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Latent Infection with Cytomegalovirus Is
Associated with Poor Memory CD4
Responses to Influenza A Core Proteins in the
Elderly
Evelyna Derhovanessian, Andrea B. Maier, Karin Hähnel,
Janet E. McElhaney, Eline P. Slagboom and Graham
Pawelec
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 193:3624-3631; Prepublished online 3
September 2014;
doi: 10.4049/jimmunol.1303361
http://www.jimmunol.org/content/193/7/3624
The Journal of Immunology
Latent Infection with Cytomegalovirus Is Associated
with Poor Memory CD4 Responses to Influenza A Core
Proteins in the Elderly
Evelyna Derhovanessian,* Andrea B. Maier,†,‡,x Karin Hähnel,* Janet E. McElhaney,{
Eline P. Slagboom,‡,‖ and Graham Pawelec*
S
easonal epidemics of influenza cause serious illness and
death throughout the world. The likely global disease
burden is estimated by the World Health Organization at up
to 1 billion infections, 3–5 million cases of severe disease, and
between 300,000 and 500,000 deaths annually (1). In developed
countries, the elderly account for .90% of influenza-related
deaths. This is believed to be due to the diminished state of immunity in the elderly (2, 3) generally referred to as “immunosenescence,” as well as the probably related low success rate of
prophylactic vaccination (4), still the most effective tool for
*Department of Internal Medicine II, Centre for Medical Research, University of
Tubingen, 72072 Tubingen, Germany; †Department of Gerontology and Geriatrics,
Leiden University Medical Centre, 2333 ZC Leiden, the Netherlands; ‡Netherlands
Consortium for Health Aging, Leiden University Medical Centre, 2333 ZC Leiden,
the Netherlands; xDepartment of Internal Medicine, Section of Gerontology and Geriatrics, VU University Medical Centre, 1081 HV Amsterdam, the Netherlands; {Advanced Medical Research Institute of Canada, Sudbury, Ontario, Canada P3E 5J1; and
‖
Section of Molecular Epidemiology, Leiden University Medical Centre, Leiden, the
Netherlands
Received for publication December 17, 2013. Accepted for publication July 28, 2014.
This work was supported by European Commission (Grant FP6 036894, “LifeSpan”;
Grant FP7 259679, “IDEAL”), Grant DFG-PA 361/14-1, and the Bundesminsterium f€ur
Bildung und Forschung project “Gerontoshield” (Grant 0315890F). The Leiden Longevity Study was funded by the Innovation Oriented Research Program on Genomics
(SenterNovem; Grants IGE01014 and IGE5007), the Centre for Medical Systems Biology, and the Netherlands Genomics Initiative/Netherlands Organization for Scientific
Research (Grants 05040202 and 050-060-810). The study in Vancouver, BC, Canada, was
sponsored by the Canadian Institutes of Health Research (Grant MOP-89729 to J.E.M.).
Address correspondence and reprint requests to Dr. Evelyna Derhovanessian at the
current address: BioNTech AG, Freiligrathstrasse 12, 55131 Mainz, Germany. E-mail
address: [email protected]
Abbreviations used in this article: EMA, ethidium monoazide; HI, hemagglutination
inhibition; IAV, influenza A virus; MP, matrix protein; NP, nucleoprotein; NR, nonresponder; R, responder.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303361
controlling influenza and influenza-related complications. Immunosenescence is thought to be more marked in the adaptive arm of
the immune system and is characterized by reduced numbers and
percentages of circulating naive T cells reciprocated by accumulations of late-differentiated memory T cells as well as reduced
B cell diversity and function (5). Some of these parameters, especially the accumulation of late-differentiated memory CD4 and
CD8 T cells, have been shown to correlate with poor humoral
responses of the elderly to influenza vaccination (6–8).
Besides chronological age, a latent infection with CMV has been
repeatedly demonstrated to be a major force driving T cells toward
a more late-differentiated phenotype, one of the hallmark features
of immunosenescence (9, 10). CMV is a ubiquitous b herpes virus,
the seroprevalence of which rises with age. In many countries,
.70% of individuals over the age of 60 are infected by CMV (11).
