Download Memory Cells in Old Age T Cells Are Potent + CD25

CD25-Expressing CD8+ T Cells Are Potent
Memory Cells in Old Age
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of August 3, 2017.
Dietmar Herndler-Brandstetter, Susanne Schwaiger, Ellen
Veel, Christine Fehrer, Daniel P. Cioca, Giovanni Almanzar,
Michael Keller, Gerald Pfister, Walther Parson, Reinhard
Würzner, Diether Schönitzer, Sian M. Henson, Richard
Aspinall, Günter Lepperdinger and Beatrix
Grubeck-Loebenstein
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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 © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:1566-1574; ;
doi: 10.4049/jimmunol.175.3.1566
http://www.jimmunol.org/content/175/3/1566
The Journal of Immunology
CD25-Expressing CD8ⴙ T Cells Are Potent Memory Cells in
Old Age1
Dietmar Herndler-Brandstetter,2* Susanne Schwaiger,2* Ellen Veel,* Christine Fehrer,†
Daniel P. Cioca,* Giovanni Almanzar,* Michael Keller,* Gerald Pfister,* Walther Parson,‡
Reinhard Würzner,§ Diether Schönitzer,¶ Sian M. Henson,储 Richard Aspinall,储
Günter Lepperdinger,† and Beatrix Grubeck-Loebenstein3*
I
nfectious diseases are frequent and severe in elderly persons
(1), and the efficacy of vaccinations is low (2). This is due to
age-related changes within the immune system (3). The almost complete involution of the thymus in early adulthood (4)
leads to a progressive and dramatic decrease in the numbers of
naive T cells (5). In contrast, CD8⫹CD28⫺ effector T cells have
been shown to accumulate with age (6). These cells are clonally
restricted and produce large amounts of IFN-␥, but no IL-2 or IL-4
(7, 8). CD8⫹CD28⫺ effector T cells have mainly been attributed
detrimental effects in old age. Their accumulation has been reported to trigger chronic inflammatory processes in elderly persons
(9, 10). High numbers of CD8⫹CD28⫺ cells have also been shown
to be associated with insufficient efficacy of vaccines to induce Ab
production in old age (8, 11) and to predict higher mortality (12).
*Immunology Division and †Extracellular Matrix Research Division, Institute for
Biomedical Aging and Research, Austrian Academy of Sciences, Innsbruck, Austria;
‡
Institute for Legal Medicine and §Department of Hygiene, Microbiology and Social
Medicine, Innsbruck Medical University, Innsbruck, Austria; ¶Institute for Blood
Transfusion and Immunological Department, Innsbruck, Austria; and 储Department of
Immunology, Imperial College London, Chelsea & Westminster Hospital, London,
United Kingdom
Received for publication March 16, 2005. Accepted for publication May 16, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Austrian Science Fund (Project P16205-B01).
B.G.L. is head of the Institute of Vaccination Immunology of the Austrian Green
Cross Society for Preventive Medicine. G.L. is an Austrian Programme for Advanced
Research and Technology fellow of the Austrian Academy of Sciences. He is also
supported by the Jubilee Fund of the Austrian National Bank (Project 10481) and the
Austrian Science Fund (Project S9309-B09).
2
D.H.B. and S.S. contributed equally to this paper.
3
Address correspondence and reprint requests to Dr. Beatrix Grubeck-Loebenstein,
Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg
10, 6020 Innsbruck, Austria. E-mail address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
There is strong circumstantial evidence that CMV may be a
dominant factor to drive CD8⫹ T cell differentiation and hereby
induce premature immune senescence (13). CMV-specific CD8⫹
T cells have also been shown to occur as large expanded clones
that may dominate the repertoire (14). In a recent publication (15),
we demonstrated that aging as well as CMV infection lead to a
decrease in the size of the naive CD8⫹ T cell pool, but to an
increase in the number of IFN-␥-producing CD8⫹CD28⫺ effector
T cells. The size of the CD8⫹ memory T cell population that
produces IL-2 and IL-4 also increases with aging, but this increase
is missing in CMV carriers. Lifelong latent CMV infection thus
seems to diminish the size of the naive and early memory T cell
pool and to drive a Th1 polarization within the immune system. A
recent publication demonstrates that the humoral immune response
to influenza vaccination is reduced in CMV carriers (16). This
finding indicates that lifelong CMV infection may restrict immunological diversity and thus compromise immunological memory
in old age.
We have recently described a population of IL-2/IL-4-producing
CD8⫹ T cells that display a central memory-like phenotype and
constitutive CD25 expression, yet without regulatory function
(17). This population characteristically occurs in a subgroup of
healthy elderly persons still capable of raising a protective humoral
immune response after influenza vaccination. CD8⫹CD25⫹ T
cells are virtually absent in elderly persons with CD8⫹CD28⫺
effector cell accumulation and in young individuals, who characteristically have high numbers of naive CD8⫹ T cells and low
memory/effector counts (15).
We now demonstrate that nonregulatory CD8⫹CD25⫹ T cells
represent a memory T cell reservoir of great diversity in old age.
This population may therefore be an important prerequisite for
intact immune responses in elderly persons, in whom naive T cells
can no longer be regenerated.
0022-1767/05/$02.00
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We have recently described an IL-2/IL-4-producing CD8ⴙCD25ⴙ nonregulatory memory T cell population that occurs in a
subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study
addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We
demonstrate that CD8ⴙCD25ⴙ memory T cells frequently exhibit a CD4ⴙCD8ⴙ double-positive phenotype. The expression of the
CD8 ␣␤ molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells.
