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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2860-2868
IMMUNOBIOLOGY
Shortage of circulating naive CD8+ T cells provides
new insights on immunodeficiency in aging
Francesco F. Fagnoni,
Rosanna Vescovini,
Giovanni Passeri,
Giovanni Bologna,
Mario Pedrazzoni,
Giampaolo Lavagetto,
Amos Casti,
Claudio Franceschi,
Mario Passeri, and
Paolo Sansoni
From the Department of Internal Medicine and Biomedical Sciences and
the Institute of Biological Chemistry, University of Parma, Parma,
Italy; the Department of Experimental Pathology, University of Bologna,
Bologna, Italy; and INRCA (the National Institute for
Research and Care for Elderly), Ancona, Italy.
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Abstract |
Clinical observations indicate that elderly people are prone to
severe, often lethal infectious diseases induced by novel pathogens.
Since the ability to mount primary immune responses relies on the
availability of naive T cells, the circulating naive T-cell reservoir
was evaluated throughout the human life span. Naive T cells were
identified as CD95 T lymphocytes for their phenotypic
and functional features. Indeed, the lack of CD95 marker is sufficient
to identify a population of naive T cells, as defined by coincidence
with previously characterized CD45RA+ CD62L+ T cells. Naive
CD95 T cells, as expected, require a costimulatory
signal, such as CD28, to optimally proliferate after anti-CD3
stimulation. Cytofluorimetric analysis of circulating T lymphocytes
from 120 healthy subjects ranging in age from 18 to 105 years revealed
that naive T cells decreased sharply with age. The younger subjects had
a naive T-lymphocyte count of 825 ± 48 cells/µL, and the
centenarians had a naive T-lymphocyte count of 177 ± 28 cells/µL.
Surprisingly, the naive T-cell count was lower in CD8+
than in CD4+ subsets at any age, and the oldest
individuals were almost completely depleted of circulating naive
CD8+ T cells (13 ± 4 cells/µL). Concomitantly, a
progressive expansion of CD28 T cells occurs with age,
which can be interpreted as a compensatory mechanism. These data
provide new insights into age-related T-cell-mediated immunodeficiency
and reveal some analogies of T-cell dynamics between advanced aging and
human immunodeficiency virus (HIV) infection. In conclusion, the
exhaustion of the naive CD8+ T-cell reservoir, which has
never been reported before, suggests that this T-cell pool is a major
target of the aging process and may define a parameter possibly related
to the life span of humans.
(Blood. 2000;95:2860-2868)
© 2000 by The American Society of Hematology.
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Introduction |
Many studies have suggested a correlation between
immune function and age-related risk of morbidity and mortality,
although the contribution of the immune system to survival and human
life span has not been fully elucidated.
Clinical evidence indicates that with advancing age, immune responses
against recall antigens may still be conserved,1 but the
ability to mount primary immune responses against novel antigens
declines significantly.2 The impaired ability to mount immune responses to new antigens may result in a high susceptibility to
infectious diseases and may limit the efficacy of vaccination strategies in elderly people. Although several age-related
modifications of the immune system have been described so far, no
single immune parameter seems to account for this age-related
failure.3-6
The immune responses to novel antigens rely on the availability of
naive T cells. In order to mount primary responses to new antigens even
in advanced age, ie, a long time after the onset of thymic
atrophy,7 naive T cells have to survive as long-lived resting cells from the time of their initial release from the thymus.
Based on this assumption, the life span of these T cells in humans
should reach several decades, especially when considering very old
people such as centenarians. Alternatively, it should be hypothesized
that in advanced age, naive T cells may still be provided by thymic
remnants8-10 and/or by other organs which take over the
thymus or by peripheral thymic-independent pathways.11 In
any event, the evaluation of a naive T-cell reservoir represents a
major task and should give insights into the mechanisms underlying the
changes occurring in the immune system with age.
The phenotypic identification of truly naive T cells is still
controversial. Several markers have been alternatively proposed for
this task. The minimal requirement for the phenotypical definition of
naive T cells seems to be the resting state, ie, lack of activation antigens and low-level expression of adhesion
molecules.12,13 On the other hand, the most reliable marker
to distinguish naive from nonnaive cells should be specifically and
irreversibly acquired (or lost) after productive
T-cell-receptor (TCR) engagement followed by clonal
expansion, but not after exposure to cytokines.14 Until a
few years ago, this requirement seemed to be met by CD45 isoforms,15 and indeed these markers have been widely used
for this purpose. However, recent findings indicate that the CD45RO isoform can back-revert to CD45RA,16,17 whereas CD45RO can be induced after prolonged exposure to interleukin-2 (IL-2) in vitro.18,19 These data indicate that the distinction
between naive and memory T cells, according to CD45 isoforms, is not
satisfactory. Recently, double labeling with CD45RA and CD62L was shown
to accurately identify naive T cells in HIV-infected
subjects,20-22 and currently this double staining procedure
may be considered as a standard for the phenotypic assessment of naive
T cells in humans.
