Mechanisms of Ageing and Development
96 (1997) 127 – 136

T lymphocyte proliferative capability to defined
stimuli and costimulatory CD28 pathway is not
impaired in healthy centenarians
Paolo Sansoni a,*, Francesco Fagnoni a, Rosanna Vescovini a,
Marco Mazzola a, Vincenzo Brianti a, Giovanni Bologna a,
Enrica Nigro a, Giampaolo Lavagetto a, Andrea Cossarizza b,
Daniela Monti b, Claudio Franceschi b,c, Mario Passeri a
Istituto di Clinica Medica Generale e Terapia Medica, Uni6ersity of Parma, 6ia Gramsci 14,
Parma 43 100, Italy
b
Dipartimento di Scienze Biomediche, Sezione di Patologia Generale, Uni6ersity of Modena,
6ia Campi 287, Modena 41 100, Italy
c
INRCA, 6ia Birarelli, 8, 60 100 Ancona, Italy

a

Received 12 September 1996; received in revised form 27 January 1997

Abstract
It is generally assumed that T cell proliferation is impaired in aged individuals. We report
data on the proliferative capability of peripheral blood mononuclear cells (PBMC) and T
lymphocytes from 40 healthy people of different ages, (19 – 107 years), including 14 centenarians, to defined mitogenic stimuli. We observed no age-related proliferative impairment both
in PBMC and in purified T cells stimulated by anti-CD3 mAb or phorbol myristate acetate
(PMA). Furthermore, T cells stimulated by anti-CD3 mAb or PMA and costimulated by
CD28 mAb did not proliferate differently among young, middle aged subjects and centenarians. Thus, short term T cell proliferation is not affected even at extreme age when well
defined stimuli are used on cells deriving from carefully selected healthy subjects. © 1997
Elsevier Science Ireland Ltd.
Keywords: Centenarians; T cells; CD28; Proliferation

* Corresponding author. Tel.: + 39 521 290783/4; fax: +39 521 290776.
0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 1 8 8 7 - 3

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1. Introduction
An old tenet in medicine is that aging is accompanied by a deterioration of the
immune system that could contribute to increased risk of morbidity and mortality
[1 – 4] However, the study of carefully selected healthy aged subjects showed that
most immune functions are well preserved even in advanced age [5,6]. We have
shown that several immune responses are well preserved in healthy centenarians in
comparison to middle-aged and young subjects[7,8].
A very important function which has been suggested to deteriorate with age and
to play a major role in the aging process is the capability of cells from aged subjects
to respond to mitogenic stimuli and, consequently, to undergo cell proliferation [9].
A defect in this function is very important for immune cells, whose capability to
undergo clonal expansion is critical to set up an effective response to antigenic
stimuli [10]. Accordingly, we thought it worthwhile to assess the proliferative
capability of peripheral blood mononuclear cells (PBMC) and purified T
lymphocytes from healthy centenarians in comparison to middle-aged and young
subjects. Our results demonstrate that this function is well preserved when defined
stimuli, such as anti-CD3 mAb or phorbol 12-myristate acetate (PMA), are used.
This conclusion is further supported by experiments showing that the costimulatory
pathway via the CD28 molecule is well preserved in centenarians.

2. Materials and methods

2.1. Subjects.
We studied a total of 14 centenarians with a mean age of 100.89 0.3 years
(range, 100 – 107 years), 3 males and 11 females. All subjects were in relatively good
clinical condition without relevant acute or chronic disease affecting the immune
system and mentally competent to give informed consent. In particular, none of the
subjects had cancer, were suffering from serious cardiac, brain or kidney disease or
taking drugs known to affect the immune system. Along with a complete social and
medical history, we performed a physical examination. As control groups we
studied 10 healthy middle-aged subjects with a mean age of 58.2 9 2.1 years (range,
48 – 68 years) and 16 healthy young donors with a mean age of 28.19 0.6 years
(range, 19 – 36 years). All these control subjects were selected according to the
SENIEUR protocol [5,6].

2.2. Monoclonal antibodies.
MAb to CD3 (OKT3) was generously provided by Dr E. Engleman, Stanford
University (Stanford, CA); anti-CD8 conjugated with fluorescein (FITC), antiCD28 conjugated with phycoerythrin (PE) and isotype-matched control mAb were
purchased from Becton Dickinson (San Jose, CA).
´

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129

2.3. Cell preparation and enrichment of T lymphocytes.
PBMC were obtained by Ficoll-Hypaque gradient centrifugation from freshly
drawn venous blood collected around 08:00 from a centenarian, a middle-aged and
a young control on the same day. After washings with PBS, the cells were
suspended in RPMI-1640 with 10% AB serum, 2mM L-glutamine, 100 vg/ml
streptomycin and 100 U/ml penicillin, hereafter referred to as complete medium.
PBMC were fractionated into T and non-T cells by a single step rosetting method
[11]. T cells were separated from sheep red blood cells by hypotonic lysis of the
latter. T cells were further depleted of monocytes by adherence (1 h) to plastic. The
purity of T cells preparation was always \95% as assessed by cytofluorimetric
analysis.

