Journals of Gerontology: Biological Sciences
cite as: J Gerontolog A Biol Sci Med Sci, 2015, 917–923
doi:10.1093/gerona/glu128
Advance Access publication April 7, 2015
Original Article
CCR4+ Regulatory T Cells Accumulate in the
Very Elderly and Correlate With Superior 8-Year
Survival
Evelyna Derhovanessian,1 Sijia Chen,2 Andrea B. Maier,3 Karin Hähnel,1
Anton J. M. de Craen,2 Helene Roelofs,4 Rudi Westendorp,2,5 and
Graham Pawelec1
Department of Internal Medicine II, Centre for Medical Research, University of Tübingen, Tübingen, Germany.
Department of Gerontology and Geriatrics and 3Department of Internal Medicine, Section of Gerontology and
Geriatrics, VU University Medical Centre, Amsterdam, the Netherlands. 4Department of Immunohematology and
Blood Transfusion, Leiden University Medical Centre, Leiden, the Netherlands.5Netherlands Consortium for Health
Aging, Leiden University Medical Centre, Leiden, the Netherlands.
1
2
Address correspondence to: Evelyna Derhovanessian, Dr. rer. nat., Biontech AG, Freiligrathstraße 12, 55131 Mainz, Germany.
Email: [email protected]
Received January 21 2014; Accepted July 1 2014.
Decision Editor: Rafael de Cabo, PhD
Abstract
CD4+ regulatory T cells (Tregs) are a distinct population of T cells involved in maintaining peripheral
tolerance to self-antigens. Several studies have shown increased frequency and number of Tregs in
the elderly. Whether such an increase has any clinical relevance has not been addressed. Here, we
have analyzed circulating Tregs in 114 donors between the ages of 18 and 89 years and assessed
their implications for survival of the very elderly. In line with previously published data, we
observed higher proportions of Tregs in the elderly. Expression of chemokine receptor 4 (CCR4) by
Tregs has been shown to characterize antigen-primed activated Tregs with immediate suppressive
function. Thus we further analyzed Tregs expressing or lacking this chemokine receptor. There were
more CCR4+ and CCR4− Tregs in the elderly than the young. Finally, using a subset of 48 elderly
donors participating in the Leiden 85-plus study we documented that people with greater median
frequencies of CCR4+ Tregs enjoyed a better 8-year survival rate than those with lower frequencies
of these cells. Our data, demonstrating for the first time a positive correlation between increased
frequency of Tregs and survival in the elderly, imply an increasing importance of controlling
inappropriate immune responses and inflammation as we grew old.
Key Words: Tregs—Cytomegalovirus—Elderly—Mortality
The distinct lineage of CD4 regulatory T cells (Tregs), constitutively expressing the transcription factor Foxp3, plays an essential role in protection against autoimmunity and maintenance of
immune homeostasis (1,2), evidenced by severe autoimmune and
inflammatory disorder in individuals with a loss of function mutation in the Foxp3 gene (3). Accordingly, these Tregs are reduced
in number and/or function in patients with different systemic
auto-immune diseases (4). Reciprocally, they accumulate in both
© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved.
For permissions, please e-mail: [email protected].
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peripheral blood and solid tumors in cancer patients and can be
associated with poor prognosis (5).
Tregs are characterized by high expression levels of the α-chain
of the interleukin-2 (IL-2) receptor (CD25) as well as by expressing the transcription factor Foxp3. The majority of Tregs are generated during T-cell development in the thymus as antigen-primed
mature T cells specialized for suppression. The main task of these
cells is to migrate to inflamed tissues and suppress effector T cells (1).
Accordingly, they express a variety of chemokine receptors, which
allows their homing to different tissues. The expression of one of
these receptors, chemokine receptor 4 (CCR4), has been shown to
characterize Tregs, which are functionally primed and exert suppressive function without requiring activation through the T-cell receptors, contrary to their CCR4-negative counterparts (6).