In this paper we have focused on the CD95 activation antigen (Ag),
which is a member of the tumor necrosis factor/nerve growth factor-R
(TNF/NGF-R) family.23,24 Most of the literature on CD95 is
devoted mainly to cells expressing this molecule. CD95 is expressed on
peripheral blood T cells after TCR/CD3 stimulation in
vitro.25,26 Moreover, in vivo studies indicate that after antigen stimulation, T cells accumulate within the CD95+
subset.27 Furthermore, in adults, CD95 is preferentially
expressed by CD45RARO+ T cells but not
by CD45RA+RO T cells.27 An
increase of CD95+ T cells has been observed in
adulthood,28 aging,29-32 and pathological conditions such as HIV infection33 and systemic lupus
erythematosus (SLE).34 Thus, CD95 Ag expression might
reflect previous or ongoing in vivo antigen-specific activation, and
its role in physiological and pathological conditions has been
extensively studied. Surprisingly, very little is known regarding
lymphocytes that do not express CD95 and their biological role.
We have hypothesized that the absence of CD95 expression could
represent a novel tool able to discriminate naive T cells. This
possibility was tested according to the above-mentioned and restrictive
criteria to distinguish naive from nonnaive T cells in humans. Because
the lack of CD95 Ag resulted as a reliable marker of naive
T cells, we have evaluated the changes of naive CD95
T cells during aging. Our study demonstrates a decrease of naive T
cells within the CD4 subset and an almost complete loss of naive T
lymphocytes within the CD8 T-cell subset in old people, particularly in
centenarians. This exhaustion of naive T cells within the CD8 T cells
could account for the reduced competence to face new intracellular pathogens which occurs during senescence and may define an
"immunological clock" which is possibly correlated to the life
span of humans.
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Materials and methods |
Isolations of peripheral blood mononuclear cells and T
lymphocytes
Using a sample population of 120 healthy people ranging in age from
18-105 years, peripheral blood mononuclear cells (PBMCs) were obtained
by density gradient centrifugation (Ficoll-Hypaque; Sigma,
St. Louis, MO) from freshly drawn venous blood. All subjects were in
good clinical condition, and those older than 65 years were selected
according to the Senieur protocol.35 After washings with
phosphate-buffered saline (PBS), the cells were resuspended in RPMI
1640 medium supplemented with 10% fetal calf serum (FCS), 2 mmol/L
L-glutamine, 100 µg/mL streptomycin, and 100 units/mL penicillin
(complete medium). For proliferation tests, PBMCs were further
fractionated into T and non-T cells by a single-step rosetting method
using sheep red blood cells. Enriched T cells were further depleted of
adherent cells in plastic petri dishes at 37°C, 6% carbon dioxide
(CO2). The purity of T-cell preparation, assessed by
cytofluorimetric analysis, was greater than 95%. Further isolation of
the CD3+CD95 T-lymphocyte subset was
obtained by immunoselection using coated beads (Dynabeads M-450; Dynal,
Oslo, Norway) with polyclonal sheep antimouse immunoglobulin G (IgG). T
lymphocytes were depleted from CD95+ cells by magnetic
beads coated with purified anti-CD95 monoclonal antibody (mAb).
Isolated CD3+CD95 cells comprised more
than 98% CD3+ cells.
Monoclonal antibodies
Purified monoclonal antibody (mAb) anti-CD3 (clone OKT3) was kindly
provided by Dr E. G. Engleman (Stanford University, CA). Fluorescein
isothiocyanate-conjugated (FITC-conjugated) and
phycoerythrin-conjugated (PE-conjugated) mAbs recognizing CD45RA, CD2,
CD11a, CD62L, CD69, CD71, or HLA-DR (human leukocyte antigen-DR) were
purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA).
Purified anti-CD28 mAb was also obtained from Becton Dickinson. FITC-, PE-, or CyChrome-conjugated mAbs directed against CD3, CD4, CD8, CD95,
CD28, CD25, or CD44 were obtained from PharMingen (San Diego, CA). The
negative controls, of IgG1 or IgG2a isotype and of irrelevant specificity, were purchased from Becton Dickinson and from PharMingen. They were conjugated with either FITC-, PE-, or CyChrome and were used
to set the limits of non-specific immunoglobulin cell-binding by cells
stained with mAbs conjugated with the homologous fluorochrome.
Immunofluorescence and multiparameter flow cytometric analysis
PBMCs were resuspended in PBS containing 1% FCS, 1% human serum,
10% mouse serum, and 0.01% sodium azide, then stained for 30 minutes
on ice with combinations of saturating amounts of
fluorochrome-conjugated mAbs. After staining, cells were washed
extensively and analyzed. Flow cytometry was performed using a
fluorescence-activated fluorcytometer (FACScan, Becton Dickinson). To
obtain the percentages and the absolute numbers of different T-cell
subsets, we first determined the total number of lymphocytes per µL
of peripheral blood (Coulter Counter, Coulter Electronics, Hialeah,
FL). PBMCs were stained with FITC-conjugated anti-CD95, PE-conjugated
anti-CD4 or anti-CD8, and CyChrome-conjugated anti-CD3. For each
sample, data from 104 viable cells were collected and
analyzed by flow cytometry, and lymphocytes were identified by
characteristic forward angle and side scatter profiles. The percentage
of CD3+, CD3+CD4+, and
CD3+CD8+ cells among lymphocytes allowed us to
obtain the absolute number of total T lymphocytes, CD4 T cells, and CD8
T cells, respectively. Thereafter during analysis, an electronic gate
placed on CD3+ cells and the correlated expression of CD4
or CD8 cells versus CD95 cells allowed us to obtain the percentages and
calculate the absolute numbers of CD95+ and
CD95 cells within CD3+,
CD3+CD4+, or CD3+CD8+.