2.4. Proliferation assays.
All proliferation assays were performed in round-bottomed microtitre wells in a
final volume of 0.2 ml complete medium. Stimulation of 1× 105 PBMC with
soluble anti-CD3 mAb (25 ng/ml) or 5 ng/ml PMA was carried out for 3 days at
37°C in 6% carbon dioxide – air. Purified T cells were stimulated with immobilized
mAb as follows: 100 vl anti-CD3 (0.1 vg/ml) and/or 100 vl anti-CD28 (5 vg/ml),
diluted in PBS, were placed in 96 wells microtiter plates and incubated at room
temperature overnight and then washed with PBS. T cells were challenged with
several stimuli or a combination of them: immobilized anti-CD3; immobilized
anti-CD3 + immobilized anti-CD28; 5 ng/ml PMA; PMA+immobilized anti-CD28
as specified in the figure legends. Assays were performed in triplicate and 0.5 vCi
[3H]thymidine ([3H]TdR) was added to each well 6 h before cell harvesting on glass
fiber filter paper. Uptake of [3H]TdR was measured in a liquid scintillation counter
and the results expressed as the mean counts per min 9 S.E.M. (cpm9 S.E.M.).

2.5. Cytofluorimetric analysis.
Cytofluorimetric analysis was performed on PBMC following standard methods
[12]. Briefly, 1 × 106 PBMC were incubated with 1 vg anti-CD8 FITC and
anti-CD28 PE for 20 min at 4°C, washed with PBS and suspended in 200 vl PBS
until analysis. Two colors FACS analysis was performed on a FACScan cytofluorimeter (Becton-Dickinson, CA) as previously described [12]. The expression
per cell of CD28 molecule was calculated by flow cytometry using PE-conjugated
anti-CD28 mAb, considering the mean fluorescence channel of each histogram, as
described [13].

2.6. Statistical analysis.
Statistical analysis was performed by ANOVA and Fisher test by SPSS for
Windows® software. P B 0.05 was considered significant.

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3. Results

3.1. Proliferati6e responsi6eness of PBMC and purified T cells.
When PBMC were stimulated by defined mitogenic stimuli, such as PMA or
anti-CD3 mAb, no significant difference concerning the proliferative capability was
observed among cells from young, middle-aged and centenarians (Fig. 1). PBMC
are mainly composed of T and B lymphocytes, NK cells and accessory cells such as
monocytes. It is well known that optimal T cell responsiveness requires a primary
and a costimulatory signal that usually is delivered by accessory antigen presenting
cells through the B7 molecule which is capable of interacting with the CD28

Fig. 1. Proliferative responses of PBMC from 14 centenarians, 10 middle-aged and 16 young controls to
soluble anti-CD3 mAb (25 ng/ml) or PMA (5 ng/ml).

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131

Fig. 2. Proliferative responses of purified T cells from 14 centenarians, 10 middle-aged and 16 young
controls to PMA (5 ng/ml); PMA added to immobilized anti-CD28 mAb, immobilized anti-CD3 mAb
or to coimmobilized anti-CD3 plus anti-CD28 mAb.

molecule on the T cell membrane [14]. Accordingly, purified T cells from young,
middle aged and centenarians were stimulated by the above mentioned primary
stimuli (PMA or anti-CD3 mAb) and costimulated by anti-CD28 mAb in order to
activate the most important costimulatory pathway in T cells [15]. Fig. 2 shows that

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costimulation via anti-CD28 mAb significantly increased T cell responsiveness to
PMA or anti-CD3 mAb in cells from all groups including centenarians and again
no significant age-related difference was found.

3.2. CD28 expression.
Since the target of anti-CD28 mAb is the CD28 molecule on the T cell membrane
we performed a cytofluorimetric analysis of resting lympocytes in order to study the
expression of this molecule on T lymphocytes from centenarians, middle-aged and
young controls.The results indicate that the costimulatory capability of anti-CD28
mAb is not likely to be related to the percentage of CD28 + cells in culture. Indeed
Table 1 shows that the percentage of CD28+ cells is significantly reduced in
centenarians, whose cells, however, responded well to costimulation with anti-CD28
in the presence of PMA or anti-CD3 mAb (Fig. 2). Table 1 also shows that,
notwithstanding the decrease in the percentage of CD28 + lymphocytes, the
expression of CD28 molecules per cell was, however, unchanged.

4. Discussion
Previous studies indicate that a profound remodelling of the immune system
occurs with age [7]. In particular we have reported that, in the peripheral blood of
aged people including centenarians, there is a progressive increase in serum level of
IgA, IgG1, IgG2, IgG3 but not of IgM or IgG4 [16], a decrease in absolute number
of T lymphocytes (CD3 +) involving both CD4 + and CD8 + subsets, a marked
decrease of B lymphocytes and an increase of cells with natural killer (NK) markers
[8,17]. We have also found that healthy elderly and centenarians are almost free of
organ-specific autoantibodies [18,19] and equipped with very well preserved cytotoxic activities (NK, anti-CD16 and anti-CD3 redirected killing activities) [8,17,20].
Table 1
Cytofluorimetric analysis of T lymphocytes from centenarian, middle-aged and young subjects
Centenarians
Mean age and number of subjects
% of CD28+ among T cells
Number of CD28+ T cells per vl
CD28 median fluorescence channel in linear scale