Like other components of the adaptive immune system, Tregs also
change with age. Several studies have shown a substantial increase of
Tregs in the elderly (7–12), regardless of the phenotypic markers used
for determination of these cells, although there is one study that failed
to detect any difference between the frequency of Foxp3+ T cells in
young (Y) and old (O) individuals (13). The majority of reports is
consistent with retention of function of these cells (7,9,11,13), suggesting increased levels of suppressive activity in the elderly (although
there is one study reporting decreased activity) (14). However, the
clinical implications of accumulation of this suppressive population,
and whether more immune suppression in the elderly is generally a
good or bad thing have not been addressed to date.
Here, using 10-color flow cytometry to quantify the frequency of
Tregs, defined as Foxp3+ or CD4+Foxp3+CD127loCD25hi in a large
cohort of Y, middle-aged (MA) and very O individuals, we confirm
an increased frequency of this subset in the elderly, but not in MA
individuals. In addition, we demonstrate that accumulation of a particular subset of these cells, characterized by CCR4 expression, correlates with superior 8-year survival on follow-up of very elderly
participants of the Leiden 85-plus study.
Materials and Methods
Study Participants
All individuals studied here were recruited at Leiden University
Medical Center (LUMC), Leiden, the Netherlands, after obtaining
approval from the Medical Ethics Committee of the LUMC. We
analyzed 10 Y (18–25 years, mean age: 22.2 years), 56 MA (50–65
years, mean age: 58.6 years) and 48 elderly people participating in
the Leiden 85-plus study, a population-based, prospective study of
the inhabitants of Leiden (88–89 years, mean age: 88.6 years).
Leiden 85-Plus Study
Details of this study have been published previously (15). In brief,
all inhabitants of Leiden born between 1912 and 1914 were invited
to participate in the study between September 1997 and 1999. The
blood samples analyzed here were taken in 2002. The 48 elderly participants from this study analyzed here were followed up for survival
until January 2010, by which time 87.5% of them had died. Date
and cause of death data were obtained from the Dutch civic registry.
Anti-cytomegalovirus (anti-CMV) IgG titers were measured
in plasma using a CMV IgG kit (ETI-CYTOK-G PLUS DiaSorin,
Saluggia, Italy) based on enzyme immunoassay technology.
Cognitive function was measured using the Mini-Mental State
Examination (MMSE) (16). The MMSE scores range from 0 to
30, with lower scores indicating impaired cognitive function. The
MMSE scores were assessed annually. Maximal grip strength
was measured in individuals’ dominant hand using a Jamar hand
dynamometer (Sammons Preston Inc., Bolingbrook, IL) (17). Plasma
C-reactive protein (CRP) was assayed using the Hitachi 747 system
(Hitachi, Japan).
Flow Cytometry
Cryopreserved PBMC were thawed and treated with human immunoglobulin (GAMUNEX; Talecris Biotherapeutics) and ethidium
monoazide (Invitrogen) to block Fc receptors and stain dead cells,
followed by indirect staining for CD3 using OKT3 supernatant
and a Pacific Orange-conjugated anti-mouse IgG (Invitrogen).
After blocking unbound secondary antibodies with mouse serum
(Chemicon, Millipore), cells were surface-stained with CD4-Pacific
Blue, CD127-Alexa Fluor 647 (BioLegend, San Diego, CA), CD25APC-Cy7, CD8-PerCP, CCR4-PE-Cy7 (BD Biosciences), followed by
fixation/permeabilization and staining for Foxp3 using a BioLegend
Foxp3 staining kit (in PE format) according to the manufacturer’s
instructions. Cells were measured immediately using an LSR-II (BD).
For data analysis, EMA+ dead cells were excluded. CD3+ T cells
were gated from the viable cells, followed by gating of CD4 T cells
(CD4+CD8−) in a CD4 versus CD8 dot plot (Supplementary Figure 1).
The frequency of Foxp3+ T cells as well as Foxp3+CD127loCD25hi
cells was determined within total CD4 T cells.