For other subset frequencies, PBMCs were stained with
CyChrome-conjugated anti-CD3, anti-CD4, or anti-CD8 mAbs and different combinations of FITC- and PE-conjugated mAbs. To define single subsets
we used an acquisition gate of 104 viable cells, uniquely
identifying CD3+, CD4+, or (to
exclude CD3CD8+ cells) CD8hi
T cells. Quadrants and gates were set based on isotype control mAbs
(not shown in the figures). Samples were acquired and analyzed at
different times for different donors; thus, given the variation of
background intensity, quadrant and gate positions vary among donors.
The absolute number of individual CD3, CD4, or CD8 T-cell subpopulations expressing a particular phenotype were calculated by
multiplying their percentages derived from FACS gating by the absolute
count per µL of each population.
Proliferation assay
For proliferation assays, purified total T cells and isolated
CD95 T cells were stimulated with immobilized mAbs
as follows. In 96-well round-bottom microtiter plates, either 12.5, 25, or 50 ng/mL anti-CD3 alone or 1 ng/mL anti-CD3 plus 200 ng/mL anti-CD28 were added to PBS. After incubation at room temperature for 90 minutes,
the wells were washed with PBS, and 1 × 105 cells
were added to 0.2 mL complete medium. Triplicate well cultures were
incubated for 3 days, and 0.0185 MBq of
3H-thymidine (3H-TdR) was added to each well 18 hours before cell harvesting on glass fiber filter paper. Uptake of
3H-TdR was measured in a liquid scintillation counter, and
the results were expressed as the mean counts per minute plus or minus standard error of the mean (cpm ± SEM).
Statistical analysis
Comparisons between groups of different ages were done by ANOVA.
Changes of percentages and absolute numbers of cells with age were
tested by simple regression analysis, and relationships between
variables were tested by multiple regression analysis. Statistical
analysis was conducted using Statistical Package for Social Sciences
for Windows (SPSS, Chicago, IL), and P < .05 was considered significant.
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Results |
Absence of the CD95 Ag as a marker for the phenotypic identification
of CD45RA+CD62L+ naive T cells
Using flow cytometry, we analyzed freshly isolated PBMCs in 3-color
fluorescence with CD45RA and CD62L coexpression (or CD11a, not shown)
either with CD4 or CD8 T cells. Evaluation of CD45RA and CD62L
coexpression, which may be considered the standard phenotype to define
naive T cells, was tested for the first time in donors of different
ages. The results in Figure 1, from
stainings of a representative 18-year-old and an 80-year-old subject,
indicate that double-positive naive T cells were reduced in old
subjects (40% versus 77% among CD4+ T cells, and 5.5%
versus 42% among CD8+ T cells). Moreover, the percentage
of these cells was higher within the CD4 subset than within the CD8
subset in both age groups. Enumeration of circulating naive T cells by
this method indicated that old people maintain less naive T cells than
was previously reported following staining with the classical marker
CD45RA. In fact in both subjects, considering CD45RA+
within CD8+ cells, a large subset of CD45RA+ T
cells not stained by CD62L was evident (Figure 1).
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| Fig 1.
Evaluation of CD45RA+CD62L+
naive T cells in donors of different ages.
Freshly isolated PBMCs were stained with the following combinations of
FITC-, PE-, and CyChrome-conjugated mAbs: CD45RA, CD62L, and CD4 or
CD8. A representative comparison of 5% probability contour plots from
a young (18-year-old) and an old (80-year-old) subject is shown. An
electronic gate was set on CD4+ or CD8+ T
lymphocytes as indicated, and expression of CD45RA (X axis) was
correlated to CD62L (Y axis). In both the CD4 and CD8 subsets,
CD45RA+CD62L+ (double-positive) naive T cells
(upper-right box) were profoundly reduced in the old subject compared
with the young subject; within each of these subjects, the percentage
of double-positive cells was higher in the CD4 subset than in the CD8
subset. Moreover, within the CD8 T cells, the proportion of
CD45RA+CD62L cells
(lower-right box) showed an important age-dependent increase.