Although usually asymptomatic in immunocompetent carriers, this
virus poses a major challenge to the immune system of the host,
evidenced by devotion of a very large proportion of memory CD4
and CD8 T cells to maintaining this virus in a latent state in healthy
individuals (12). The chronic encounter of the immune system with
the virus leads to accumulation of late-differentiated CD8 cells,
lacking expression of CD28, a parameter that has been associated
with poor humoral and cellular responses to influenza vaccination
(7, 8, 13). Accordingly, seropositivity for CMV has been demonstrated to correlate with poor humoral responsiveness of the elderly
to influenza vaccination in several studies (6, 14–16).
Humoral immune responses are required to prevent infection;
they depend on Ag-specific CD4+ T cell help for antiviral B cell
responses, Ab class switch, affinity maturation, and long-lived
plasma cell generation. In addition, both CD4 and CD8 T cells,
which directly kill and clear already virus-infected cells, are essential for protecting the organism (17). Accordingly, pre-existing
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Influenza remains a major pathogen in older people. Infection with CMV and the accumulation of late-differentiated T cells associated with it have been implicated in poor Ab responsiveness to influenza vaccination in the elderly, most of whom are CMV
positive. However, whether CMV infection also affects memory T cell responses to influenza remains unknown. To investigate this,
we assessed T cell responses to influenza A matrix protein and nucleoprotein ex vivo in 166 Dutch individuals (mean age 62.2 y, range
42–82) and validated the results in a second cohort from North America (mean age 73.1 y, range 65–81, n = 28). We found that less
than half of the CMV-infected older subjects mounted a CD4 T cell response to influenza Ags, whereas ∼80% of uninfected elderly
did so. A similar proportion of younger subjects possessed influenza A virus–responsive CD4 T cells, and, interestingly, this was
the case whether they were CMV-infected. Thus, the effect of CMV was only seen in the older donors, who may have been exposed
to the virus for decades. The percentage of donors with CD8 responses to influenza A virus was lower than those with CD4; this
was not influenced by whether the subjects were CMV seropositive or seronegative. CMV-seropositive responders had significantly higher frequencies of late-differentiated CD4 T-cells (CD45RA+/2CCR72CD272CD282) compared with CMV-infected
nonresponders. These data add to the accumulating evidence that infection with CMV has profound but heterogeneous effects
on responses to the products of other viruses and have implications for the design of influenza vaccines, especially in the
elderly. The Journal of Immunology, 2014, 193: 3624–3631.
The Journal of Immunology
larity. After exclusion of duplets and EMA-negative dead cells, T cells
were gated as CD3+, followed by gating of CD4+CD82 and CD42CD8+
cells. Production of each cytokine was then determined in CD4+ and CD8+
cells separately. On average 300,000 CD4+ and 100,000 CD8+ cells were
acquired. A donor was considered to have a positive CD4 or CD8 response
(responder) to CMV or IAV when the percentage of CD4 or CD8 cells
producing IFN-g, TNF, or IL-2 in the sample stimulated with the respective peptide-pool was at least twice the percentage of cells producing
the same cytokines in the unstimulated negative control. In addition,
a clear population of cytokine-producing cells needed to be visible in the
FACS plots. These criteria were adopted on the basis of data from repeated
validation tests as well as recommendations of international harmonization
panels in which we have been participating (23, 24). Production of IFN-g
and/or TNF and/or IL-2 was considered as a positive response.
Analysis of T cell phenotypes
Differentiation phenotypes of T cells were determined in cohort 1 as
published previously (21). Briefly, PBMCs were thawed, Fc receptors
blocked and dead cells were stained as described above. Cells were
then stained with anti–KLRG-1 primary Ab (provided by Prof. Dr.