They also have longer telomeres than their CD8ⴙCD25ⴚ memory counterparts, thus indicating a shorter replicative history.
CD8ⴙCD25ⴙ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags,
whereas the CD8ⴙCD25ⴚ memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not
proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same
clones occur in CD8ⴙCD25ⴙ and CD8ⴙCD25ⴚ memory T cell populations, demonstrating a lineage relationship between CD25ⴙ
and CD25ⴚ memory CD8ⴙ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the
differentiation of CD8ⴙ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune
responsiveness in the absence of naive T cells in old age. The Journal of Immunology, 2005, 175: 1566 –1574.
The Journal of Immunology
1567
Materials and Methods
CFSE labeling
Volunteers
The fluorescent dye CFSE (Molecular Probes) was used to determine proliferation and differentiation. Cells were suspended in PBS at a concentration of 1 ⫻ 106/ml. An equal volume of 1 ␮M CFSE in PBS was added,
and cells were incubated in the dark at room temperature for 10 min.
Unbound or deacetylated CFSE was quenched by adding 5% FCS, followed by two washing steps with culture medium. CD8⫹CD45RO⫹
CD25⫹ or CD8⫹CD45RO⫹CD25⫺ T cells were cultured together with
autologous, irradiated (30 Gy) PBMCs as APCs (106 cells/well) at a density of 106 cells/well in 24-well plates and were stimulated with PHA (1
␮g/ml; Sigma-Aldrich) for 4 days. CFSE-labeled cells were costained with
PE-, PerCP-, and allophycocyanin-conjugated mAbs and analyzed on a
FACSCalibur flow cytometer.
Peripheral blood samples were obtained from a total of 19 apparently well,
healthy, elderly persons (mean age, 75 ⫾ 7 years; range, 64 – 85 years).
Only subjects who were known to have ⬎15% CD25⫹ cells in their CD8⫹
T cell population and to have a good humoral immune response after influenza vaccination were chosen for the present study. Before bleeding, a
health check was performed for each participant. All participants had given
their informed written consent, and the study was approved by the local
ethical committee. HLA typing was performed routinely. Serum EBV and
CMV IgG titers were analyzed by ELISA using the commercially available
kit ENZYGNOST (Dade Behring). Anti-hepatitis B core protein and antihepatitis B surface protein Abs were determined using a microparticle enzyme immunoassay in an AXSYM Diagnostic unit (Abbott Laboratories).
Flow cytometry
Cell purification
Preparation of PBMCs was performed by density gradient centrifugation
(Ficoll-Hypaque; Amersham Biosciences). CD8⫹CD45RO⫹CD25⫹ and
CD8⫹CD45RO⫹CD25⫺ T cells were enriched from PBMCs in a series of
separations using magnetic beads as described previously (17). Briefly,
PBMCs were first depleted of naive and NK cells by the application of
CD45RA and CD56 microbeads (Miltenyi Biotec) and a LD depletion
column (Miltenyi Biotec). Subsequently, CD8⫹ T cells were positively
selected using CD8 MultiSort microbeads (Miltenyi Biotec). After removal
of the CD8 MultiSort microbeads, the CD8⫹ T cells were stained with an
allophycocyanin-conjugated mAb recognizing CD25 (BD Pharmingen).
Cells were washed twice with PBS supplemented with 0.5% BSA and 2
mM EDTA, pH 7.2, and CD25⫹ cells were obtained by positive selection
using anti-allophycocyanin microbeads (Miltenyi Biotec) and applying a
LS column (Miltenyi Biotec). The purity of the obtained population was
⬎90%. The CD25⫺ fraction was depleted of residual CD25⫹ cells in an
additional LD column (Miltenyi Biotec), resulting in a purity of ⬎95%. We
also performed cell sorting of PBMCs by staining with mAbs for CD45RA
(FITC), CD3 (PE), CD4 (PE-Cy7), and CD25 (allophycocyanin) using a
FACSVantage (BD Biosciences). The purity of the CD25⫹ and CD25⫺
CD8⫹CD45RO⫹ populations assessed by FACS was ⬎93%.
[3H]Thymidine incorporation
All cell culture experiments were performed in RPMI 1640 (Invitrogen
Life Technologies) supplemented with 10% FCS (Sigma-Aldrich) and 1%
penicillin-streptomycin (Invitrogen Life Technologies). Isolated
CD8⫹CD45RO⫹CD25⫹ or CD8⫹CD45RO⫹CD25⫺ T cells were cultured
together with autologous irradiated (30 Gy) PBMCs as APCs (105 cells/
well) at a density of 105 cells/well in 96-well plates and were stimulated
with anti-CD3 mAb (OKT3; 30 ng/ml; Orthoclone; Transplant), OKT3 and
rIL-2 (20 ng/ml; Novartis), or rIL-2 alone. For MLR cultures, irradiated (30
Gy) allogeneic PBMCs were used instead. The growth status of the cells
was investigated daily using the color of the medium as an indicator and
microscopic analysis. The viability of the cells was determined by trypan
blue staining on the last day of culture. There was virtually no difference
between CD25⫹ and CD25⫺ CD8⫹CD45RO⫹ T cells. After 1 wk of culture, the proliferation rate was determined by quantification of DNA synthesis after incubation with 1 ␮Ci [3H]thymidine (ICN Pharmaceuticals)
for the last 8 h of culture. Cells were harvested onto glass-fiber filters
(Wallac), and [3H]thymidine incorporation was measured using a liquid
scintillation counter. The results were expressed as the mean cpm ⫾ SD of
triplicate analyses.
