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*
Department of Immunology, The Royal Free Hospital School of Medicine, London, United Kingdom;
Imperial Cancer Research Fund Tumour Immunology Unit and Department of Oncology and Sexually Transmitted Diseases, University College London Medical School, London, United Kingdom; and
Department of Rheumatology, Birmingham University Medical School, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of the IL-2 receptor. IL-7 prevented the
decrease in Bcl-2 and Bcl-xL and induced cell cycling in up
to 20% of cord T cells after 8 days, resulting in a threefold increase
in cord T cell numbers. However, the expanded cells retained a
CD45RA+CD45RO- phenotype. Similar results were
obtained with adult CD45RA+ T cells. IL-7-expanded
CD45RA+RO- T cells expressed CD45RO after
stimulation through the TCR. Investigations into the regulation of
replicative senescence showed that after 12 days in culture with IL-7,
cord blood CD45RA+ T cell proliferation resulted in
telomere shortening. Nevertheless, IL-7-expanded cord blood T cells
still maintained longer telomeres than unstimulated adult T cells. IL-7
but not IL-2 could directly induce high telomerase activity which
probably retarded the rate of telomere shortening in cord blood T
cells. These results suggest that proliferation induced by IL-7 may be
important for extrathymic expansion of neonatal CD45RA+ T
cells and may also contribute to the maintenance of the adult
CD45RA+ T cell pool. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two possibilities for naive CD45RA+ T cell maintenance throughout life have been suggested. In the first, reversion from CD45RO+ to CD45RA+ expression may occur in some cells (9). However, data from patients with viral infections suggest that the majority of the expanded CD45RO+ population perish by apoptosis rather than revert to CD45RA+ expression (10). A second possibility is that expansion of T cells within the CD45RA+ T cell pool may occur in the presence of certain cytokines that induce proliferation without acquisition of CD45RO expression (11, 12).
Another homeostatic constraint on the T cell pool is telomere shortening as a result of proliferation (13, 14, 15). Telomeres are unique terminal chromosomal structures consisting of hexameric repeats ((TTAGGG)n) and are involved in the maintenance of chromosomal integrity (16). Cell division leads to the loss of telomeric DNA; therefore telomere shortening acts as a mitotic clock, restricting the number of divisions that a cell can undergo (17, 18). Critically short telomeres signal growth arrest, a process known as replicative senescence (15). Previous studies have shown that CD45RO+ T cells have shorter telomeres than CD45RA+ T cells, suggesting that the former population has decreased replicative potential (19).
To investigate mechanisms that may regulate homeostasis of the naive T cell pool, we have studied both adult and cord blood CD45RA+ T cells, the latter population being truly naive, not complicated by the presence of putative "back-converted" CD45RO+ T cells (4, 5, 9). In adults, CD45RO+ T cells are particularly prone to apoptosis because of their low expression of survival genes such as bcl-2 but high expression of apoptosis-inducing molecules such as CD95 (Fas/Apo-1) (5, 7, 10, 20). Resting adult CD45RA+ T cells are also susceptible to apoptosis but to a much lesser extent than CD45RO+ T cells in either a resting or an activated state (21) (D. Pilling, A. N. Akbar, and M. Salmon, manuscript in preparation). Freshly isolated resting cord blood CD45RA+ T cells, however, were highly susceptible to spontaneous apoptosis in culture, suggesting that neonatal and adult unprimed populations have some functional differences (22). Both cord and adult CD45RA+ T cells can be induced to proliferate by IL-7, without conversion to a CD45RO+RA- phenotype. Furthermore, we show for the first time that IL-7 alone can induce strong telomerase activity in CD45RA+ T cells without the requirement for TCR stimulation. Although a decrease in the mean telomere length was observed after culture, telomerase up-regulation by IL-7 may enable bystander expansion of naive T cells by cytokines without significant telomere loss, thus preserving the replicative potential of the expanded cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Umbilical cord blood samples were obtained after uncomplicated births. Blood samples were collected into heparinized syringes and processed immediately. Heparinized venous peripheral blood samples obtained from healthy laboratory staff were used as adult control cells.
Cytokines used in the study
The cytokines used in this study were obtained from R&D Systems (Abingdon, U.K.) and used at pretitrated optimal concentrations of 5 ng/ml.
Lymphocyte subset purification
PBMC were separated using Ficoll-Hypaque (Nycomed, Oslo, Norway)
density gradient centrifugation as previously described (10).