H.-P. Pircher, Freiburg, Germany) for 20 min at 4˚C, followed by staining
with Pacific Orange–conjugated goat anti-mouse IgG (Invitrogen) for another 20 min on ice. After blocking with mouse serum (see above) the
following directly-conjugated Abs were added: CD3-PE (Caltag; Invitrogen), CD4-PerCP, CD8-allophycocyanin-H7, CCR7-PE-Cy7 (BD Biosciences, Heidelberg, Germany), CD27-allophycocyanin, CD45RA-Pacific
Blue, CD28-Alexa Fluor 700 (BioLegend, San Diego, CA), and CD57FITC (Immunotools, Freiburg, Germany). After 20-min incubation on ice,
cells were washed and analyzed immediately on an LSR II cytometer. For
a detailed gating strategy, please see Ref. 21.
Materials and Methods
Study population
CMV serology
Cryopreserved PBMCs from two different cohorts were included in this
study. Cohort 1 consisted of 166 subjects participating in the Leiden
Longevity Study. Detailed characteristics of these donors have been published previously (21). They were between the ages of 42 and 82 y with
a mean age of 62.2 y and a CMV seropositivity rate of 45.2% overall.
Study cohort 2 was recruited in Vancouver, BC, Canada and consisted of
28 donors between the ages of 65 and 81 y with a mean age of 73.1. The
CMV prevalence was 75%.
CMV serostatus and CMV-specific IgG titers were determined by ELISA,
using the CMV-IgG-ELISA PKS assay (Medac, Wedel, Germany), according
to the manufacturer’s instructions. IgG specificity for different Ags was
determined by means of a recombinant CMV IgG immunoblot (Mikrogen,
Neuried, Germany), according to the manufacturer’s instructions.
Analysis of T cell responses
T cell responses to different Ags were tested as described previously (22).
Briefly, PBMCs were thawed and allowed to rest for 8 h in X-Vivo 15
medium (Cambrex) at 1 3 106/ml at 37˚C. Cells were then stimulated with
overlapping peptides covering the whole sequence of MP and/or NP (cohort 1/cohort 2, respectively) from the influenza A virus (IAV). These
PepMixes were purchased from JPT Technologies (Berlin, Germany) and
were used at a concentration of 1 mg/ml as recommended by the manufacturer. Cells from CMV-seropositive donors were additionally stimulated
with pp65 and IE-1 PepMix. A total of 1 ml/ml GolgiPlug (BD Biosciences, Heidelberg, Germany) was added together with the peptides, and
the cultures were incubated for 16 h. This provides a direct ex vivo readout
of responding memory cell frequencies without any intervening long-term
culture period, which could possibly lead to expansion of naive T cells.
Medium alone and 50 ng/ml PMA (Sigma-Aldrich, Munich, Germany)
together with 750 ng/ml ionomycin (Merck, Darmstadt, Germany) were
used as negative and positive controls, respectively. The cells were then
harvested, washed, and treated with human Ig, Gamunex (Bayer, Leverkusen, Germany), and ethidium monoazide (EMA) (Invitrogen, Karlsruhe, Germany) for 10 min on ice to block FcRs and label nonviable cells.
The cells were then stained first indirectly for CD3 (OKT3 primary and
Pacific Orange–conjugated goat anti-mouse IgG secondary Ab [Invitrogen]). After the cells were blocked with mouse serum (Chemicon/
Millipore), they were stained with CD4-PerCP and CD8-allophycocyanin-H7 mAbs (BD Biosciences). Following fixation and permeabilization
using the BD Cytofix-Cytoperm solution (BD Biosciences), the cells were
stained with IFN-g-PE-Cy7 (BD Biosciences), TNF-FITC, and IL-2-Alexa
Fluor 700 (BioLegend). Samples were measured directly using a BD-LSRII with FACSDiva software (BD Biosciences). The spectral overlap between all channels was calculated automatically by the BD FACSDiva
software, after measuring negative and single-color controls. Data were
analyzed using FlowJo software (Tree Star, Portland, OR). Gating strategy
is shown in Fig. 1. First events acquired during a nonconstant flow of the
machine were excluded in a forward light scatter-versus-time dot plot
followed by gating the lymphocyte population based on size and granu-
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 5.
Differences between groups for the categorical variables were assessed with
the x2 test. Mann–Whitney testing was used for comparison of two independent groups. The p value was set at 0.05.