4
Abbreviations used in this paper: CRTH2, chemoattractant receptor of Th2 cell;
flow FISH, flow cytometric fluorescence in situ hybridization; HBV, hepatitis B virus;
PNA, peptide nucleic acid; sjTREC, signal-joint TCR rearrangement excision circle.
To determine the expansion potential of CD8⫹CD45RO⫹CD25⫹ and
CD8⫹CD45RO⫹CD25⫺ T cells upon stimulation with specific Ags, cells
from HLA-A2-positive donors were seeded at a concentration of 2.5 ⫻ 105
cells/well together with the same number of autologous, irradiated (30 Gy)
PBMCs as APCs, antigenic peptides (2 ␮g/ml), and rIL-2 (20 ng/ml). All
subjects had positive EBV, but negative anti-HBc and anti-HBs, Ab serology. Only one person had a positive CMV Ab serology. The peptides used
were FLPSDFFPSV (hepatitis B virus (HBV) core), CLGGLLTMV
(EBVLMP2; Proimmune), GILGFVFTL (influenza virus matrix FLU
M158 – 66; Fundacion Instituto de Inmunologia de Colombia), and NLVPMVATV (human CMVpp65; Bachem). After 1 wk of culture, the cells were
harvested, and the frequencies of Ag-specific cells in the two subpopulations were determined by tetramer staining (Proimmune). Propidium iodide
(1 ␮g/ml; BD Pharmingen) was added directly before analysis to exclude
dead cells.
RNA isolation and TCR CDR3 spectratyping
RNA isolation and first-strand cDNA synthesis were performed as previously described (8). Briefly, total RNA was extracted from purified
CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T cells using TriReagent (Sigma-Aldrich). Glycogen (Roche) was added as a carrier for
RNA precipitation at a concentration of 1 ␮g/ml. Total RNA (1 ␮g) was
used for first-strand cDNA synthesis using a RT system (Promega). TCR
V␤ transcripts were amplified by PCR using a HotStart Taq Master Mix kit
(Qiagen) and primers (MWG) specific for each of the human V␤ families
and a specific primer for the C region of the ␤-chain (labeled with the
fluorescent dye marker 6-FAM) as described previously (18). After an
initial incubation at 95°C for 15 min, optimal cycling conditions were 95°C
for 1 min, 58°C for 1 min, and 72°C for 1 min for 34 cycles, followed by
a final extension period at 72°C for 20 min. For analysis of CD25 mRNA
expression, additional RNA from PBMCs, stimulated with PHA for 48 h,
was used for control purpose. PCR amplification was performed using a
CD25 forward primer (5⬘-GAG AAA GAC CTC CGC TTC AC-3⬘) and
reverse primer (5⬘-CGA GTG GCT AGA GTT TCC TG-3⬘; MWG) (19)
and 31 cycles.
CDR3 spectratyping was performed with some modifications as previously described (20, 21). An aliquot of the PCR product was diluted in 16
␮l of deionized formamide and 1.2 fmol of internal lane standard GeneScan-350 Tamra (PerkinElmer). The samples were denatured at 90°C for 2
min and snap-cooled on ice before loading onto a CE 310 Genetic Analyzer
(PerkinElmer). Each sample was injected for 5 s at 15 kV and electrophoresed for 24 min at 10 kV using a 36-cm capillary and POP4
(PerkinElmer). Analysis of the raw data was performed applying the
GeneScan 2.1 analysis software package (Applied Biosystems) with the
Local Southern method for fragment size estimation. In the case of a
normal distribution of individual clones within the V␤ family, a Gaussian
profile was depicted. The appearance of a dominant peak suggested the
presence of an oligoclonal or clonal T cell population. The occurrence of
dominant clonal expansion within the different V␤ families was quantified
using a diversity score between 1 and 3 (20); 1 was assigned to a Gaussian
distribution, 2 corresponded to a pattern with one to three peaks above the
Gaussian background, and 3 corresponded to one predominant peak above
the Gaussian distribution.
Sequencing and molecular tracking of TCR V␤ clones
cDNA of purified CD8⫹CD45RO⫹CD25⫺ T cells was used to amplify the
V␤3 and V␤16 regions including a single predominant peak. For ease of
ligation into a sequencing vector, the V␤3 and V␤16 primers were synthesized with EcoRI restriction sites at their 5⬘ terminus. Hence, the purified PCR products were digested with EcoRI before subcloning into pBSSK⫹. Sequence analyses were performed by MWG, and adequate primers
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Immunofluorescence surface staining was performed by adding a panel of
conjugated mAbs to freshly prepared PBMCs. The Abs used were CD3
(FITC or PE), CD4 (PE-Cy7 or allophycocyanin), CD8 (PerCP), CD25 (PE
or allophycocyanin; clone 2A3), CD28 (PE or allophycocyanin), CD44
(FITC), CD45RO (FITC, PE, or allophycocyanin), CD62L (allophycocyanin), CCR7 (PE), HLA-DR (PE), CD94 (FITC), CD95 (PE), NKB1
(FITC), CD158a/h (FITC), and CD158b/j (FITC; all from BD Pharmingen)
and chemoattractant receptor of Th2 cells (CRTH2;4 (PE; Miltenyi Biotec).