CD45RA+ T cells were separated by negative selection by
immunomagnetic bead depletion. Cells were firstly incubated in a
mixture of mAbs directed at the populations to be eliminated (CD16,
CD14, CD19, and CD45RO; see Table I
for
details). Cord blood samples were preincubated with
anti-glycophorin A (Dako, High Wycombe, U.K.) to eliminate
nucleated RBC precursors. Cells were then incubated with Dynabeads
(Dynal, Oslo, Norway) coated with affinity-purified goat anti-mouse
Ig M450) at room temperature for 30 min. A magnetic particle
concentrator allowed the recovery of unbound CD45RA+ T
cells. Two rounds of depletion were performed. Purified populations
contained 85–95% CD3+/CD45RA+. In cord blood
samples, contaminating CD3-/RA- cells
constituted <4% of gated populations, whereas the percentage of
CD3-/RA+ ranged from 1 to 10%.
CD45RA+-enriched T cells were cultured, in the presence or
absence of cytokines, in RPMI 1640 (Life Technologies, Paisley, U.K.)
containing 10% FCS (M. B. Meldrum, Bourne End, U.K.), antibiotics
(Life Technologies), and supplemented with L-glutamine (ICN
Biomedicals, High Wycombe, U.K.).
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FACScan (Becton Dickinson, Oxford, U.K.) analysis of T cell
phenotype was performed as previously described (23) using different
combinations of the Abs shown in Table I
. Cells were incubated with Abs
for 10 min and fixed in 2% paraformaldehyde in PBS before analysis.
Analysis of the intracellular expression of Bcl-2 and
Bcl-xL required cell permeabilization with Permeafix (Ortho
Diagnostic Systems, High Wycombe, U.K.). Data analysis was performed
using LYSIS II Software.
Detection of apoptosis and cell cycle analysis
Apoptosis was detected as described previously (7, 10), first in cytocentrifuge preparations of cells by morphologic changes such as chromatin condensation, nuclear fragmentation, and decreased nuclear-cytoplasmic ratio after May-Grünwald-Giemsa staining. Secondly, the nucleus of cells in suspension was labeled with propidium iodide (PI3, Sigma-Aldrich, Poole, U.K.) after permeabilization with 90% ethanol. PI binds to DNA, allowing the identification of apoptotic, resting, and proliferating cells by their variable DNA content. Apoptotic cells characteristically have lower DNA content and are smaller than resting viable cells which in turn have one-half the DNA content of proliferating cells.
Enumeration of the absolute numbers of lymphocytes
The number of viable cells surviving in culture, treated or
untreated with the IL-2R common -chain cytokines, was assessed using
the Cytoron Absolute Cytometer (Ortho). Samples were collected at
different time points, fixed with 1% paraformaldehyde in PBS, and run
through the Cytoron Absolute using preset gates for viable cells.
Activation of cytokine-expanded populations by anti-CD3 Ab
Anti-CD3-coated plates were prepared by overnight incubation of
anti-CD3 (UCHT1, 10 ng/ml) at 37°C in coating buffer
(Sigma-Aldrich). The plates were then washed twice with medium (RPMI
1640, containing 10% FCS), and cells were added at 106/ml.
Cells cultured with or without anti-CD3 and in the presence of IL-2
R common -chain cytokines were harvested at 1 and 2 days after
anti-CD3 stimulation. Response to anti-CD3 stimulation was
evaluated by cell viability and PI staining to determine the relative
degree of apoptosis and proliferation.
Assessment of the mean terminal restriction fragment (TRF) length
CD45RA+-enriched cord blood T cells were analyzed
before and after culture with IL-7. Cell samples (2 x
106 to 4 x 106 cells) were pelleted by
centrifugation, transferred into Eppendorfs, and washed in PBS. The
pellets were then snap-frozen in liquid nitrogen and stored at
-70°C. Genomic DNA was extracted from frozen samples using the
WIZARD genomic DNA purification kit (Promega, Southampton, U.K.) and
digested for 6 h at 37°C with restriction enzymes
MspI and RsaI (both from Pharmacia Biotech, St.
Albans, U.K.). The digests were then electrophoresed in a 0.7% agarose
gel in Tris-acetate for 48 h at 20 V. The gel was then subjected
to depurination, denaturation in an alkaline solution, and
neutralization. The DNA was transferred onto a nitrocellulose membrane
(Hybond N+, Amersham Life Science, Amersham, U.K.) by
Southern blotting and fixed by baking at 80°C. Before hybridization
with the telomeric probe, the membranes were prehybridized with Rapid
Hyb Buffer (Amersham). Hybridization with the telomeric specific probe
(TTAGGG)3, which had previously been labeled with
[-32P]ATP using polynucleotide kinase (Pharmacia), was
performed at 42°C for 1 h. Autoradiographs were obtained by
exposing the autoradiography film (Hyperfilm MP, Amersham) to the
hybridized membrane at -70°C for the appropriate amount of time. TRF
length was calculated by densitometry of autoradiographs within the
linear range using Molecular Analyst software (Bio-Rad, Hemel
Hempstead, U.K.). The mean TRF length was calculated as described (24).