Results
Ex vivo detection of IAV-specific T cells in cohort 1
We first determined the frequency of CD4 and CD8 T cells
responding to a mixture of both NP and MP peptides after an
overnight stimulation with peptide pools spanning the whole sequence of these Ags in a cohort of 166 middle-aged and elderly
individuals (Fig. 1). Peptide-specific CD4 responses were detected
in 129 individuals (77.7%), whereas only 97 had CD8+ IAVspecific T cells (58.4%). Fig. 2A shows representative FACS
plots from one donor with a CD4-dominant (donor 1, left) and
another donor with a CD8-dominant response (donor 2, right) to
these influenza peptides. The frequency of IAV-reactive CD4+
cells ranged between 0.015 and 0.46% of total CD4+ T cells
(Fig. 2B) and was generally lower than the frequency of IAVreactive CD8+ T cells (range: 0.032–1.33% of total CD8+
T cells; Fig. 2B). In 23 individuals (13.9%), no responses of either
CD4 or CD8 T cells could be detected. Comparing these individuals (nonresponders, NR) with the remainder (responders, R)
regarding age and CMV seropositivity, two parameters known to
impact the functional status of T cells, we observed that NR were
slightly but not significantly older (median age 65 versus 60.7 y
[ p = 0.17]). In addition, NR tended to be more often CMV-infected
than R: 13 of 23 (56.5%) versus 62 of 143 (43.3%), respectively,
but this was not significant (p = 0.24). Despite a lack of cytokine
response after stimulation with influenza Ags, NR did not differ
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CD4 memory T cells have been found to be associated with disease protection and limiting disease severity in an influenza
challenge model in humans (18). Cellular responses are mainly
targeted against matrix protein (MP) and nucleoprotein (NP), two
core proteins of the virus, which are conserved between strains;
hence, memory T cells specific for these proteins can mediate
cross-protective immunity, as was demonstrated recently (19).
This is especially very important in the context of pandemics and
in the absence of neutralizing Abs.
Whether cellular responses to influenza are affected by aging
or the presence of a latent infection with CMV has not been explored to date. Given the fact that CMV infection has been associated with poor cellular responses to EBV (20), we aimed to
analyze whetehr this was also the case for influenza. In this study,
we demonstrate in two independent cohorts that CD4 responses to
influenza core proteins are absent in almost half of the CMVseropositive elderly, whereas older people not infected with this
virus respond as well as the young. Hence, advanced chronological age plays a role in depressed responses to influenza but only
in concert with CMV infection. Our data also suggest that contrary
to the widely accepted concept, a more late-differentiated CD4
compartment is not detrimental but is associated with better CD4
responses to the influenza virus and, hence by implication, better
protection in vivo.
3625
3626
from R in their CD4 and CD8 T cell production of IFN-g, TNF, or
IL-2 on stimulation with PMA/ionomycin (Fig. 2C).
CMV seropositivity is associated with lower CD4 T cell
responses to IAV Ags only in the elderly
Having observed a trend toward a possible negative association
between age and CMV-seropositivity with memory responses to
IAV in vitro, we sought to determine whether there was an additive
effect between these parameters. For this, individuals were stratified according to CMV serostatus and age (,65 or .65 y). In the
,65 y group, CD4 responses were detected in 52 of 61 (85.2%) of
CMV-seronegative and in 41 of 48 (85.4%) of CMV-seropositive
CMV AND T CELL MEMORY TO IAV
donors (Fig. 3A). However, in individuals over the age of 65 y,
CMV seropositivity was significantly associated with fewer CD4
responders; while older CMV-seronegative individuals had a similar CD4 response rate compared with the younger donors (23
of 30, 76.7%), only 13 of 27 (48.1%) CMV-seropositive elderly
mounted a memory CD4 response to IAV (p = 0.026 compared
with CMV-seronegative age-matched donors and p = 0.0006
compared with CMV-matched younger donors; Fig. 3A). IAVspecific CD8 responses were detected in similar proportions of
donors in each age group, regardless of CMV infection and with
no difference between younger or older donors (Fig. 3B). Thus,
neither age nor CMV affected the frequency of individuals capable
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FIGURE 1. Gating strategy for detection of peptide-specific T cells. After an overnight incubation period with or without Ags, cells were harvested,
washed, and treated with human Ig, Gamunex, and EMA. The cells were then stained for CD3, CD4, and CD8-allophycocyanin-H7 followed by fixation,
permeabilization, and intracellular staining for IFN-g, TNF, and IL-2. For the data analysis, first events acquired during a nonconstant flow of the machine
were excluded (A), followed by gating the lymphocyte population based on size and granularity (B). After exclusion of duplets (C and D) and EMAnegative dead cells (E), T cells were gated as CD3+ (F), followed by gating of CD4+CD82 and CD42CD8+ cells (G). Production of each cytokine was then
determined in CD4+ (H) and CD8+ (I) cells separately.