After staining, cells were fixed in 2% formaldehyde, and fluorescence was
measured with a FACSCalibur flow cytometer (BD Pharmingen).
Tetramer staining
1568
CD25-EXPRESSING CD8⫹ T CELLS IN OLD AGE
detecting the predominant clone within TCR V␤3 (5⬘-CAG TTT CGG
CGG GGG GGC CAC-3⬘) and V␤16 (5⬘-CAG CAG CCA AGA GGG AAG
ACA GTA C-3⬘) were chosen using the ClustalW program (具www.ebi.ac.uk/
clustalw/典)
(22, 23). To determine whether these predominant clones also occur in CD25⫹
T cells, RNA and cDNA preparations of purified CD8⫹CD45RO⫹CD25⫹
were performed. A first round of PCR using the same conditions as those
described above was performed before amplification using the two clonotypespecific primers. The amplification products were analyzed by 2.5% agarose
gel electrophoresis.
Analysis of signal-joint TCR rearrangement excision circles
(sjTRECs)
Telomere length analysis by flow cytometric fluorescence in situ
hybridization (flow FISH)
The telomere lengths of CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T
cells were determined using flow FISH with a fluorescent-labeled peptide nucleic
acid (PNA) telomere probe. The lymphoblastic leukemia T cell line 1301,
which has unusually long telomeres and is tetraploid (26), was used as a
standard for each telomere length measurement (27). The lymphocytes
were fixed and permeabilized using the standard procedure with the Cytofix/Cytoperm kit (BD Biosciences). Three milliliters of the Cy5-labeled
PNA telomere probe (Applied Biosystems) or an equivalent quantity of
correspondingly labeled control Ab (BD Pharmingen) was added to hybridization buffer. The cells were incubated in a hybridization buffer containing 70% formamide at 82°C for 10 min, then snap-cooled on ice and
incubated at room temperature for 90 min. After three rounds of washing
in a posthybridization buffer and three rounds in PBS, the fluorescence of
the Cy5-labeled PNA telomere probe was analyzed for the two T cell
subsets on a FACSCalibur flow cytometer. Together with the fluorescence
of the lymphoblastic leukemia cell line 1301, the individual telomere
length was calculated.
Statistical analysis
An independent-samples t test was performed to compare
CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T cells using SPSS
software for Windows version 11.5. A value of p ⬍ 0.05 was considered
statistically significant.
Results
CD8⫹CD25⫹ T cells have an early memory surface phenotype
and can coexpress CD4
As a first step we analyzed CD25 mRNA expression in
CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T cells after cell sorting, as described in Materials and Methods (Fig. 1A).
CD8⫹CD25⫹ cells displayed a constitutive expression of CD25
mRNA, whereas CD25⫺ cells failed to do so (Fig. 1B). CD25
mRNA analysis was performed using PCR and CD25-specific
primers (15). Surface phenotype analysis of CD25⫹ and CD25⫺
CD8⫹ T cells by four-color flow cytometric analysis additionally
revealed that both subpopulations were Ag-experienced T cells,
because they expressed CD45RO, CD44high, and CD95 (Fig. 1D).
CD25⫹ T cells also expressed the lymph node-homing markers
CD62L and CCR7, thus displaying a central memory-like phenotype, whereas CD25⫺ T cells were CCR7⫺ and had mostly a low
expression of CD62L. In contrast to CD25⫺ cells, a major fraction
of CD25⫹ cells expressed CRTH2, indicating a Tc2 phenotype. A
FIGURE 1. Phenotypic characterization of CD8⫹CD25⫹ and CD8⫹
CD25 ⫺ T cells. A, CD8 ⫹ CD45RO ⫹ CD25 ⫹ and CD8 ⫹ CD45RO ⫹
CD25⫺ T cells were isolated from elderly persons using magnetic beads as
described in Materials and Methods. Only persons with ⬎15% CD25expressing CD8⫹ T cells, who characteristically have a good humoral
immune response after influenza vaccination, were studied. B, CD25
mRNA expression of purified CD25⫹ and CD25⫺ T cells was analyzed by
PCR using CD25-specific primers. Stimulated PBMCs were used as a
positive control. C, Surface expression of CD8 vs CD4 on purified CD8⫹
CD45RO⫹CD25⫹ T cells. One of four experiments is shown. D, Surface
expression of CD62L, CCR7, CD44, CD95, CD57, HLA-DR, CRTH2, and
CD4 on CD8⫹CD45RO⫹CD25⫹ (black line) and CD8⫹CD45RO⫹CD25⫺
T cells (gray line). One of eight experiments is shown.
substantial proportion of CD8⫹CD25⫹ cells also coexpressed the
CD4 molecule. These cells truly expressed a CD4⫹CD8⫹ doublepositive phenotype, because there were no contaminating
CD4⫹CD8⫺ T cells (Fig. 1C). Because CD25 and CD4 surface
molecules are thought to be increased only on activated CD8⫹
cells, we determined additional activation markers. However,
CD8⫹CD25⫹ T cells did not express the early activation marker
CD69, the chronic activation marker HLA-DR, or the late differentiation marker CD57 (Fig. 1D). They were also negative for
receptors such as CD56, CD16, CD94, NKB1, CD158a/h, and
CD158b/j and were thus not NK T cells.