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Looking for an alternative single naive T-cell marker, we tested
whether, in addition to the lineage markers, staining with CD95 alone
could be used instead of CD45RA/CD62L double staining. To this end,
using the same subjects, we tested CD95 coexpression with
CD45RA and CD62L in CD4 or CD8 subsets (Figure
2A and 2B). The results indicated that all
CD95 T cells express both CD45RA and CD62L (and low
levels of CD11a, not shown). These findings were unequivocal for any
donor tested and were detectable both in CD4 and CD8 T-cell subsets
(Figure 2A and 2B). Furthermore, because PBMC stainings shown (Figures 1, 2, and 3) were obtained from the same
representative donors, it must be noted that the percentages of
CD95 T cells were substantially coincident with
those of the double-positive CD45RA+CD62L+ T
cells. These same staining patterns were verified in 10 donors ranging
from 18-100 years of age (Table 1).
Globally, these results indicate that the absence of CD95 Ag can define
putative naive T cells currently identified by double staining with
CD45RA and CD62L.
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| Fig 2.
Analysis of CD45RA and CD62L expression versus CD95
expression in both CD4 and CD8 subsets.
Freshly isolated PBMCs were stained with the following combinations of
FITC-, PE-, and CyChrome-conjugated mAbs: CD95, CD45RA or CD62L, and
CD4 or CD8. Analysis of CD45RA and CD62L expressions (Y axis) versus
the CD95 expression (X axis) was carried out using lymphocytes from the
same donors of Figure 1. (A) Within CD4 T cells, all
CD95 cells (square box) were CD45RA+ and
CD62L+ in both young and old donors. (B) Similarly, all
CD8+CD95 cells (square boxes) were
CD45RA+ and CD62L+. It is important to note
that in both CD4 and CD8 cells, the percentages of
CD95 T cells were substantially identical to those
of the double-positive CD45RA+CD62L+ T cells
shown in Figure 1.
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| Fig 3.
Comparison of CD28 expression versus CD95 or CD45RA
expression in both CD4 and CD8 T cells.
Freshly isolated PBMCs were stained with the following combinations of
FITC-, PE-, and CyChrome-conjugated mAbs: CD95 or CD45RA, CD28, and CD4
or CD8. (A) In both the young and old subjects, 5% probability contour
plots of CD28 expression (Y axis) versus CD95 expression (X axis)
showed that both CD4 and CD8 T cells were made of 3 different subsets:
CD95CD28+ (upper-left boxes),
CD95+CD28+ (ungated), and
CD95+CD28 (lower boxes). In all
stainings, CD28 T cells expressed CD95 at lower
intensity than the CD28+CD95+ counterparts. (B)
Analysis of CD28 expression (Y axis) versus CD45RA expression (X axis).
In both the young and old donors, not all CD45RA+ T cells
expressed CD28. Within the CD4+ T cells, all
CD45RA+ were CD28+ (upper-right box), whereas
in the CD8+ T cells, CD45RA+ T cells comprised
both CD28+ (upper-right box) and CD28 T
cells (lower-right box). Note that the data were obtained using
lymphocytes from the same donors as in Figure 1 and 2.
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To further test if CD95 T cells consist of putative
naive T cells, we investigated the phenotype of this T-cell subset by using 3-color flow cytometry analysis. The absence of CD95 activation Ag was specific for resting T cells because early-, intermediate-, and
late-activation antigens, such as CD69, CD25, CD71, HLA-DR, and CD45RO,
when expressed, were consistently confined among CD95+ T
cells (data not shown). Furthermore, adhesion molecules, such as CD2,
CD11a, and CD44, were constantly and homogeneously at lower intensity
on CD95 T cells than on CD95+ T cells
(data not shown).
For clonal expansion, naive T cells need both TCR engagement and
costimulatory signals mainly provided by the CD28 Ag on their membrane.
Thus, it is expected that naive T cells are CD28+. Indeed,
we found that all CD95 T cells bear CD28 Ag, whereas
CD95+ T cells comprise both CD28+ and
CD28 T cells, and CD28 T cells
express CD95 at lower intensity than the CD28+ counterpart
(Figure 3A). On the other hand, we found that not all
CD45RA+ T cells expressed the CD28 Ag. Indeed, separate
analysis of CD4 and CD8 subsets showed that CD45RA+ T cells
comprised both CD28 and CD28+ T cells
(Figure 3B) within the CD8 subset, whereas in the CD4 subset,
all CD45RA+ were CD28+.
In both young and old subjects, the imbalance between the CD4 and CD8
subsets regarding the number of CD45RA+ cells exceeding the
CD95 naive cell was due to a subset of the
CD8+CD45RA+ T cells not stained by CD28. The
proportion of this still uncharacterized T-cell subset, which bears a
CD45RA isoform but is no longer defined as naive T cells, augmented
heavily among aged people. These results, obtained from the
same representative young and old donors, indicated that the imbalance
between CD4 and CD8 subsets, as well as an overestimate of naive T
cells within the CD8 subset, was due to a subset of
CD45RA+(CD62L) CD28 T cells.