Middle aged

Young adults

101.1 9 0.4 (10)
50.5 9 3.5**
583 9 47**
385 913.9

63.5 9 1.9 (17)
77.3 93.8**
1138 970**
358 9 21

28.89 0.9 (24)
90.259 1.4
1558 947
372 9 18

Data are reported as mean values9 S.E.M.
PBMC were double stained with FITC-conjugated mAb anti-CD3 and PE-conjugated mAb anti-CD28
as described in Section 2.
Analysis of CD28 expression was obtained by Lysis II® software.
Statistical analysis was done by using the ANOVA Fischer test.
**PB0.01 vs. either of the two other groups.

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133

An important and extensively studied immune function is lymphocyte capability
to undergo cell proliferation. Several reports indicate that this function is impaired
with age [1,2]. The reason for this defect has been variably referred to among T cell
subsets, an imbalance [21], a reduced proliferation of CD8 + T cells [22], a failure
to produce IL-2 [23] or to a decreased number of cells capable of completeing the
S phase of the cell cycle [24]. However, most of the previous studies refer to the
capability of PBMC to proliferate when stimulated with PHA, a mitogen used to
assess such a function since 1960 but whose fine mechanism is not completely
understood [25]. The available data indicate that PHA induces activation of T cells
in a complex way through cross-linking of several surface structures, including
CD2, CD3, CD5 molecules [26,27] and perhaps others. Thus, we thought it
worthwhile to study the proliferative capability of either PBMC or purified T cells
from people of different ages using more defined stimuli acting at different levels of
the signal transduction cell machinery, i.e. plasma-membrane (anti-CD3) or
transmembrane (PMA). When this approach was used no proliferative defect was
evident with both stimuli in cells from any group, including people who are close
to the maximum life span such as subjects over 100 years of age. These data are in
accord with earlier reports in unseparated PBMC from subjects aged between 70
and 82 years [28,29].
We tried to further dissect the pathways known to be involved in lymphocyte
proliferation and particularly the main costimulatory T-cell pathway via the CD28
molecule. To this end we used anti-CD28 mAb to act as surrogate for this most
important contribution by accessory cells to T cell activation [14]. This system has
the advantage of optimizing the lymphocyte proliferative conditions, indeed the
ligation of CD28 molecules increases the expression of IL-2 receptors and IL-2
availability [15,30,31] which, in turn, are thought to be defective in lymphocyte
cultures from elderly subjects [23,32]. Under these conditions, costimulation via
CD28 significantly increased T lymphocyte proliferation in middle-aged and young
controls, as well as in centenarians, and this phenomenon was of the same extent in
the three groups.
According to the two signal theory, T lymphocyte activation derives from
MHC-peptide specific engagement of TCR-CD3 and contestual crosslinking of non
antigen-specific accessory molecules such as CD28 [33,34]. Our data indicates that
both CD3 and CD28 pathways are well preserved in T cells from aged people
including centenarians. Our results suggest that the cell machinery responsible for
the transduction of primary and costimulatory signals is functioning well even in
cells from these subjects of very advanced age. The results presented are in apparent
contrast with the data in the literature indicating that lymphocytes from elderly
people show a proliferative defect when complex stimuli such as PHA [2] or
alloantigens are used [35]. The defective response to PHA could be related to the
complexity of this stimulus (proliferation of different T cell subsets? Activation-induced cell death? Induction of cell nonresponsiveness?).
The results shown here suggest that, when cells from carefully selected aged
subjects are used and stimulated with well defined stimuli T lymphocyte proliferation is not impaired as previously thought and that some important activation

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pathways, such as those involving CD3, are well preserved into the last decades of
life. Furthermore, the efficient costimulation through CD28 molecules in PMA and
in anti-CD3 stimulated cultures indicate that the costimulation through the CD28
pathway is not impaired in centenarians despite the reduced number of CD28
positive cells with age.
About 1/3 of centenarians, despite their very advanced age, are still in good
mental and physical condition. These subjects are the best example of successful
aging, being free of the major age-related diseases. Therefore, healthy centenarians
are a model of human longevity and constitute a very select group of exceptional
individuals. The current observation of a well preserved T cell proliferative capability involved in adaptive immune responses, together with previous observations
concerning well preserved innate immunity, suggests that such a well equipped
immune system may contribute to longevity in human and, on the other hand,
indicate that the immune system of healthy centenarians is still apparently capable
of coping with infectious diseases.
In summary, we suggest that a generalized age-related defect in the cell proliferative machinery can be excluded and that important activation pathways are intact
in lymphocytes even in the last decade of life. The data presented here fit the
hypothesis that aging is not characterized by unidirectional deterioration but by a
complex remodelling of immune functions [7,36].

Acknowledgements
This work was supported by the National Research Council (CNR) target project
‘Ageing’, M.U.R.S.T. (40 and 60%) to PS and CF and from Fondazione Cassa di
Risparmio di Parma

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