Measurement of telomerase activity
A modified version of the telomeric repeat amplification
protocol (TRAP) was used (Oncor, Gaithersburg, MD) as previously
described (25). Extracts from varying cell numbers were used for
telomeric elongation, using a [-33P]ATP-end-labeled
primer. These samples were used for PCR amplification (Perkin-Elmer
Cetus, Norwalk, CT), using 25 to 28 cycles of 30 s at 94°C and
30 s at 59°C. The PCR products were run on a 12% polyacrylamide
(Amersham) gel which was vacuum dried for exposure to autoradiography
film (Hyperfilm MP, Amersham). Telomerase activity was calculated using
the optical density of the telomeric repeat bands, divided by the
strength of the internal PCR control band (which also served to
indicate the absence of Taq inhibitors). Extracts from the
immortalized 293 cell line provided the positive control. The negative
controls were obtained by heat inactivation of the RNA template for
each cell extract used. In addition, lysis buffer was used in place of
cell extract in one reaction tube.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes from 10 umbilical cord blood samples and 10
normal adult individuals were prepared and cultured under the same
conditions. Cord blood T cell populations contain <3% (range,
0.5–6%) CD45RO+ T cells, whereas T cell populations from
healthy adults contain 40% (range, 30–50%) of these cells (22). When
CD45RA+ T cells from adult and cord were isolated and
cultured without added exogenous stimuli, similar survival rates for
the first 72 h were observed in culture (Fig. 1). This was followed by a substantial
decrease in the viability of cord blood T cells. By day 8, virtually
all the cells in cord cultures were dead, whereas 75% of the cells in
adult cultures were still viable. These data clearly show a deficiency
in the ability of CD45RA+ cord blood T cells to survive in
culture compared with their adult counterparts.
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The occurrence of apoptosis in cord blood CD45RA+ T
cells after 6 days in culture was demonstrated by increased PI
incorporation as a sub-G0 peak (Fig. 2B). The level of apoptosis in
cultured cord T cell samples was greater than that observed in freshly
isolated cord blood T cells (Fig. 2A) and adult
CD45RA+ T cells (Fig. 2C). The occurrence of
apoptosis was also confirmed by the typical morphologic changes
detected by May-Grünwald-Giemsa staining (data not shown). Fig. 2D represents the mean percentage apoptosis of cord and
adult CD45RA+ T cell populations cultured for 6 days
showing a statistically significant increase in apoptosis in cord blood
T cells (p < 0.0002).
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-chain cytokines
In view of previous studies showing the ability of a group of
cytokines sharing the -chain of the IL-2 receptor to prevent resting
and activated T cell death (23, 26, 27), we investigated the effect of
these cytokines (IL-2, IL-4, IL-7, and IL-15) on cord blood T cell
apoptosis. Supplementing cord blood T cell cultures with these
cytokines resulted in a significant increase in cell recovery at day 8
relative to the initial cell input (Fig. 3A). Both CD4+ and
CD8+ T cells were expanded (data not shown). IL-7 produced
the most striking results inducing a threefold increase in cell numbers
compared with the initial input of cells. The greater effect of IL-7
compared with the other IL-2R common -chain-signaling cytokines was
still observed when up to 10-fold higher concentrations of IL-2, IL-4,
and IL-15 were used (data not shown). IL-6 was used as a control
cytokine, and no rescue was achieved. The increase in cell recovery
induced by IL-2 and especially IL-7 in both adult and cord blood T
cells resulted in part from proliferation (Fig. 3B). Control
adult and especially cord T cells cultured without added cytokines for
8 days showed a considerable decrease in viability (see Fig. 1). In
four cord and four adult CD45RA+ T cell samples studied,
only 4.4 ± 1.4% (mean ± S.E.M.) and 2.2 ± 1.5%,
respectively, of residual control cells cultured without cytokines for
8 days were in cycle.
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Adult and cord CD45RA+RO-T cells have
been shown to convert to a "primed" phenotype
(CD45RO+RA-) upon stimulation (4, 6, 22). We
investigated whether proliferation induced by cytokines alone caused a
similar switch. Cells cultured with or without IL-2 or IL-7 were
harvested at several time points and analyzed for CD3, CD45RA, and
CD45RO expression. The mean results from six cord blood samples are
shown in Fig. 4A. The absolute
numbers of CD3+/RA+ cells were always increased
in cytokine-supplemented cultures as compared with control cultures.
Interestingly, proliferation in these cells was not accompanied by a
switch to CD45RO+RA- (Fig. 4D).
IL-7 produced the most pronounced effect, not only in terms of absolute
numbers of viable cells recovered but also in the extent of
CD45RA+ T cell proliferation induced. Similarly, adult
CD3+CD45RA+ T cells cultured in the presence of
these cytokines did not switch to CD45RO+ expression (data
not shown).