Statistical Analysis
Data from Y, MA, and O donors were compared using the Kruskal–
Wallis test and Dunn’s multiple comparison test. Two independent
groups were compared using the Mann–Whitney U test. Cox regression analysis was performed in the Leiden 85-plus study to assess the
associations between different Treg subsets and survival after adjusting
for potential confounders, including gender, CMV serostatus, sum of
comorbidities, total number of medication, and levels of CRP. In addition, MMSE score and grip strength were also included as confounders based on published data suggesting that these parameters should be
considered as predictors of mortality (18–20). For number of comorbidities we summed the presence of cardiovascular comorbidities (hypertension, angina pectoris, myocardial infarction, stroke, and intermittent
claudication), diabetes, chronic obstructive pulmonary disease (COPD),
cancer, and arthritis. p values < .05 were considered statistically significant. Data analysis was performed using GraphPad Prism5 and SPSS.
Results
Frequency of Circulating Tregs Is Greater in the
Elderly Than the Young
First we compared the frequency of Foxp3+ and Foxp3+CD127loCD25hi
CD4+ T cells in a cohort of Y (n = 10, mean age: 22.2 years), MA
(n = 56, mean age: 58.6 years), and elderly (n = 48, mean age: 88.6
years) individuals from Leiden. As shown in Figure 1A, although
the Y tended to have lower frequencies of CD4+ FoxP3+ T cells than
the MA, this difference was not statistically significant. However,
the difference between MA and O, as well as between Y and O, was
highly significant (p < .0001). Data shown in Figure 1B confirms that
Y and MA have the same levels of Tregs as defined by the “classical”
extended phenotype CD4+CD25hi CD127lo, in both age groups significantly fewer than in the elderly. Thus, Y and MA individuals have
similar levels of circulating Tregs, whereas these cells are enriched in
the very elderly. Furthermore, we compared the frequency of CCR4+
and CCR4− Foxp3+CD127loCD25hi CD4+ T cells between the three
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 70, No. 8
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Figure 1. The impact of age on the frequency of CD4+ regulatory T cells. The proportion of Foxp3+ (A), Foxp3+CD127loCD25hi (B), CCR4−Foxp3+CD127loCD25hi
(C), and CCR4+Foxp3+CD127loCD25hi (D) from total CD4 cells was compared between 10 young (Y, 18–25 years, mean age: 22.2 years), 56 middle-aged (MA,
50–65 years, mean age: 58.6 years) and 48 elderly (O, 88–89 years, mean age: 88.6 years) from Leiden. Each symbol represents one individual. Horizontal bars
represent the median of each group. p values were calculated using the Dunn’s multiple comparison test. *p < .05, **p < .01, ***p < .001.
groups. The majority of the Tregs (>70%) in all individuals tested
expressed CCR4, and elderly had significantly higher levels of both
CCR4+ and CCR4− Tregs in the peripheral blood when compared
with MA and Y individuals (Figure 1C and D).
Latent Infection With Cytomegalovirus Has No
Impact on the Frequency of Tregs
Considering the strong impact of a latent infection with CMV on
the frequency and differentiation phenotypes of CD4 and CD8 T
cells (21), we sought to determine whether the prevalence of CMV
was different in the three age groups, reflecting the different frequencies of Tregs. In the Y group, only one of the 10 donors was CMVseropositive (CMV+). However, in the MA group, 52% (29/56) of
participants were infected with CMV, and as high as 81% (39/48) in
the very elderly (p for trend < .0001). Thus, we investigated whether
the increased level of Tregs observed in the very elderly when compared with the Y and the MA groups correlated with significantly
higher prevalence of this virus. For this, individuals were stratified
according to CMV-serostatus and age. Because almost all of the Y
individuals tested were CMV−, this analysis was performed only in the
MA-versus-O participants. As shown in Figure 2, CMV− and CMV+
individuals in both age groups had equal frequencies of Foxp3+ and
Foxp3+CD127loCD25hi CD4+ T cells in the peripheral blood. The frequency of CCR4+ or CCR4− Tregs did not differ between the groups
either. We also explored whether the level of anti-CMV IgG antibodies correlated with the frequency of different Treg subsets. As shown
in Figure 2E, there was no difference between CMV+ donors with
lower than median (low) or higher than median (high) levels of antiCMV IgG titers. This analysis was performed in MA donors only.