Activation requirement of CD95 T cells is typical of
naive T cells
Naive T cells, but not memory or effector T cells, require
costimulation to activate and proliferate optimally. We tested the
responsiveness of CD95 T cells to stimulation either
through the CD3/TCR complex alone or together with a costimulation. To
this end we tested the proliferative response of
CD95 T cells to increasing doses of anti-CD3 mAb
alone or to a combination of submitogenic doses of anti-CD3 plus a
costimulation provided by anti-CD28 mAb (Figure
4). Unseparated T cells responded to anti-CD3 mAb in a dose-dependent manner, indicating that T cells contain both naive and memory cells. By contrast,
CD95 T cells did not proliferate to increasing doses
of plastic-bound anti-CD3 mAb without costimulation, and they showed an
optimal response only when costimulation was provided. This finding
indicates that CD95 T cells are indeed resting cells
and supports the hypothesis that they may be unprimed T cells.
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| Fig 4.
Activation requirement of CD95 T cells.
Data are from a representative experiment of 3 similar experiments. T
cells showed dose-dependent proliferative responses to immobilized
anti-CD3 mAb. On the contrary, purified CD95 T cells
did not respond to increasing doses of anti-CD3 mAb alone and showed an
optimal proliferation only when costimulated by immobilized anti-CD28
(1 mg/mL) and anti-CD3 mAb.
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CD95 naive T cells decrease with age
Based on findings that CD95 T cells consist of
naive T cells and that there are less CD95 T cells
among old donors than young donors, we further investigated their
absolute number from young adulthood to the extreme limit of human
life. To this end, we analyzed peripheral blood T cells from 120 healthy donors ranging in age from 18-105 years for expression of the
CD95 Ag. Confirming previous reports, we found that absolute numbers of
total circulating lymphocytes (data not shown), as well as percentages
and absolute numbers of CD3+ T lymphocytes, decreased
significantly (R = .58, P = .0001) and progressively with age (Figure 5A), whereas
non-T lymphocytes remained unchanged (R = .13, P =
not significant). Once we plotted CD95 and
CD95+ T-cell subsets in the different donor ages, we found
a progressive decrease of CD95 T cells, both in
percentage (R = .64, P = .0001) and absolute number
(R = .72, P = .0001) (Figure 5B and 5C). This
age-related reduction was particularly evident in the oldest donors, in
whom circulating naive T cells almost disappeared. By contrast,
CD95+ T cells symmetrically increased in percentage with
age (R = .64, P = .0001) (Figure 5B), but their
absolute counts, given the reduction of total lymphocyte absolute
number, did not change significantly (R = .065,
P = .48) (Figure 5C). Therefore, these changes imply that the
most impressive age-related modification within circulating CD3+ T cells consists of a sharp loss of
CD95 naive T cells. The trend and the magnitude of
the CD95 T-cell decrease are apparently
superimposable to that of total T cells, suggesting that the
age-related T lymphopenia may largely depend on the marked decrease of
this subset.
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| Fig 5.
Effect of age on absolute numbers of T and non-T
lymphocytes.
Data from 120 healthy donors, 18-105 years of age, were plotted as
individual data points. R and P values were calculated
by linear regression analysis. (A) Absolute numbers of T lymphocytes
(defined as CD3+,  ) declined progressively with age,
whereas non-T lymphocytes (defined as CD3,  )
remained unchanged. (B, C) The percentages and absolute numbers of
circulating CD95 ( ) and CD95+ ( )
cells among CD3+ T cells were calculated as
described in "Materials and methods." The percentages and
absolute numbers of circulating CD95 cells
progressively decreased with age, whereas CD95+ cells
increased only in percentages but did not significantly
change in absolute numbers until the last decades of life.
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The age-related loss of CD95 T cells was further
investigated within CD4 and CD8 T-cell subsets by analyzing a cohort of
71 subjects. The results showed that both percentages (Figure
6A and 6B) and absolute numbers (Figure 5C
and 5D) of CD95 T cells declined significantly
(P = .0001) with age within both CD4 and CD8 T-cell subsets.
We also observed that in very old people, CD95 T
cells almost disappeared within the CD8 T-cell subset but not within
the CD4 T-cell subset. This finding might represent an amplification of
an imbalance already present in young adulthood. Indeed the ratio
between the number of circulating
CD4+CD95 and
CD8+CD95 cells was about 2 in young people; the mean plus or minus the standard error of the mean
of CD6+CD95 cells was 533 ± 33,
whereas CD8+CD95 cells were 213 ± 23
in the older donors. This difference became more evident with age,
reaching its peak in centenarians, with a ratio of about 11 (137 ± 24 CD4+CD95 cells versus
13 ± 4 CD8+CD95 cells). Therefore,
these data indicate that far advanced age is characterized by a
profound reduction of CD4+ circulating naive T cells
(Figure 6C) and by an almost complete loss of circulating naive cells
within class I-restricted CD8+ T cells (Figure 6D).
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| Fig 6.
Effect of age on CD95 expression on T-cell subsets.
The percentages and absolute numbers of CD95 ()
and CD95+ () cells within both CD4 and CD8 T cells were
plotted as individual data points from 71 subjects. R and
P values were calculated by linear regression analysis. The
percentage of CD95 cells decreased with age, and
symmetrically the percentages of CD95+ T cells
increased with age in both the (A) CD4 subset and the (B)
CD8 subset. (C, D) Considering absolute numbers, only
CD95CD4+ and
CD95CD8+ cells significantly declined.