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Bcl-2 and Bcl-xL expression in cord blood T cells
To determine whether the susceptibility of cord blood T cell to
apoptosis was caused by changes in the constitutive expression of Bcl-2
or Bcl-xL which prevent apoptosis (28, 29), freshly
isolated cord and adult T cells were stained for these proteins and
analyzed by flow cytometry. It was found that both proteins were
expressed at the same concentrations in cord blood and adult peripheral
blood T cells before culture (data not shown). Bcl-2 and
Bcl-xL levels were then measured after culture in the
presence or absence of the survival-promoting cytokines (Fig. 5). After 7 days in culture without
exogenous addition of cytokines, Bcl-2 and Bcl-xL levels
decreased, when compared with day 0 (Fig. 5, A and
B). A comparison of the mean percentage decrease in Bcl-2
levels after culture in cord and adult CD45RA+ T cells
showed that the mean expression by cord T cells decreased by 42.9%,
whereas that of adults decreased by 3% (Fig. 5C). The mean
expression of Bcl-xL was decreased by 35% in cultured
CD45RA+ cord T cells compared with the starting population,
whereas in adults Bcl-xL expression decreased by a mean of
11% without cytokines (Fig. 5D). Although both Bcl-2 and
Bcl-xL were consistently decreased in all the cord samples
tested after culture, the magnitude of the decrease was variable. One
reason for this was that the decrease in expression of these proteins
occurred earlier in some cells than in others, which resulted in a
bimodal expression of these molecules in cultured cord T cells
(representative experiment shown in Fig. 5, A and
B). Although these bimodal profiles convincingly demonstrate
a decrease in expression of Bcl-2 and Bcl-xL in a
substantial proportion of cells, the net mean fluorescence intensity
(MFI) of these bimodal peaks showed little change compared with the
original population (Fig. 5A; MFI day 0 is 54 whereas MFI of
cells cultured for 7 days without IL-7 is 50). In addition, the
preferential death of cultured cord blood CD45RA+ T cells
with low expression of the apoptosis-regulating molecules
underestimates the extent of the decrease, since the remaining viable
cells have apparently higher concentrations of these molecules. Adult T
cells, on the other hand, do not decrease the expression of these
molecules to the same extent as cord T cells and are relatively
resistant to apoptosis under similar conditions (see Fig. 1).
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-chain cytokines, Bcl-2 and Bcl-xL
expression increased in every experiment performed (Fig. 6; representative staining profile for
CD45RA+ T cells cultured with or without IL-7 in Fig. 5, A and B). Although all the IL-2R common -chain
cytokines increase Bcl-2 and Bcl-xL expression, the greater
effect of IL-7 on cell survival and recovery suggests that signaling
components of the IL-7R, other than the -chain, also have a role in
the effects of this cytokine.
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We next investigated whether cytokine-expanded cord blood
CD45RA+ T cells could be induced to express CD45RO after
TCR ligation. Following stimulation with immobilized anti-CD3 for
48 h in the absence of APCs and costimulatory signals, cord blood
CD45RA+ RO- T cells that had been expanded by
cytokines for 6 days acquired CD45RO+ expression (Fig. 7, E–H). Although
the majority of cells at 48 h showed dual expression of both
CD45RA and CD45RO (Fig. 7), at later time points (5 days), the majority
of cells were CD45RA-,CD45R0+, confirming our
previous results (6, 7, 22). Freshly isolated cord blood
CD45RA+ T cells that were not cultured in cytokines could
also convert to CD45RO expression after anti-CD3 stimulation as
demonstrated previously (22). In addition, the small number of
CD45RA+ T cells remaining after 7 days of culture in the
absence of cytokines could also convert to CD45RO expression upon
stimulation with anti-CD3 Ab (data not shown).
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-chain cytokines induced striking increases in CD95 expression in
both CD4+ and CD8+ T cells (data not shown). It
was possible, therefore, that the major outcome of anti-CD3
stimulation was death rather than proliferation of cytokine-expanded
CD45RA+ T cells and that the cells that convert to CD45RO
were only a minor residual subset. To clarify this, we examined the
extent to which apoptosis and proliferation occurred after activation
with anti-CD3 (Fig. 8). After 24
h, there was an increase in apoptosis but proliferating cells were also
present. After 2 days (Fig. 8) and 5 days (not shown), proliferating
cells predominated, and cell numbers increased, indicating that
cytokine-expanded CD45RA+ T cells could be induced to
proliferate by TCR ligation.
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Telomere length has been used to compare the replicative
history as residual replicative capacity of T cell subpopulations,
although the modulating effects of telomerase, which is able to add
back telomeric sequences, must also be considered. We therefore looked
for telomere shortening after IL-7-induced proliferation.
CD45RA+ cord blood T cells were cultured with anti-CD3
in the presence or absence of IL-2 or IL-7. Samples were collected for
both telomere length (Fig. 9) and
telomerase activity analysis (Fig. 10).