Higher Frequency of CCR4+ Tregs Correlates With
Superior 8-Year Survival in the Very Elderly
Finally, we investigated whether the frequency of CD4+ Tregs at
baseline in the very elderly (mean age: 88.6 years) had any impact
on 8-year survival on follow-up in the Leiden 85-plus study.
Characteristics of these donors are shown in Table 1. Kaplan–Meier
analysis of survival in individuals stratified according to higher or
lower than median frequencies of Foxp3+ or Foxp3+CD127loCD25hi
CD4+ T cells revealed no difference in survival time from baseline in
these very elderly individuals (Figure 3A and B). However, subgrouping Tregs according to the surface expression of CCR4 revealed a significant survival advantage for individuals with higher than median
levels of CCR4+ Tregs when compared with those with lower than
median levels (Figure 3D). The frequency of CCR4− Tregs did not
have any correlation with survival (Figure 3C). The positive correlation between the high frequency of CCR4+ Tregs and survival
remained highly significant after adjusting for different confounders
such as gender, CMV serostatus, sum of comorbidities, total numbers of medication, CRP, MMSE and grip strength (Table 2). The
frequency of Tregs (neither total nor CCR4+ nor CCR4−) did not correlate with the sum of comorbidities (Supplementary Table 1). Finally,
exploring correlations with cause-specific mortality, we observed a
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Figure 2. The impact of a latent infection with cytomegalovirus (CMV) on the frequency of CD4+ regulatoryT cells.The proportion of Foxp3+ (A), Foxp3+CD127loCD25hi
(B), CCR4−Foxp3+CD127loCD25hi (C), and CCR4+Foxp3+CD127loCD25hi (D) from total CD4 cells was compared between 27 CMV-seronegative (CMV−) and 29 CMVseropositive (CMV+) middle aged (MO) as well as 9 CMV− and 39 CMV+ elderly (O) donors. (E) Middle-aged donors were then grouped into CMV-seronegative
(Neg) or CMVseropositive donors with lower (low) or higher than median (high) levels of anti-CMV IgG titer. Each symbol represents one individual. Horizontal
bars represent the median of each group.
significant association in the case of noncardiovascular mortality
(Table 3). Owing to the very low number of deaths caused by infectious disease, this parameter could not be analyzed in our cohort.
Frequency of Circulating Tregs Does Not Correlate
With CRP, a Marker of Systemic Inflammation
To determine whether the increased frequency of Tregs observed
to be associated with better survival in our elderly cohort was also
associated with lower systemic proinflammatory status, the level of
CRP as a marker of systemic inflammation was correlated with the
frequency of Tregs expressing or lacking the expression of CCR4.
As shown in Figure 4, we did not observe any significant correlation
between the frequency of any of the Treg subsets in the periphery (as
well as total Tregs, data not shown) with CRP in either MA donors
(Figure 4A) or the very elderly (Figure 4B).
Discussion
The higher frequency of Tregs in the elderly observed in this study
is in line with the majority of other published reports (7–12) but
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 70, No. 8
Table 1. Characteristics of Participants From the Leiden-85 Plus Study
n = 48
Women, n (%)
BMI, mean (SD)
Total number of medication, median (IQR)
Sum of all chronic diseases, median (IQR)
CMV seropositivity, n (%)
MMSE, median (IQR)
CRP mg/L, median (IQR)
Grip strength, mean (SD)
Deaths, n (%)
37 (77.1)
27.8 (4.5)
3 (2–5)
2 (1–3)
39 (81.3)
27.0 (24.0–29.0)
3.0 (1.0–5.8)
20.4 (7.9)
42 (87.5)
Notes: BMI = body mass index; CMV = cytomegalovirus; CRP = C-reactive
protein; IQR = interquartile range; MMSE = Mini-Mental State Examination;
SD = standard deviation.