The Journal of Immunology
3627
of mounting CD8 T cell responses to influenza core proteins as
measured by TNF, IFN-g, or IL 2 production. The lower proportion
of CMV-seropositive elderly individuals mounting a CD4 T cell
response to IAV Ags was not due to a lower intrinsic capacity of
their T cells to produce IFN-g, TNF, or IL-2 when stimulated with
PMA/ionomycin. On the contrary, CMV-seropositive donors produced significantly higher levels of IFN-g and TNF in both age
groups (Fig. 3C, 3D), whereas neither age nor CMV serostatus had
an impact on IL-2 levels (data not shown).
We sought to validate these findings in a second independent
cohort of individuals .65 y of age from a different country
(Canada). In this cohort, the response rate to MP and NP peptide
pools was analyzed separately. The CD4 response rate to MP in
CMV-seronegative elderly (six of seven, 85.7%) was similar to
that detected in the Dutch cohort. In this cohort too, a significantly
lower proportion of individuals mounted a CD4 response (and in
this case also a CD8 response against MP) if they were CMV
seropositive (Table I). Cellular responses against NP were detected in a much lower frequency of donors and were not influenced
by CMV status (Table I).
The frequency of cytokine-producing cells and the amount of
cytokine produced by the cells (as determined by mean fluores-
FIGURE 3. CMV seropositivity is
associated with lower CD4 responses
to IAV only in the elderly. The percentage of CMV-seronegative (N)
or CMV-seropositive (n) individuals less than or more than the age of
65 mounting a CD4 (A) or a CD8
(B) response to MP and NP proteins, measured as described in the
legends to Figs. 1 and 2. IFN-g and
TNF production in CD4 (C) and CD8
(D) cells in response to nonspecific
stimulation with PMA/ionomycin.
,65 y CMV2: n = 61, ,65 y CMV+:
n = 48, .65 y, CMV2: n = 30, .65 y,
CMV+: n = 27. Each symbol represents a single donor tested. Horizontal
lines represent the median. *p , 0.05,
**p , 0.01, ***p , 0.001.
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FIGURE 2. Ex-vivo detection of
T cells specific for core proteins
of the influenza virus. (A) Cryopreserved PBMCs were thawed and
were either left untreated (unstimulated control) or stimulated overnight with peptide pools overlapping
the whole sequence of influenza A
MP and NP. Representative FACS
plots from two donors are presented.
The cells are from the CD3+ gate.
(B) Frequency of influenza-specific
CD4 and CD8 cells in 129 and 97
healthy donors, respectively, with
detectable CD4 and CD8 responses.
(C) Frequency of CD4 (left panel)
and CD8 (right panel) T cells producing IFN-g, TNF, and IL-2 after
stimulation with PMA/ionomycin.