CD8⫹CD25⫹ cells are not gut-derived and have a shorter
replicative history than their CD8⫹CD25⫺ counterparts
To exclude that the two memory CD8⫹ populations were gut-derived,
we analyzed the expression of CD8␣␤ and the occurrence of sjTRECs
in sorted CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T
cells. Double staining of CD8⫹CD25⫹ and CD8⫹CD25⫺ T cells
with mAbs against the ␣ and ␤ CD8 receptor chains revealed that
all cells within each subpopulation were ␣␤ double-positive (Fig.
2A). None of the cells had a CD8␣␣ phenotype, which would have
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DNA isolation of purified CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺
T cells was performed using the Puregene DNA Purification Kit (Gentra Systems)
as previously described (24). The sjTRECs of CD8⫹CD45RO⫹CD25⫹ and
CD8⫹CD45RO⫹CD25⫺ T cells were amplified and directly quantified using by Light Cycler (Roche) using known starting numbers of standard
sjTREC molecules. Then real-time PCR was performed using Quatitect
SYBR Green (Qiagen) as previously described (25). Briefly, 100 ng of
DNA were added to a mix of 1⫻ Quatitect SYBR Green PCR Master Mix,
200 ng/ml BSA, 1.5 mM MgCl2, 0.5 ␮M forward primer (5⬘-AGG CTG
ATC TTG TCT GAC ATT TGC TCC G-3⬘), and 0.5 ␮M reverse primer
(5⬘-AAA GAG GGC AGC CCT CTC CAA GGC AAA-3⬘). The PCR conditions were an initial activation step at 95°C for 15 min, followed by 40 cycles
of denaturation at 95°C for 5 s, annealing at 60°C for 25 s, extension at
72°C for 20 s, and a fluorescence acquisition step at 84°C for 5 s.
The Journal of Immunology
1569
thymus derived (data not shown). The sjTREC concentrations varied from person to person, but were generally relatively low. The
number of sjTRECs in CD25⫹ T cells was, on the average, still 3.5
times higher than that in CD25⫺ cells, suggesting a shorter replicative history of this specific T cell subset. This concept was
also supported by the telomere length analysis of sorted
CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T cells using flow FISH technology. In all subjects studied, CD25⫹ T cells
had longer telomeres than CD25⫺ T cells (Fig. 2B).
⫹
⫹
indicated that they were derived from bone marrow and had undergone thymus-independent differentiation in the gut mucosa
(28). SjTRECs are stable DNA episomes generated principally
during the process of TCR gene rearrangement in the thymus.
They are extrachromosomal, not replicated during mitosis, and
thus are diluted with each cell division (24). The sjTRECs were
detected in both T cell subsets, suggesting that they were both
FIGURE 3. Proliferation and differentiation of CFSE-labeled CD8⫹CD25⫹
and CD8⫹CD25⫺ T cells. Purified
CD8⫹CD45RO⫹CD25⫹ (A and C) and
CD8⫹CD45RO⫹CD25⫺ T cells (B and
D) from elderly persons with ⬎15%
CD25-expressing CD8⫹ T cells, who
characteristically have a good humoral immune response after influenza vaccination, were labeled using
the fluorescent dye CFSE and analyzed before (A and B) and after 4
days of stimulation with PHA (C and
D) with the aid of a FACSCalibur.
Cells were costained using mAbs
against CD4, CD28, CD62L, and
HLA-DR. Numbers in the graphs indicate the percentage for the respective quadrant. One of three experiments is shown.
Having established that CD8⫹CD25⫹ T cells were thymus derived
and had a shorter replicative history than their CD25⫺ counterparts, we next aimed at determining their differentiation and proliferation profile upon stimulation. Cell proliferation tracking of
purified CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T
cells was performed using the fluorescent dye CFSE, which penetrates cell membranes and couples to proteins resulting in stable,
long-term intracellular retention. CFSE segregates equally between daughter cells upon cell division, resulting in a 2-fold decrement in cellular fluorescence intensity. Upon stimulation with
PHA, both subsets divided up to five times and retained their
CD45RO (data not shown) and CD28 (Fig. 3) phenotypes. Sixtyfive percent of the CD25⫹ T cells down-regulated their initially
high CD62L expression, but did not become HLA-DR⫹ as 13% of
the CD25⫺ T cells did. CD8 single-positive CD25⫹ as well as
CD25⫺ T cells did not up-regulate the CD4 molecule upon stimulation, whereas CD8⫹CD4⫹CD25⫹ T cells maintained their
CD4⫹CD8⫹ double-positive phenotype and proliferated as well as
CD8 single-positive cells.
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FIGURE 2. CD8 CD25 T cells are not gut-derived and have longer
telomeres than CD8⫹CD25⫺ T cells. CD8⫹CD45RO⫹CD25⫹ and
CD8⫹CD45RO⫹CD25⫺ T cells were isolated from persons with ⬎15%
CD25-expressing CD8⫹ T cells, who characteristically have a good humoral immune response after influenza vaccination. A, Double staining was
performed using mAbs against the ␣ and ␤ CD8 receptor chains. All cells
within each subpopulation were ␣␤ double positive. One of eight experiments for CD8⫹CD45RO⫹CD25⫹ cells is shown. B, Telomere length was
determined for the sorted subpopulations using a fluorescent peptide nucleic acid probe and flow FISH as described in Materials and Methods. The
resulting fluorescent signal (gray curves) was blotted on a logarithmic scale
against the signal from the leukemia T cell line 1301 (empty curves). Telomere length was calculated against the known telomere length of this cell
line. One of three experiments performed is shown.