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). We found that the extensive
proliferation induced by IL-7 caused the mean TRF to decrease by only
0.5 kb (Fig. 9). Nevertheless, IL-7-cultured cord blood T cells still
maintained longer telomeres than control freshly isolated adult
CD45RA+ T cells.
We then investigated telomerase activity in these samples. Freshly
isolated cord blood CD45RA+ T cells had very low telomerase
activity (Fig. 10) which was maintained in cells cultured for 1 day
with either IL-2 or IL-7 but increased in anti-CD3-stimulated
cells. Telomerase activity was substantially higher after 4 days in
culture in both anti-CD3-treated cultures and cells cultured with
IL-7 alone. Cells cultured with IL-2 showed only a small up-regulation
of telomerase. Fig. 10 shows one representative experiment of four
performed to date. Under all experimental conditions, telomerase
activity returned to basal levels at later time points (e.g., day 11),
even when cytokines were replenished.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neonatal CD45RA+ T cells were more susceptible to
spontaneous apoptosis than adult CD45RA+ T cells. This was
associated with the inability of the former population to maintain the
expression of anti-apoptotic molecules such as Bcl-2 and
Bcl-xL. Since neonatal CD45RA+ T cells are
susceptible to apoptosis, factors that prevent death must be present in
vivo to enable expansion of the immune system in newborns (4). We have
shown that the IL-2R -chain-signaling cytokines (in particular IL-7)
are a group of factors that not only prevent apoptosis but also induce
proliferation of cord blood CD45RA+ T cells. One possible
reason for the preferential action of IL-7 in neonatal T cells is the
constitutive expression of the IL-7 receptor on cord compared with
adult CD45RA+ T cells (35, 36). These data support and
extend previous reports showing that postthymic peripheral T cells in
neonates require the presence of additional factors to attain full
maturity (22) by revealing that the presence of anti-apoptotic
factors is also required. Previous studies support the hypothesis that
IL-7 and other IL-2R common -chain-signaling cytokines may have a
central involvement in the maintenance of homeostasis of T cells at
both early and late stages of differentiation (37, 38, 39, 40). These studies
also suggest that IL-7 exerts its action on cord and adult T cell
survival in part via a Bcl-2-dependent mechanism, which supports our
current and previous observations (23, 37, 38). Our data, reported
here, suggest that IL-2, IL-4, and IL-15, which also signal via the
-chain of the IL-2R, up-regulate Bcl-2 and Bcl-xL in
cord CD45RA+ T cells. IL-7, however, consistently has a
greater effect on survival of these T cells. This suggests that Bcl-2
and Bcl-xL up-regulation is not the only way by which IL-7
exerts its effect, other signals perhaps mediated by the IL-7R
-chain may also contribute to the proliferation/survival of cord
CD45RA+ T cells (38). In addition to its role in
maintaining homeostasis, IL-7 has also been shown to be essential for
the functional development of neonatal T cells and induces the initial
expression of IL-4 by activated neonatal CD4+ T cells (35).
The non-Ag-specific expansion of CD45RA+ T cells may also
be induced by other cytokines apart from IL-7. It has been reported
that a combination of IL-2, TNF-, and IL-6 can stimulate
CD45RA+ RO- T cell proliferation without a
switch to CD45RO+RA- (11, 12). In both these
and our studies, the cytokine-expanded cells can be stimulated through
the TCR to switch to CD45RO expression (11, 12). It is of interest that
cytokine-expanded CD45RA+ T cells express increased levels
of CD95 and other adhesion molecules (12) (N. Borthwick and A. Akbar,
unpublished observations). Previous studies demonstrated that a
proportion of freshly isolated CD45RA+ T cells in adults
express increased levels of adhesion molecules (41, 42). It has been
suggested that these CD45RA+ T cells were revertant
CD45RO+ T cells (41, 42). In contrast, our results together
with those of Unutmaz et al. (12) suggest the possibility
that these cells may be cytokine-expanded CD45RA+ T cells.
We have also shown that IL-4 alone could induce the proliferation of
cord CD45RA+ T cells in agreement with previous reports
(43) and have extended these observations by showing that that
conversion to CD45RO+ did not occur. Other cytokines that
induce bystander T cell proliferation (44) should now also be
investigated for their effect on the CD45RA+ T cell pool.