3628
CMV AND T CELL MEMORY TO IAV
Table I. Memory responses to influenza A MP and NP in cohort 2
Response to MP, n (%)
CD4 response
CD8 response
Response to NP, n (%)
CD4 response
CD8 response
CMV2 (n = 7)
CMV+ (n = 21)
p Value
6 (85.7)
6 (85.7)
9 (42.9)
9 (42.9)
0.04
0.04
2 (28.6)
4 (57.1)
7 (33.3)
8 (38.1)
ns
ns
cence intensity) did not differ between CMV-seronegative and
CMV-seropositive responders (data not shown). We then analyzed
the pattern of cytokine response of IAV-specific T cells. Because of
the low number of donors producing IL-2, this cytokine had to be
excluded from the polyfunctionality analysis. The majority of IAVspecific CD4 and CD8 T cells produced only IFN-g but not TNF
(Fig. 4A, 4B, middle panels). Neither age nor CMV serostatus had
an impact on the proportion of cells producing both TNF and
IFN-g or only one of these cytokines (Fig. 4).
Next, we sought to determine whether CMV-seropositive NRs to
IAV Ags also failed to mount a cellular response to CMV. For this,
peptide pools of pp65 and IE-1, two immunodominant Ags of
CMV, were used. These experiments were performed in 71 CMVseropositive individuals from cohort 1. CMV-specific CD4 and/or
CD8 responses were detected in 70 (98.6%) donors; the majority of
whom (92.8%) mounted both a CD4 and a CD8 response against
these Ags, confirming that the absence of CD4 responses against
IAV in some CMV-seropositive donors is not due to a general lack
T cell functionality. Next, we stratified the donors according to the
presence or absence of IAV-specific CD4 or CD8 T cells and
compared the frequency of CMV-specific CD4 and CD8 cells
between these individuals. The frequency and also the absolute
numbers per unit of blood of CD4 and CD8 T cells specific for
CMV did not differ between individuals without a CD4 response
to IAV (CD4-NR n = 18, median age 68.9 y, interquartile range
8.2) and those mounting a CD4 response (CD4-R, n = 47, median
age 61.3 y, interquartile range 8.6) (Fig. 5). CMV-specific CD4 or
FIGURE 4. The quality of cytokine response to IAV core proteins is
similar in young and old CMVseronegative and CMV-seropositive
donors. Frequency of CD4+ (A) and
CD8+ (B) cells producing only TNF
(left panels), only IFN-g (middle
panels), and both TNF and IFN-g
(right panels) was compared between CMV-seronegative and CMVseropositive donors below or above
the age of 65 y. This analysis was
limited to those donors with a
detectable IAV-specific T cell response. Each dot represents a single
donor tested. Horizontal lines represent the median.
The absence of a CD4 response to IAV in CMV-seropositive
individuals is not associated with lower serum anti-CMV
IgG titers, but more CD4-responders have IgG specific
for CMV-gB2
Next we sought to determine whether the humoral response to
CMV was different between CMV-seropositive individuals who
did not have CD4 T cell responses to IAV (CD4-NR) and those who
did (CD4-R). Both groups had similar titers of anti-CMV IgG
Abs (Fig. 5C). Almost half of the donors in both groups had IgG
titers higher than the detection limit of the kit. These donors have
been excluded from Fig. 5C. We then characterized the specificity of IgG responses to different CMV Ags. As shown in Fig. 5B,
a significantly higher proportion of CD4-R compared with CD4NR donors had serum IgG reactivity to gB2, one of the main
targets of neutralizing Abs against CMV (Fig. 5D).
CMV-seropositive individuals mounting a CD4 response to IAV
possess T cells with a more late-differentiated CD4 phenotype
It is well established that a higher proportion of peripheral blood
CD4 and CD8 T cells from CMV-seropositive than -negative
donors has a late-differentiated phenotype, the accumulation of
which has been correlated with poor cellular responses to Influenza
vaccination in the elderly. Thus, we next sought to determine
whether a more late-differentiated T cell compartment was associated with poor CD4 responses to influenza core proteins. For this,
the frequency of different naive and memory phenotypes was
determined according to the surface expression of CD45RA,
CCR7, CD27, and CD28. A differentiation index was calculated
by dividing the frequency of the most late-differentiated effector
memory and effector populations (CD45RA+/2CCR72CD272
CD28 2 ) by the frequency of naive cells (characterized as
CD45RA+CCR7+CD27+CD28+). CMV-seropositive individuals
were again grouped according to the presence or absence of a CD4
response to IAV as described in the previous section. CD4 T cells
had a significantly higher differentiation index in CD4-R compared
with the NR (Fig. 6A). This was not due to a lower frequency of
naive CD4 cells because the frequency and absolute number of
cells with this phenotype was similar between CD4-R and CD4-NR
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The absence of CD4 response to IAV in CMV-seropositive
individuals is not reflected in a lower T cell response
to CMV Ags
CD8 responses were detected at equal levels between individuals
with or without a CD8 response to IAV (data not shown).