Upon PHA stimulation, CD8⫹CD25⫹ T cells proliferate and
partially down-regulate CD62L, but retain their HLA-DR⫺
CD4⫹ phenotype
1570
CD25-EXPRESSING CD8⫹ T CELLS IN OLD AGE
FIGURE 4. In vitro growth of CD8⫹CD25⫹ and
CD8⫹CD25⫺ T cells. A, Isolated CD8⫹CD45RO⫹CD25⫹
and CD8⫹CD45RO⫹CD25⫺ cells from three elderly donors
with ⬎15% CD25-expressing CD8⫹ T cells, who characteristically have a good humoral immune response after influenza vaccination, were stimulated with IL-2, irradiated, HLA-mismatched, allogeneic PBMCs, OKT3,
or OKT3 in combination with IL-2 as described in Materials and Methods. After 1 wk of culture, [3H]thymidine incorporation was measured. The results are expressed as the mean cpm ⫾ SD from three independent
experiments. ⴱ, p ⬍ 0.05, CD25⫹ vs CD25⫺ cells. B, On
the last day of culture, a photograph of CD25⫹ and CD25⫺
cells was taken to demonstrate the different growth characteristics after stimulation with OKT3 and IL-2.
We also investigated the proliferative capacity of purified
CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ cells upon
stimulation with IL-2, alloantigen, OKT3, and a combination of
OKT3 and IL-2. CD8⫹CD25⫹ T cells grew substantially better
than their CD25⫺ counterparts when stimulated with IL-2 alone or
alloantigen (Fig. 4A). However, CD25⫹ cells grew less well than
CD25⫺ cells when stimulated with OKT3 or a combination of
OKT3 and IL-2. The two subsets also had different growth patterns
after stimulation with PHA or OKT3 and IL-2. Although CD25⫹
cells had a polyclonal proliferation pattern leading to the formation
of many small cell clusters, only a few large clones were observed
in the CD25⫺ population (Fig. 4B).
FIGURE 5. CD8⫹CD25⫹ T cells
recognize a panel of different Ags.
Isolated CD8⫹CD45RO⫹CD25⫹ and
CD8⫹CD45RO⫹CD25⫺ cells from
HLA-A2-positive elderly persons
with ⬎15% CD25-expressing CD8⫹
T cells who characteristically have a
good humoral immune response after
influenza vaccination were stimulated with immunodominant peptides
derived from HBV, influenza virus,
EBV, or CMV (2 ␮g/ml) in the presence of IL-2 (20 ng/ml). Irradiated
autologous PBMCs were used as
APCs. After 1 wk, the cells were
stained for CD8, and the frequency of
Ag-specific cells was analyzed using
tetramer staining. A, The graph
shows a representative FACS profile.
Numbers in the graphs indicate the
percentages of Ag-specific cells after
culture. B, CD25⫹ and CD25⫺ T cell
responses to antigenic peptide stimulation in different subjects. Three experiments were performed for each
Ag. Results from subjects who did
not have a response to the specific Ag
were not included in the graphs.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
CD8⫹CD25⫹ T cells proliferate better upon stimulation with
IL-2 or alloantigen than their CD25⫺ counterparts
The Journal of Immunology
1571
CD8⫹CD25⫹ T cells recognize a panel of different Ags
To obtain additional information about the Ag reactivity of
CD8⫹CD25⫹ T cells, we compared the propagation of cells of
different Ag specificities in CD8⫹CD45RO⫹CD25⫹ and
CD8⫹CD45RO⫹CD25⫺ T cells by stimulating the two subpopulations with a panel of antigenic peptides (Fig. 5). The HBV core
protein-derived peptide FLPSDFFFPSV induced a weak response
in CD25⫹ and no response in CD25⫺ cells. Stimulation of CD25⫹
cells with the influenza virus matrix protein M1-derived peptide
FLU M158 – 66 led to the propagation of a relatively large number
of Ag-specific cells. In contrast, only a few M158 – 66 peptide-specific cells were found when the CD25⫺ population was stimulated
with the M158 – 66 peptide. Stimulation with immunodominant peptides from EBV (LMP2) and CMV (pp65) led to the propagation
of T cells with respective Ag specificities in both subpopulations.
T cell expansion in response to these two Ags, however, was more
pronounced in the CD8⫹CD25⫺ than in the CD8⫹CD25⫹ subpopulation. It was of interest that most of the elderly persons selected for the present study on the basis of a relatively high number
of CD25⫹ T cells in their CD8⫹ population did not have
CMVpp65-specific cells. This is in agreement with recently published data showing that CD8⫹CD25⫹ cells rarely occur in subjects with latent CMV infection (15).