Cell proliferation is generally associated with telomere shortening, which results in replicative senescence (13, 14, 15, 16). Telomeric repeats are synthesized by telomerase, a riboprotein that uses its RNA component as a template (45, 46). Telomerase is constitutively expressed on germline and tumor cells (15). Although most somatic cells do not express telomerase, the low activity seen in resting T cells is increased upon activation in vitro (14, 47, 48, 49, 50, 51) and in vivo4. Up-regulation of telomerase activity in activated T lymphocytes may retard the loss of telomeres and the development of senescence (13, 14, 15, 16). We therefore investigated whether cytokine-driven expansion of CD45RA+ T cells induced changes in telomerase activity and telomere length. We found low telomerase activity in ex vivo naive T cells derived from cord blood, confirming previous observations on peripheral blood T cells (48, 52, 53). Surprisingly, we found that upon culture with IL-7, cord blood T cells strongly up-regulated telomerase activity to the same extent as anti-CD3-stimulated cells. After an initial burst of strong telomerase activity which lasted for 4–6 days, it declined dramatically. In agreement with previous reports, we found no significant telomerase up-regulation upon culture with IL-2 alone. Cord blood T cells cultured with IL-7 were found to have decreased telomere length after a 12-day period, suggesting that under these in vitro conditions, telomerase activity may not sufficient to completely abrogate the progress toward replicative senescence. Nevertheless, the mean TRF length was still longer in IL-7-expanded CD45RA+ T cells than in adult CD45RA+ T cells. It would be predicted that excessive expansion by CD45RA+ T cells would eventually result in senescence and growth arrest. Since IL-7-expanded T cells acquire resistance to apoptosis, this may be an important mechanism, which protects against uncontrolled cytokine-mediated expansion of the naïve/unprimed T cell pool. These results are compatible with observations that in aged subjects, CD45RA+ T cells have shorter telomeres than similar cells from young individuals, the former cells having progressed further toward senescence than the latter (19). Although this may be the result of reversion of CD45RO+ T cells with shorter telomeres to CD45RA+ expression, our current data suggest that bystander activation by cytokines, which aged cells have experienced more frequently, may also contribute to these observations. Collectively, these results suggest that IL-7 contributes to the maintenance of the naive T cell pool by slowing down rather than completely abrogating the development of senescence of expanded CD45RA+ T cells.
The factors that regulate the size of the CD45RA+ T cell pool in adults are not well defined, but clearly, mechanisms that enable the replenishment of CD45RA+ T cells throughout life are required to prevent the eventual loss of this subset through recruitment, by activation, into the CD45RO+ pool followed by removal through apoptosis or replicative senescence (14, 54). Apart from reversion of cells from CD45RO to CD45RA (9), we now suggest that expansion of CD45RA+ T cells by IL-7 may be a mechanism for extrathymic expansion of the developing T cell system in neonates in a non-Ag-specific manner and may also contribute to the maintenance of the CD45RA+ T cell pool in adults.
An important question is whether there is sufficient IL-7 production in vivo to enable CD45RA+ T cell stimulation to occur. It has been shown that IL-7 is found in adult human intestinal epithelial cells and is intimately involved in the regulation of mucosal lymphocyte proliferation (36). Also, the recently reported development of chronic colitis in IL-7-transgenic mice indicates that chronic inflammation at this site may be mediated by excessive colonic epithelial cell-derived IL-7 (55). We have also recently demonstrated that there is high level of IL-7 expression by epithelial cells in neonatal gut (L. Poulter, D. Howie, T. T. MacDonald, and A. Akbar, unpublished observations) and that considerable levels of T cell proliferation can be observed in this tissue (56). These data are compatible with the idea that IL-7-driven proliferation may take place in certain anatomic sites in both adults and neonates.
In summary, we have provided evidence that CD45RA+ T cell proliferation induced by cytokines such as IL-7 which induce proliferation without a switch to CD45RO expression. This cytokine-mediated expansion may have an important role in extrathymic expansion of neonatal cells during growth and may also contribute to the maintenance of a naive T cell repertoire in adults. Further investigations into the mechanisms that regulate the homeostasis of the CD45RA+ T cell pool are important to rationalize ways in which the naive T cell compartment may be reconstituted in patients who are immunodeficient for various reasons.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arne N. Akbar, Department of Clinical Immunology, The Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. E-mail address:
3 Abbreviations used in this paper: PI, propidium iodine; MFI, mean fluorescence intensity; TRAP, telomeric repeats amplification protocol; TRF, terminal restriction fragment.
4 Maini, M. K., M. V. D. Soares, C. F. Zilch, A. N. Akbar, and P. C. L. Beverley. Telomerase up-regulation in an acute viral infection: a mechanism to maintain the replicative capacity of CD8+ T cells undergoing clonal expansion. Submitted for publication.