In contrast, the CD95+CD4+ and
CD95+CD8+ cells did not change.
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T-cell dynamics in aging: relationship between loss of
CD95 and increase of CD28 T cells
The functional and clinical implications of distinct changes in the
T-cell compartments suggested that we verify whether the progressive
loss of naive T cells, here described, might be related to another
profound modification that we recently reported: the expansion of
CD28 T cells during aging.36 As
previously shown (Figure 3), the reduction of CD95 T
cells was associated with an increase of CD28 T
cells in the older subjects, and this phenomenon was particularly evident within CD8+ T cells. However, CD95 and CD28 are not
mutually exclusive markers, as indicated by the presence of a
CD95+CD28+ subset. Thus, we separately analyzed
and compared CD4 and CD8 subsets for the expression of the CD95 and
CD28 markers among 2 extreme age groups of our study: young subjects
(n = 15; 20 ± 0.5 years) and centenarians (n = 15;
100.5 ± 0.3 years). Results showed the relative contribution of
CD95+CD28+,
CD95+CD28, and
CD95CD28+ subsets to the composition of
total circulating CD4+ and CD8+ T cells (Figure
7). When considering only percentages
(Figure 7A and 7B) the age-related decrease of
CD95CD28+
(P = .0001) was associated with a significant
increase (P < .05) with age of both
CD95+CD28+ and
CD95+CD28 subsets, either within CD4 or
CD8 T cells. By contrast, when considering absolute counts (Figure
7C and 7D), we found no significant differences in the
CD95+CD28+ subsets between young and old
subjects, either within CD4 or CD8 T cells (P = .78 and
P = .52, respectively), whereas
CD95+CD28 T cells increased
significantly in both subsets (P = .05 among CD4+
T cells, and P = .02 among CD8+ T cells).
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| Fig 7.
Contribution of CD95+CD28+,
CD95+CD28, and
CD95CD28+ to CD4+ or
CD8+ T-cell subsets in young subjects compared with
centenarians.
Data from 15 young subjects and 15 centenarians represent the means of
different cell subpopulations in the 2 groups and were tested by
analysis of variance (ANOVA). (A) In both CD4+
and CD8+ subsets of centenarians, the
percentage reduction of CD95CD28+ ()
(P = .0001) was mirrored by an
increase of CD95+CD28+ () and
CD95+CD28 ( ) cells
(P < .05 for both). (B) Considering absolute numbers among
either CD4 or CD8 T cells, the counts of
CD95+CD28+ did not change
significantly. Thus, the loss of
CD95CD28+ cells was only
associated with an increase of CD95+CD28
cells (P = .05 among CD4+ T cells, and
P = .02 among CD8+ T cells).
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An overall picture of this phenomenon in total CD3+
circulating lymphocytes from 96 subjects of different ages is reported in Figure 8. We again found that both
subsets statistically changed with age (R = .75 and
P = .0001 for CD95CD28+, and
R = .26 and P = .009 for
CD95+CD28), however the magnitude of the
CD95CD28+ T-cell loss was more prominent
than the expansion of CD95+CD28, as
shown by the slopes of the 2 regression lines ( 8.4 ± 0.7 cells
per year for CD95CD28+ T cells and
1.6 ± 0.6 cells per year for
CD95+CD28 T cells). To assess the
relationship between CD95CD28+ and
CD95+CD28 T cells and age, we used
multiple linear regression analysis. Using
CD95CD28+ as the dependent
variable and age and CD95+CD28 as the
predictors, we found that the absolute number of
CD95CD28+ was related to both variables
(R2 = .58 for the model, P = .0001 for
age, and P = .02 for CD95+CD28
T cells). These 2 factors together explained 58% of the variability of
CD95CD28+, but the effect of age was
more pronounced than the effect of CD95+CD28 T cells (standardized
coefficients: 0.71 versus 0.16). By contrast, we found
that the absolute number of CD95+CD28 T
cells was weakly related only to
CD95CD28+ (P = .02) and not to
age (P = .9). In such a wide age range, more complex
relationships may exist between these variables. However, by limiting
the multiple linear regression analysis only to people over 55 years of
age (n = 52), the statistical findings were virtually identical to
those reported above. In conclusion, the T-cell dynamic emerging from
these results is that first, aging mainly induces a decrease in the
number of CD95CD28+ T cells, and this in
turn is associated with an increase of
CD95+CD28 T cells.
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| Fig 8.
Age-related changes of absolute numbers of naive
CD95CD28+ () and
CD95+CD28 ( ) T cells.
Absolute numbers of CD95 and CD28
T cells among CD3+ cells from 96 donors were plotted as
individual data points. R and P values were calculated
by linear regression analysis. While CD95 T cells
significantly decreased with age, CD28 T cells
simultaneously increased.
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Discussion |
In this study we demonstrate that far advanced age is characterized
by a profound reduction of circulating naive T cells. These results
were obtained by labeling naive T cells with either the CD45RA/CD62L
double-staining standard method or the CD95 single-staining method.