CD8⫹CD25⫹ cells have a highly diverse TCR repertoire
To analyze the clonal composition of the CD8⫹CD45RO⫹CD25⫹
and CD8⫹CD45RO⫹CD25⫺ T cell subsets, we used immunoscope technology. TCR CDR3 spectratyping demonstrated that the
CD25⫹ population was mostly polyclonal, whereas the CD25⫺
subset had a more restricted clonality (Fig. 6A). Peaks of a certain
size, which were dominant in the CD25⫺ population, could frequently also be identified as slightly enlarged in the CD25⫹ fraction. In the CD25⫹ population, 83 ⫾ 5% of the amplified V␤
FIGURE 7. The same clones that dominate the CD8⫹CD25⫺ T cell
repertoire also occur in the CD8⫹CD25⫹ T cell population. A, Having
analyzed the clonal composition of CD8⫹CD45RO⫹CD25⫹ and
CD8⫹CD45RO⫹CD25⫺ T cells by CDR3 spectratyping (Fig. 6), we performed molecular tracking of dominant clones. We therefore chose two V␤
regions, displaying a dominant clone within the CD25⫺ population, and
performed sequence analysis to ascertain the identities of these dominant
clones and to create adequate primers. Arrows indicate clones with the
same CDR3 length, which occur in the CD25⫹ as well as in the CD25⫺
population. B, Clone V␤3- and clone V␤16-specific primers were then
used to amplify the cDNA of CD25⫹ and CD25⫺ T cells. PCR products
were analyzed by 2.5% agarose gel electrophoresis.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 6. CD8⫹CD25⫹ T cells have a highly diverse repertoire. A, The clonal composition of the 24 V␤ families was analyzed by CDR3 spectratyping
in isolated CD8⫹CD45RO⫹CD25⫹ and CD8⫹CD45RO⫹CD25⫺ T cells. Only persons with ⬎15% CD25-expressing CD8⫹ T cells, who characteristically
have a good humoral immune response after influenza vaccination, were studied. Eight randomly chosen V␤ families are shown. The graph represents one
of three identical experiments. B, Deviations from the Gaussian profile were classified using a diversity score (of 1–3). The number of V␤ families
corresponding to a diversity score of 1, 2, or 3 was assessed in CD25⫹ and CD25⫺ cells in three different individuals. In each person the number of clones
of one diversity type was expressed as a percentage of all 24 V␤ families (100%). The bars represent mean values of the percentage of families with a certain
diversity score in three subjects. CD25⫹ and CD25⫺ T cells are compared.
1572
families had a Gaussian or close to Gaussian CDR3 size distribution pattern (diversity score, 1), 14 ⫾ 6% had minor deviations,
and 3 ⫾ 2% had pronounced deviations from the Gaussian profile
(Fig. 6B). In contrast, a Gaussian profile was found in only 24 ⫾
8% of V␤ families in the CD25⫺ population, whereas 42 ⫾ 3%
displayed minor deviations from the Gaussian profile (diversity
score, 2), and 34 ⫾ 10% were dominated by a single dominant
peak (diversity score, 3).
The same clones that dominate the CD8⫹CD25⫺ T cell
repertoire also occur in the CD8⫹CD25⫹ T cell population
Discussion
We recently reported that IL-4-producing CD8⫹ T cells with a
CD25⫹ memory phenotype accumulate in a subgroup of healthy
elderly persons who still have an intact humoral immune response
after influenza vaccination (17). These apparently beneficial
CD8⫹CD25⫹ T cells are rare or even absent in elderly persons
with latent CMV infection, who characteristically have high percentages of CD8⫹CD28⫺ effector cells (15, 29 –31). CD8⫹CD25⫹
T cells are also rare in young individuals who still have high numbers of naive T cells (15, 17). This indicates that shrinkage of the
naive T cell population could increase the available space and
facilitate nonspecific memory T cell turnover (29), resulting in a
larger CD8⫹CD25⫹ memory T cell population in elderly adults
(15, 17). However, a change in the composition of the CD8⫹ T cell
pool toward cells of a higher differentiation stage in the elderly
(14, 32, 33), as observed in patients with latent CMV infection,
negatively correlates with the occurrence of CD8⫹CD25⫹ cells
(15). CD8⫹CD28⫺ T cells that expand in response to persistent
antigenic challenge thus seem to have a survival advantage over
CD25⫹ memory T cells (34, 35), indicating loss of diversity and
loss of intact memory in the presence of chronic antigenic
stimulation.
The analysis of CD8⫹CD25⫹ and CD8⫹CD25⫺ memory T
cells in the present study provides novel insights into TCR repertoire diversity, replicative history, and lineage relationship of
CD8⫹ T cells in old age. CD8⫹CD25⫹ T cells display a central
memory-like phenotype, as shown by their expression of
CD45RO, CD28, and the lymph node-homing markers CD62L and
CCR7. These cells display a constitutive expression of the IL-2R
␣-chain (CD25) and CD25 mRNA. They have not been recently
activated, because they do not express CD69 and HLA-DR. Apart
from regulatory T cells, CD25 expression has been detected on
human CD4⫹CRTH2⫹ cells (36). CD25 has also been reported to
be expressed at higher levels on Th2 than on Th1 cells upon stimulation (37). Moreover, IL-2, apart from IL-4, plays a central role
in Th2 differentiation and has been shown to stabilize the accessibility of the Il4 gene (38). Thus, constitutive expression of the
high affinity IL-2R on memory Tc2 cells may have a functional
assignment in the maintenance of type 2 cells and seems not to be
exclusively expressed on regulatory T cells. Furthermore,
CD8⫹CD25⫹ T cells grow well after stimulation with IL-2 only.
This may allow them to survive and divide in a bystander manner
in vivo, even in the absence of their specific Ag. IL-7 and IL-15
could play additional roles in this process (34) as could IL-4 and
IL-9 (39). Turnover in response to nonspecific stimuli allows
CD8⫹CD25⫹ cells to persist for extended periods and ensures the
maintenance of a diverse memory T cell pool in old age. Due to
their IL-4 production, CD8⫹CD25⫹ cells can prevent loss of the
CD28 molecule (40), assist memory cell generation (41), induce
MHC class II up-regulation on Ag-presenting B cells, and promote
Ab isotype switching to IgG1 and IgE (42).
