infection in kidney transplanted patients (50).
Differences in Ag load and in costimulatory signals at the time of
priming would therefore determine the functional capacity and
avidity of CMV-specific CD4+T lymphocytes. According to this
model, controlling Ag load in CMV-infected patients with antiviral
therapy would allow the emergence of CD4+T cell clones with
more potent antiviral properties and the reconstitution of anti-
CMV immunity. Longitudinal analysis of the functional capacities
and repertoire of CD4+T cell clones over the natural course of pri-
mary CMV infection and following anti-CMV therapy are needed
to test this hypothesis.
In conclusion, our study shows that during primary CMV infec-
tion, CD4+T lymphocytes express a phenotype of functional ex-
haustion, including reduced proliferative and cytokine responsesand
increased expression of the inhibitory receptor PD-1. These results
indicate that CMV is part of the group of viruses including HIVand
HCV that induce functional exhaustion of CD4+T lymphocytes in
humans. Exhaustion of CD4+T lymphocytes during primary CMV
infection may limit the control of CMV replication and therefore
represent a target for therapeutic interventions.
We thank the patients and healthy volunteers who participated in the study.
We thank the investigators Dr. Roland Devlieger (Universitair Ziekenhuis
Gasthuisberg, Leuven, Belgium), Dr. Jacques Francotte (Centre Hospitalier
Universitaire de Tivoli, La Louvie `re, Belgium), Dr. Pierre Leblicq (Ho ˆpital
Ambroise Pare ´, Mons, Belgium), Dr. Jack Levy (ImmuneHealth Research
Centre, Gosselies, Belgium; Centre Hospitalier Universitaire Saint-Pierre,
Brussels, Belgium), Dr. Philip Loquet (AZ St. Augustinus, Wilrijk, Bel-
gium), Dr. Marc Mare ´chal (Centre Hospitalier Universitaire de Charleroi
Andre ´ Ve ´sale, Montigny-Le-Tilleul, Belgium), Dr. Dominique Thomas
(Ho ˆpitaux Iris Sud Ixelles, Brussels, Belgium), and Dr. Michel Van Rys-
selberge (Centre Hospitalier Universitaire Saint-Pierre, Brussels, Belgium)
as well as GlaxoSmithKline Biologicals for providing samples from the
ongoing sponsored study NCT01251744.
The authors have no financial conflicts of interest.
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8EXHAUSTION OF CD4+T LYMPHOCYTES IN PRIMARY CMV INFECTION
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