Both approaches allowed us to recognize an age-dependent naive T-cell
depletion that was much more significant than previously estimated by
the CD45RA isoform staining method.37 This sharp age-related loss of naive CD95CD3+ cells
paralleled the rate of total T-cell loss (Figure 5), suggesting that
the well-known age-related T lymphopenia may largely depend on the
marked loss of CD95 naive T cells. A detailed
analysis of age-related changes within the CD4 and CD8 subsets showed
that naive T-cell reduction was profound in both T-cell subsets, but
the reduction was deeper within the CD8+ T cells, where an
almost complete loss in the oldest donors was observed. From the data
here reported, it appears that the most profound age-related change in
the T-cell compartment is the loss of circulating naive
CD8+ T cells. The use of markers thus far available has
largely underestimated this phenomenon.
Class I-restricted CD8+ T cells play a major role in
infectious diseases caused by pathogens living inside cells, and they constitute an important effector arm for immune surveillance against tumors.38 While few studies have found a shortage of naive
CD8+ T cells in HIV-infected adults,20-22 most
studies on human senescence have neglected the naive CD8 T-cell count
as an indicator for evaluating the risk of immune system failure.
Infectious diseases, such as influenza and pneumonia, and cancer are
major health problems in older people and represent leading causes of
death in this population.39 Therefore, naive CD8 T cells
constitute an important reservoir, and their shortage could predict
lack of protection against novel class I-restricted antigens. Based on
our results of the loss of naive T cells within the CD8+
compartment, it can be predicted that very old subjects have low
protection against infectious diseases, especially viruses, and
malignant cells. Moreover, given the direct correlation between the
decrease of CD95 T cells and the increase of age,
with an almost complete exhaustion in very old subjects such as
centenarians, it can also be predicted that a very low number of
CD95 T cells correlates with a shorter life
expectancy. Although these 2 predictions need to be verified in a
perspective study, it must be noted that a high susceptibility to viral
infections and a current short life expectancy both characterize far
advanced aged people.
A theory on the aging process by F.M. Burnet40 proposed,
among other points, that most organs or physiological systems undergo weakening and reduce their physiological reservoirs with aging. However, they "...differ widely in the time needed to use up
their quota of cells. Many systems may never approach the limit, but that system, vital to life, which first uses up its quota, will be the
chief secondary mediator of aging."40 Our current
results, showing the progressive decline of CD8 naive T cells until
exhaustion in centenarians, may fit with this interpretation, and
accordingly, we propose that the shortage of CD8 naive T cells may
represent a secondary biological clock related to the human life span.
Our results are consistent with most previously published data on the
expression of CD95/Fas/Apo-1 by T lymphocytes. Initially, Miyawaki et
al28 demonstrated that T lymphocytes from newborns do not
bear CD95, whereas percentages of CD95+ T cells increase
gradually until 50 years of age. More recently, other studies have
confirmed significant increases of CD95 expression with age, either as
percentages of T cells29,30 or as intensity of mean
fluorescence,31 by comparing a group of aged people versus
young controls. Moreover, Aggarwal and Gupta32 found up-regulation of the CD95-CD95L system along with reduced expression of
Bcl-2 and increased susceptibility to apoptosis both in CD4 and CD8 T
cells from aged donors. Our current study, although focused on the
characterization of CD95 T cells, extends those
previous results regarding percentages of T cells expressing CD95 well
beyond middle age and reports a gradual increase by each decade, until
over 100 years of age. On the other hand, the integrate analysis,
including for the first time absolute counts, evidenced not simply the
well-known increase of CD95+ T-cell percentages, but rather
a sharp age-related loss of CD95 T cells paralleling
the rate of total T-cell loss.
Somewhat surprisingly, 2 papers41,42 have reported opposing
results regarding T-cell expression of CD95. Lechner et
al41 found negligible expression of CD95 on freshly
isolated T cells at any age, whereas Aspinall et al42
described a decrease of percentages and absolute counts of
CD95+ T cells both in CD4 and CD8 T cells in a group of 12 older females compared with young controls. The possible explanations
for these outlying results are not fully understandable, but in our
opinion the divergent classification of CD95+dim
T cells, which we characterized as
CD28 T cells and which increase with age, may
justify at least some of these discrepancies.
Our results indicate that the absence of the CD95 Ag by itself can
define putative naive T cells currently identified by a standard
double-staining method with CD45RA and CD62L. Therefore, we propose to
identify naive T cells phenotypically as CD95 cells.
This approach is further supported by the demonstration that
CD95 T cells do not express activation antigens, but
they express adhesion antigens at low intensity, express CD28 Ag, and
have a costimulatory requirement for activation.