Phenotypic characterization revealed that up to 20% of the
CD8⫹CD25⫹ T cell population coexpressed the CD4 molecule.
The origin, function, and role of circulating CD4⫹CD8⫹ T cells
are still a matter of debate. However, the expression of the CD8␣␤
molecule suggests that CD4⫹CD8⫹ double-positive T cells were
not derived from the gut, but were most likely thymus derived.
Performing cell proliferation tracking using the fluorescent dye
CFSE, we demonstrated that purified memory CD8⫹ T cells do not
acquire CD4 coexpression upon stimulation with PHA (Fig. 3) or
OKT3 and IL-2 (unpublished observation), but maintain their double-positive phenotype within the CD8⫹CD25⫹ population. It is
therefore likely that peripheral double-positive cells represent a
distinct memory T cell population that increases with age (43– 45).
Due to the coexpression of CD4, double-positive cells can enhance
the interaction with APCs by serving as an adhesion and a costimulatory molecule, interacting with MHC class II (46).
We also demonstrate that the TCR repertoire of CD8⫹CD25⫹
memory cells is highly diverse, whereas the CD8⫹CD25⫺ subset
has a more restricted clonality. This is in agreement with previous
results showing that CD8⫹ central memory cells have a greater
TCR diversity than CD8⫹ effector memory cells (47). Together
with telomere length analysis, our results demonstrate a shorter
replicative history for CD8⫹CD25⫹ T cells. This has also been
shown with respect to Ag specificity, because CD8⫹CD25⫹ T
cells contain greater numbers of different specificities, which proliferate rapidly upon Ag encounter (Fig. 5). Our results suggest
that continuous differentiation of CD8⫹ T cells throughout life
leads to the loss of certain Ag specificities, but to the unproportional accumulation of others, for instance for persistent Ags, and
appears to be associated with the inability to raise a sufficient cellular immune response to pathogens such as influenza (11, 17).
Moreover, it seems likely that the relatively oligoclonal
CD8⫹CD45RO⫹CD25⫺ T cell population contains precursors of
the relatively nonresponsive effector cells described by many
groups (7, 48). Studies presently being performed in our laboratory
are aimed at elucidating this possibility.
We also performed molecular tracking of clones, a technique
used for the identification of putatively pathogenetic T cell clones
in aplastic anemia (23) or rheumatoid arthritis (49) and to monitor
the persistence and localization of adoptively transferred T cells in
tumor immunotherapy (50). We used this method to ascertain that
clones dominating certain TCR V␤ families within the
CD8⫹CD25⫺ population also occur in the CD8⫹CD25⫹ subset.
The data shown in Fig. 7 are in accordance with our results
achieved by CDR3 spectratyping and telomere length analysis and
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Using immunoscope technology, peaks with the same CDR3
length were found in both CD8⫹CD25⫹ and CD8⫹CD25⫺ cells
(indicated by arrows in Fig. 7A), but within some V␤ families one
such clone dominated only the CD8⫹CD25⫺ population, whereas
the CD8⫹CD25⫹ population displayed a polyclonal repertoire. We
therefore wanted to ascertain the identity of these dominant clones
by extracting the RNA of isolated CD8⫹CD45RO⫹CD25⫺ T cells
from a donor previously shown to exhibit such dominant clones
within the V␤3 and V␤16 regions (Fig. 7A). Sequencing of these
two TCR V␤ regions including the dominant clones was performed as described in Materials and Methods. Our results revealed that each of the two single peaks analyzed contained only
one T cell clone. Using clonotype-specific primers, these clones
were also detected in CD8⫹CD25⫹ T cells (Fig. 7B). The results
of molecular tracking of dominant clones demonstrate a lineage
relationship between CD25⫹ and CD25⫺ memory CD8⫹ T cells.
In view of the fact that CD25⫹ T cells have a larger TCR repertoire
and a shorter replicative history, we propose a differentiation pathway from CD25⫹ to CD25⫺ memory CD8⫹ T cells as they
proliferate.
CD25-EXPRESSING CD8⫹ T CELLS IN OLD AGE
The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
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enabled us to propose a lineage relationship from CD25⫹ to
CD25⫺ memory CD8⫹ T cells.
New vaccination approaches aimed at supporting the long-term
survival of immune-competent CD8⫹CD25⫹ memory T cells
should be considered, because they would have multiple beneficial
effects for the elderly population. Booster immunization approaches for elderly persons could, for instance, take advantage of
recent progress in DNA vaccine research. By inducing both humoral and cellular immune responses, cytokine DNA, especially
IL-2, but also IL-7 or IL-15, could be used as an adjuvant specifically targeting CD8⫹CD25⫹ memory T cells and thus augmenting immunity even in subjects with a low CD8⫹CD25⫹ T cell
frequency.
In conclusion, our results suggest that CD25-expressing CD8⫹
cells represent a memory T cell reservoir of great diversity in old
age. We propose that CD8⫹CD25⫹ cells represent an early stage
in the differentiation of CD8⫹ T cells and demonstrate a lineage
relationship from CD25⫹ to CD25⫺ memory CD8⫹ T cells. Loss
of the physiologically occurring CD8⫹CD25⫹ memory population
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