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Table 2. Predictive Value of Dichotomized T-Cell Subsets for AllCause Mortality
CD127loFoxp3+CD25hi/CD4+
Foxp3+/CD4+
CCR4+Tregs/CD4+
CCR4−Tregs/CD4+
Model 1
Model 2
Model 1
Model 2
Model 1
Model 2
Model 1
Model 2
HR (95% CI)
p
0.78 (0.40–1.51)
0.66 (0.32–1.36)
0.77 (0.41–1.46)
0.79 (0.36–1.72)
0.38 (0.18–0.78)
0.33 (0.15–0.73)
1.03 (0.55–1.91)
0.79 (0.40–1.54)
.46
.26
.43
.55
.009
.006
.94
.46
Notes: CI = confidence interval; HR = hazard ratio; MMSE = Mini-Mental
State Examination. Cox regression analysis adjusted for possible confounders.
All T-cell subsets were dichotomized in low or high according to the median
of the whole group. Model 1 (n = 48): adjustment for gender. Model 2 (n =
47): Model 1 and adjustments for cytomegalovirus serostatus, sum of comorbidities, total numbers of medication, log transformed C-reactive protein, log
transformed MMSE score, and grip strength.
Figure 3. Correlation analysis between the frequency of different circulating CD4+ Tregs and survival in participants of the Leiden 85-plus study. Kaplan–Meier
survival curves of elderly donors stratified according to lower than median (Low, black curves) or higher than median (High, gray curves) frequencies of Foxp3+
(A), Foxp3+CD127loCD25hi (B), CCR4−Foxp3+CD127loCD25hi (C), and CCR4+Foxp3+CD127loCD25hi (D) cells are shown. p values were calculated using the Log-rank
(Mantel–Cox) test. *p < .05. (E) The frequency of Tregs and different subsets lacking or expressing CCR4 were compared between nonsurvivors (n = 34, black
symbols) and survivors (n = 14, gray symbols).
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 70, No. 8
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Table 3. Predictive Value of CCR4+ Tregs for Cause-Specific Mortality
CCR+ Tregs/CD4+
Deaths, n
B
SE
p
HR (95% CI)
All-cause mortality
Cardiovascular mortality
Noncardiovascular mortality
Cancer
39
14
22
5
−1.002
−0.840
−1.176
0.673
0.380
0.676
0.521
1.188
.008
.214
.024
.571
0.367 (0.174–0.774)
0.423 (0.115–1.625)
0.309 (0.111–0.858)
1.960 (0.191–20.107)
Notes: CI = confidence interval; HR = harzard ratio; SE = standard error. Cox regression analysis adjusted for possible confounders. CCR+Tregs/CD4+ were
dichotomized in low or high according to the median of the whole group. Model 2 is shown with adjustments for cytomegalovirus serostatus, body mass index,
sum of comorbidities, total numbers of medication, log transformed C-reactive protein, log transformed MMSE score, and grip strength.
Figure 4. Correlation analysis between the frequency of Tregs in the periphery and C-reactive protein levels. Data from 44 middle-aged donors from the Leiden
Longevity Study (LLS) (A) and 33 elderly donors from the Leiden 85-plus (B) study are shown.
not with the report of Hwang and his coworkers (13), showing no
increase in the frequency of Tregs in the elderly. This discrepancy
might be due to the younger age of the elderly donors studied in the
latter report. The mechanisms leading to increased levels of Tregs
in the elderly are not well understood. It has been elegantly demonstrated in one study in humans that Tregs can be derived from highly
proliferative memory cells and it has been proposed that conditions
associated with an increase in the memory pool might therefore lead
to an increase in the regulatory pool (11). Thus, clonal expansion in
response to chronic viral infection has been postulated as a possible
mechanism and was observed to take place in mice (22). However,
we did not see higher frequencies of Tregs in individuals latently
infected with CMV when compared with those not harboring the
virus, in agreement with the findings of Chidrawar and his coworkers (12). This might be due to the fact that clonal expansion of CD4
cells in the face of a chronic infection with CMV does not occur as
strongly as in CD8 cells in the same donors (23,24). Additionally,
if such an expansion were to occur, it would also happen in the
non-Treg effector fraction of CD4 T cells. Indeed, data from kidney
transplant recipients demonstrate that the dominant CMV-specific
clonotypes are shared between Tregs and non-Tregs (25). In this
case, the frequency of Tregs within total CD4 cells (the parameter
studied here) would not change, as the proportion of non-Tregs also
increases.