The present finding reinforces the notion that naive T-cell frequency
cannot be evaluated on the basis of CD45RA expression alone. Indeed, we
have shown that physiological aging was coupled by an expansion of a
subset of CD95+CD45RA+CD28 T
cells paradoxically coexpressing putative markers of virgin and
activated cells. The coexpression of CD95 activation Ag in CD45RA+ T cells extends previous findings showing that (1)
the CD8+CD45RA+ T-cell subpopulation contains
cells with the phenotype of activated T cells expressing high levels of
CD11a and CD29 Ags43 and (2) these cells are significantly
increasing with age.44 Similarly, it has been shown that
the CD8+ T-cell subset contains expanded clones which are
very frequent in old donors and are present not only in
CD45RO+ T cells but also in CD45RA+ T
cells.45 Moreover, our results show that this activated
CD8+CD45RA+CD95+ T-cell subset
consists of CD28 T cells, which have been
extensively studied in humans. CD28 T cells have
phenotypic and functional features of terminally differentiated armed
effector T lymphocytes,36,46,47 have a telomere length
suggestive of a long replicate history,47,48 and contain
clonal expansions.49 Taken together, these findings support
the notion that CD28 T cells cannot be considered as
thymus-derived naive T cells. Accordingly, we propose that staining
with CD95 or any other equivalent approach, excluding
CD28 T cells, is required whenever an expansion of
CD28 T cells occurs. Consequently, the
interpretation of previous findings about CD45RA stainings in clinical
settings, characterized by an expansion of CD28 T
cells including viral infection (HIV, cytomegalovirus, Epstein-Barr virus, etc.) and/or chronic inflammatory diseases (rheumatoid arthritis, SLE, etc.), should be reconsidered.
To date, CD28 T cells have been described only in
humans, and their origin is still controversial. They may be generated, as such, from extra-thymic lymphopoiesis, or alternatively, they may
derive from loss of the CD28 molecule by thymus-derived T cells. As
shown here and in a previous report,36 aging is associated with expansion of CD28 effector T cells. By using
multiple linear regression fitting, we show that during the aging
process, there is a significant correlation between
CD28 expansion and CD95
reduction. In particular, the hypothesis emerging from our results is
that aging induces, in the first place, a decrease in the number of
CD95CD28+ T cells, and this in turn is
associated with an increase of CD95+CD28
T cells, which could represent a compensatory mechanism of the immune system.
Some recent studies on T-cell regeneration in different clinical
settings in humans have similarly shown that thymic lymphopoiesis is
severely limited in adults, and thymic-independent expansion of a
mature T-cell population may represent the primary pathway by which T
cells are regenerated.7,11,50,51 Notably, several distinctions have been observed between CD8+ and
CD4+ T cells. In patients undergoing intensive
chemotherapy, Mackall et al50 described that
CD8+ T cells recover more rapidly than CD4+ T
cells, but there is a prolonged alteration in the composition of the
CD8+ subset, with a predominance of
CD8+CD28 cells. Consistently, a
longitudinal study demonstrated that in HIV+
patients at different stages of the disease, variations of
CD8+CD28 T cells inversely correlated
with CD4+ and CD8+CD28+ T
cells.52 This suggests that in these patients,
CD3+ T-cell homeostasis is maintained by the continuous
production of CD8+CD28 T
cells.52 Moreover, in HIV-infected patients, Gorochov et al53 found no significant enhancement of the
CD8+ T-cell V repertoire after highly active
antiretroviral therapy. Reminiscent of what is seen after
toxic insults from chemotherapy, irradiation, or infections such as
HIV, we hypothesize that during aging, when the ability to replenish
the naive pool via thymopoiesis is reduced, the immune system tries to
compensate for the progressive loss of naive T cells by increasing
thymic-independent pathways, such as the peripheral expansion of mature
CD28 T cells, especially within the
CD8+ subset.
Taken together, these age-dependent changes in the T-cell
subpopulations indicate that advanced age, per se, is a condition characterized by lack of adaptive immune response to new intracellular pathogens. These changes also strongly support the notion that aging
shares similarities with persistent and chronic stimulatory conditions
of the immune system by infectious agents such as HIV. In particular,
the progressive loss of naive T cells within the CD8+
subset,20-22,54 the expansion of
CD8+CD28 T cells,52 as well
as the restriction in the CD8+ T-cell
repertoire53,55 suggest a typical perturbation of the CD8+ T-cell subset that occurs in both HIV disease and
advanced aging. The mechanisms underlying the CD8+ naive
T-cell loss in aging, as well as in HIV disease, may be unique or
reflect common immunoregulatory processes that HIV could accelerate.
In any event, we are suggesting that the loss of naive T cells,
particularly within the CD8+ T-cell compartment, represents
a hallmark of immunosenescence as well as some immunodeficiencies and
could provide a useful biomarker in both conditions.
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Acknowledgment |
We thank Lucia Orlando for her excellent technical assistance.
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Footnotes |
Supported by grant 9806171821-003 from MURST (Ministry for
University & Scientific Research, Rome, Italy); grant for the project "Predictive Molecular Markers of Osteoporosis," Ministry of
Health, Rome, Italy; and grant 1998/99 from Foundation of the Cassa di Risparmio, Bank of Parma and Piacenza, Parma, Italy.
F.F.F. and R.V. contributed equally to this work. F.F.F. is
currently affiliated with the Division of Medical Oncology, Maugeri Foundation Medical Center,Loc. Cravino, Pavia, Italy.
Submitted August 13, 1999; accepted December 30, 1999.
Reprints: Paolo |
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Abstract
Introduction