Received for publication May 11, 1998. Accepted for publication July 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells. Eur. J. Immunol. 19:771.[Medline]
-chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185:189.This article has been cited by other articles:
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  A. Andersson, S.-C. Yang, M. Huang, L. Zhu, U. K. Kar, R. K. Batra, D. Elashoff, R. M. Strieter, S. M. Dubinett, and S. Sharma IL-7 Promotes CXCR3 Ligand-Dependent T Cell Antitumor Reactivity in Lung Cancer J. Immunol., June 1, 2009; 182(11): 6951 - 6958. [Abstract] [Full Text] [PDF]
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 R. I. Azevedo, M. V. D. Soares, J. T. Barata, R. Tendeiro, A. Serra-Caetano, R. M. M. Victorino, and A. E. Sousa IL-7 sustains CD31 expression in human naive CD4+ T cells and preferentially expands the CD31+ subset in a PI3K-dependent manner Blood, March 26, 2009; 113(13): 2999 - 3007. [Abstract] [Full Text] [PDF]
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S. O. Alpdogan, S. X. Lu, N. Patel, S. McGoldrick, D. Suh, T. Budak-Alpdogan, O. M. Smith, J. Grubin, C. King, G. L. Goldberg, et al. Rapidly proliferating CD44hi peripheral T cells undergo apoptosis and delay posttransplantation T-cell reconstitution after allogeneic bone marrow transplantation Blood, December 1, 2008; 112(12): 4755 - 4764. [Abstract] [Full Text] [PDF]
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M. Lavanya, S. Kinet, A. Montel-Hagen, C. Mongellaz, J.-L. Battini, M. Sitbon, and N. Taylor Cell Surface Expression of the Bovine Leukemia Virus-Binding Receptor on B and T Lymphocytes Is Induced by Receptor Engagement J. Immunol., July 15, 2008; 181(2): 891 - 898. [Abstract] [Full Text] [PDF]
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 S. M. Churchman and F. Ponchel Interleukin-7 in rheumatoid arthritis Rheumatology, June 1, 2008; 47(6): 753 - 759. [Abstract] [Full Text] [PDF]
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Y. Yang, J. An, and N.-p. Weng Telomerase Is Involved in IL-7-Mediated Differential Survival of Naive and Memory CD4+ T Cells J. Immunol., March 15, 2008; 180(6): 3775 - 3781. [Abstract] [Full Text] [PDF]
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R. D. Kilpatrick, T. Rickabaugh, L. E. Hultin, P. Hultin, M. A. Hausner, R. Detels, J. Phair, and B. D. Jamieson Homeostasis of the Naive CD4+ T Cell Compartment during Aging J. Immunol., February 1, 2008; 180(3): 1499 - 1507. [Abstract] [Full Text] [PDF]
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A. K. Damjanovic, Y. Yang, R. Glaser, J. K. Kiecolt-Glaser, H. Nguyen, B. Laskowski, Y. Zou, D. Q. Beversdorf, and N.-p. Weng Accelerated Telomere Erosion Is Associated with a Declining Immune Function of Caregivers of Alzheimer's Disease Patients J. Immunol., September 15, 2007; 179(6): 4249 - 4254. [Abstract] [Full Text] [PDF]
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A. N. Akbar and M. Vukmanovic-Stejic Telomerase in T Lymphocytes: Use It and Lose It? J. Immunol., June 1, 2007; 178(11): 6689 - 6694. [Abstract] [Full Text] [PDF]
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L. Swainson, S. Kinet, C. Mongellaz, M. Sourisseau, T. Henriques, and N. Taylor IL-7-induced proliferation of recent thymic emigrants requires activation of the PI3K pathway Blood, February 1, 2007; 109(3): 1034 - 1042. [Abstract] [Full Text] [PDF]
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L. Swainson, E. Verhoeyen, F.-L. Cosset, and N. Taylor IL-7R{alpha} Gene Expression Is Inversely Correlated with Cell Cycle Progression in IL-7-Stimulated T Lymphocytes. J. Immunol., June 1, 2006; 176(11): 6702 - 6708. [Abstract] [Full Text] [PDF]
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K.-i. Yamanaka, R. Clark, B. Rich, R. Dowgiert, K. Hirahara, D. Hurwitz, M. Shibata, N. Mirchandani, D. A. Jones, D. S. Goddard, et al. Skin-derived interleukin-7 contributes to the proliferation of lymphocytes in cutaneous T-cell lymphoma Blood, March 15, 2006; 107(6): 2440 - 2445. [Abstract] [Full Text] [PDF]
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J. S. Sutherland, G. L. Goldberg, M. V. Hammett, A. P. Uldrich, S. P. Berzins, T. S. Heng, B. R. Blazar, J. L. Millar, M. A. Malin, A. P. Chidgey, et al. Activation of Thymic Regeneration in Mice and Humans following Androgen Blockade J. Immunol., August 15, 2005; 175(4): 2741 - 2753. [Abstract] [Full Text] [PDF]