The Journal of Immunology
3629
(data not shown). However, we observed significantly higher frequencies and absolute numbers of late-differentiated effector memory and effector cells in the CD4-R group (Fig. 6B). Consistent with
this, cells expressing markers of very late or “terminal” differentiation, sometimes also termed markers of “senescence” (CD57 and
KLRG-1), were enriched in the CD4-R group (Fig. 6C, 6D).
Discussion
In this study, we report that memory CD4 responses to influenza
virus core protein MP are compromised in a subset of CMVseropositive elderly individuals, whereas almost all CMVseronegative elderly possess influenza-reactive CD4 memory
T cells. Unexpectedly and counterintuitively, this was not due to
CMV-associated immunosenescence, because individuals lacking
a CD4 response had significantly lower levels of late-differentiated CD4 T cells expressing the putative senescence markers
CD57 and KLRG-1, and a less-differentiated CD4 subset, relative
to people mounting a CD4 response to IAV Ags. This is interestingly in contrast to our recent data demonstrating a negative
impact of the presence of such late-differentiated CD4 T cells on
humoral responses to influenza vaccination (6). However, our in
vitro memory T cell response assay may not be reflecting T cell
help for Ab formation. It is also consistent with findings that the
presence of prevaccination late-differentiated IAV-specific memory T cells correlates with poor Ab responsiveness after vaccination (25). In addition, responsiveness to influenza vaccination is
well known to negatively correlate with prevaccination hemagglutination inhibition (HI) titers. In other words, donors who already have high amounts of HI Ab, thus a stronger immune
response to the virus, respond less well to vaccination in terms of
fold increase in HI titer, the parameter of which is used to estimate
vaccine responsiveness. Hence, the presence of late-differentiated
CD4 T cells correlating with better cellular responses to IAV could
be indicative of higher HI titers and therefore poorer responsiveness to vaccination.
What proportion, if any, of the late-differentiated CD4 T cells
observed in our study to be more abundant in CD4-Rs is specific for influenza, could not be tested. We have previously demonstrated in three different cohorts of different ages that latedifferentiated CD4 T cells lacking CCR7, CD27, and CD28 and
expressing KLRG-1 are directly associated with CMV seropositivity and are almost completely absent in CMV-seronegative
donors (6, 22, 26); thus, it is more likely that these cells are
specific for CMV and not influenza. However, it has been shown
that in CMV-seropositive individuals, especially (but not only)
when they are old, CD4 T cells specific for other viruses such as
EBV, Varicella Zoster virus, and HSV also have lost the expression
of CD27 and CD28 and are significantly more late-differentiated
compared with cells of the same specificity in CMV-seronegative
donors (27). In fact, the phenotype of CD4 T cells specific for all
non-CMV Ags (also the recall Ag purified protein derivative) was
very similar to that of CMV-specific CD4+ T cells, suggesting
a bystander effect mediated through differentiation-inducing
factors secreted as a result of CMV infection (27). It has been
reported that CD4+ T-cells reactive to CMV and lacking the
expression of CD27 and CD28 have regulatory properties (28).