Whatever the mechanisms accounting for the observed age-associated increased frequency of Tregs might be, the potential implications of the accumulation of these anti-inflammatory, suppressive
cells have not been investigated so far. Here, we report that elderly
people with higher than median levels of a particular subset of Tregs,
namely those carrying the chemokine receptor CCR4, have better
8-year survival rates than people with lower levels of CCR4+ Tregs.
Expression of this chemokine receptor characterizes a subset of
primed memory-type Tregs, which are readily suppressive without
any need for further stimulation through their TCR (6). Thus, our
data showing a positive impact of increased frequency of these cells
on survival in the elderly might seem unexpected at first glance, as
increased frequencies of Tregs have often been postulated to contribute to the diminished state of the immune response in the elderly and
might lead to increased incidence of cancer or reactivation of chronic
viral infections, as has been reported in mice (8). Indeed, treatment
to deplete CCR4− expressing Tregs was shown to enhance the induction of antitumor T-cell responses in one published study (26). In our
study, we did not see any correlation between increased frequency of
CCR4+ Tregs and the incidence of cancer. Whether a survival benefit
for the increased frequency of Tregs will apply to other populations,
especially those at a higher risk for cancer, remains to be explored.
On the other hand, the accumulation of these suppressive antiinflammatory cells may counteract chronic systemic inflammation,
dubbed “inflammaging,” commonly seen in the elderly and often
reported to contribute to morbidity and mortality (27). Although
the level of CRP, a marker of systemic inflammation, did not correlate with the frequency of CCR4+ Tregs in our study, the lack of any
correlation between survival and total frequency of Tregs or those
not expressing CCR4 suggest that the anti-inflammatory activity of
these cells might be of more benefit in restricting local inflammation,
for example in the lungs in the case of COPD or Influenza, which can
be detrimental to the host (28,29). Indeed, in a mouse model, CCR4+
Tregs were found to migrate to allergic airways and attenuate the
inflammatory reaction (30). Along the same lines, healthy aging and
longevity have been associated with genotypes coding for lower
proinflammatory (for interferon-γ, IL-6) and higher anti-inflammatory profiles (IL-10) (31). Thus, our data demonstrating a survival
benefit for elderly people with elevated levels of CCR4-expressing
Tregs may reflect the importance of controlling overzealous immune
responses and inflammation in community-dwelling elderly.
Supplementary Material
Supplementary material can be found at: http://biomedgerontology.
oxfordjournals.org/
Journals of Gerontology: BIOLOGICAL SCIENCES, 2015, Vol. 70, No. 8
Funding
This work was supported by the European Commission (FP6 036894
“LifeSpan”; FP7 259679 “IDEAL”); German Research Foundation
(DFG-PA 361/14-1); and the Federal Ministry of Education and
Research (0315890F “Gerontoshield”). The Leiden Longevity
Study was funded by the Innovation Oriented Research Program on
Genomics (SenterNovem; IGE01014 and IGE5007); the Centre for
Medical Systems Biology, and the Netherlands Genomics Initiative/
Netherlands Organization for Scientific Research (05040202 and
050-060-810). The Leiden 85+ study was funded by the Program
Translational Research of the Netherlands organization for health
research and development, the Leiden University Fund, and by an
unrestricted grant from the Netherlands Organization of Scientific
Research (ZonMw), the Ministry of Health, Welfare and Sports,
and the Netherlands Genomics Initiative/Netherlands Organization
for scientific research (NGI/NWO; 05040202 and 050-060-810
Netherlands Consortium for Healthy Aging).
Acknowledgments
The authors thank Dr. Robert Beck for the CMV serology, Mathias Blaurock
for assistance in statistical analysis, and Lilly Oettinger for antibody titration
and flow cytometry quality controls.
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