Whether this is true for T cells with the same phenotype specific
for other viruses has not been investigated to date but might indicate another immunosuppressive property of CMV by induction
of suppressive regulatory T cells specific for other Ags, for example influenza A. Why this negative correlation is only observed
in CD4 memory response and not CD8 is not yet clear. It has been
recognized for many years that people 65 y and older are at greater
risk of serious complications from influenza disease than younger
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FIGURE 5. CMV-specific cellular and humoral responses in CD4-Rs and NRs to IAV. PBMCs from CMV-seropositive donors were stimulated overnight
with overlapping peptides spanning the whole length of pp65 and IE-1, followed by staining for extracellular markers and intracellular cytokines as described in Fig. 2 legend. Individuals were then grouped according to absence (NR) or presence of a CD4 response (CD4-R) to influenza MP and NP. A
positive response was defined by at least a 2-fold increase in the frequency of cells producing at least one cytokine (IFN-g, TNF, or IL-2) in response to IAV
peptides compared with nonstimulated control. The frequency of CD4 (A) and CD8 (B) cells producing either IFNg and/or TNF are shown. (C) Anti-CMV
IgG titer in CD4-R sand CD4-NRs. (D) The IgG reactivity of serum of CMV-seropositive donors was tested against five different CMVAgs p65, IE-1, CM2,
gB1, and gB2 by immunoblotting. The proportion of donors with IgG reactivity to these Ags is shown in individuals without a CD4 response to IAV (CD4NR) and those harboring IAV-specific CD4 responses. Each dot represents a single donor tested. Horizontal lines represent the median.
3630
CMV AND T CELL MEMORY TO IAV
adults. Cellular responses to influenza, especially memory CD4
responses, have been shown to correlate with disease protection,
particularly in the absence of neutralizing Abs (18). Thus, the lack of
memory CD4 responses to IAV in almost half the CMV-seropositive
old individuals might help to explain the increased susceptibility
of the elderly, the majority of whom is infected with CMV, to
influenza-related complications and death. CMV seropositivity has
been associated with poor cellular responses to other viruses both in
humans (20) and in murine models of CMV infection (29, 30). One
explanation for this may be that accumulation of CMV-specific
T cells suppresses other memory T cells by filling the “immunological space” and through competition for growth factors or secretion of suppressive factors. Indeed, in our study, the lack of CD4
responses to influenza was not reflected in a poor response to CMV.
One limitation of our study is the lack of information on vaccination history in cohort 1. However, all individuals from the Canadian cohort had at least had one round of influenza vaccination
prior to the time of our analysis. This fact and the similar rate of
cellular responses observed between the two cohorts suggest that the
difference between young and elderly CMV-seropositive donors is
most probably not because of different vaccination histories. Another open question is why CD4 responses are affected but CD8
are not in these assays. It is well established by many investigators
that the distribution of different memory CD8 T cell phenotypes
is altered more dramatically than the CD4 subset in CMV-seropositive donors and in the elderly. However, the emergence of
late-differentiated CD4+ memory T cells, which is observed in only
a few CMV-seropositive donors is directly associated with a latent
infection with this virus, whereas CD8 T cells of the same phenotype also accumulate in CMV-seronegative donors. This indicates a differential impact of a latent infection with CMV on the
development of CD4 and CD8 memory T cells, which in turn might
help explain a differential impact of this virus on their function.
These data add to the accumulating evidence that infection with
CMV has profound but heterogeneous effects on responses to the
products of other viruses and have implications for the design of
influenza vaccines especially in the elderly. The perhaps unexpected finding that CMV-associated immunosenescence is not
necessarily detrimental to the host also is demonstrated in our
recent studies of the very elderly (22, 31), albeit in the CD8 subset,
suggesting that the remodeling of the T cell compartment in the
face of a latent infection with CMV might not be pathological, but
represent an adaptation of the immune system of the host in the
face of chronic challenge with CMV. Thus, continued immunosurveillance against persistent CMV is more important for host
survival than reserving immune resources for responses to other
viruses to which the individual might never be exposed.
Acknowledgments
We thank Prof. Dr. Klaus Hamprecht and Wioleta Kapis for determination
of CMV-specific IgG reactivity, Prof. Dr. Hans-Peter Pircher for providing
the anti-KRLG1 Ab, Dr. Robert Beck for the CMV serology, and Lilly
Oettinger for Ab titration and flow cytometry quality controls.
Disclosures
The authors have no financial conflicts of interest.
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