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Y. Li, W. Zhi, P. Wareski, and N.-p. Weng IL-15 Activates Telomerase and Minimizes Telomere Loss and May Preserve the Replicative Life Span of Memory CD8+ T Cells In Vitro J. Immunol., April 1, 2005; 174(7): 4019 - 4024. [Abstract] [Full Text] [PDF]
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G. Tully, C. Kortsik, H. Hohn, I. Zehbe, W. E. Hitzler, C. Neukirch, K. Freitag, K. Kayser, and M. J. Maeurer Highly Focused T Cell Responses in Latent Human Pulmonary Mycobacterium tuberculosis Infection J. Immunol., February 15, 2005; 174(4): 2174 - 2184. [Abstract] [Full Text] [PDF]
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B. Vasir, D. Avigan, Z. Wu, K. Crawford, S. Turnquist, J. Ren, and D. Kufe Dendritic Cells Induce MUC1 Expression and Polarization on Human T Cells by an IL-7-Dependent Mechanism J. Immunol., February 15, 2005; 174(4): 2376 - 2386. [Abstract] [Full Text] [PDF]
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 P. T Ngom, A. C Collinson, J. Pido-Lopez, S. M Henson, A. M Prentice, and R. Aspinall Improved thymic function in exclusively breastfed infants is associated with higher interleukin 7 concentrations in their mothers' breast milk Am. J. Clinical Nutrition, September 1, 2004; 80(3): 722 - 728. [Abstract] [Full Text] [PDF]
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 D. C. Macallan, D. Wallace, Y. Zhang, C. de Lara, A. T. Worth, H. Ghattas, G. E. Griffin, P. C.L. Beverley, and D. F. Tough Rapid Turnover of Effector-Memory CD4+ T Cells in Healthy Humans J. Exp. Med., July 19, 2004; 200(2): 255 - 260. [Abstract] [Full Text] [PDF]
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J. R. Reed, M. Vukmanovic-Stejic, J. M. Fletcher, M. V. D. Soares, J. E. Cook, C. H. Orteu, S. E. Jackson, K. E. Birch, G. R. Foster, M. Salmon, et al. Telomere Erosion in Memory T Cells Induced by Telomerase Inhibition at the Site of Antigenic Challenge In Vivo J. Exp. Med., May 17, 2004; 199(10): 1433 - 1443. [Abstract] [Full Text] [PDF]
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A. Muthukumar, A. Wozniakowski, M.-C. Gauduin, M. Paiardini, H. M. McClure, R. P. Johnson, G. Silvestri, and D. L. Sodora Elevated interleukin-7 levels not sufficient to maintain T-cell homeostasis during simian immunodeficiency virus-induced disease progression Blood, February 1, 2004; 103(3): 973 - 979. [Abstract] [Full Text] [PDF]
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S. O. Schonland, J. K. Zimmer, C. M. Lopez-Benitez, T. Widmann, K. D. Ramin, J. J. Goronzy, and C. M. Weyand Homeostatic control of T-cell generation in neonates Blood, August 15, 2003; 102(4): 1428 - 1434. [Abstract] [Full Text] [PDF]
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S. Cavalieri, S. Cazzaniga, M. Geuna, Z. Magnani, C. Bordignon, L. Naldini, and C. Bonini Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence Blood, July 15, 2003; 102(2): 497 - 505. [Abstract] [Full Text] [PDF]
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S. Jaleco, L. Swainson, V. Dardalhon, M. Burjanadze, S. Kinet, and N. Taylor Homeostasis of Naive and Memory CD4+ T Cells: IL-2 and IL-7 Differentially Regulate the Balance Between Proliferation and Fas-Mediated Apoptosis J. Immunol., July 1, 2003; 171(1): 61 - 68. [Abstract] [Full Text] [PDF]
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R. M. O'Neill, J. Hassan, and D. J. Reen IL-7-Regulated Homeostatic Maintenance of Recent Thymic Emigrants in Association with Caspase-Mediated Cell Proliferation and Apoptotic Cell Death J. Immunol., May 1, 2003; 170(9): 4524 - 4531. [Abstract] [Full Text] [PDF]
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E. Verhoeyen, V. Dardalhon, O. Ducrey-Rundquist, D. Trono, N. Taylor, and F.-L. Cosset IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes Blood, March 15, 2003; 101(6): 2167 - 2174. [Abstract] [Full Text] [PDF]
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C. Bello-Fernandez, J. Stasakova, A. Renner, N. Carballido-Perrig, M. Koening, M. Waclavicek, O. Madjic, L. Oehler, O. Haas, J. M. Carballido, et al. Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties Blood, March 15, 2003; 101(6): 2184 - 2190. [Abstract] [Full Text] [PDF]
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T. J. Fry, M. Moniuszko, S. Creekmore, S. J. Donohue, D. C. Douek, S. Giardina, T. T. Hecht, B. J. Hill, K. Komschlies, J. Tomaszewski, et al. IL-7 therapy dramatically alters peripheral T-cell homeostasis in normal and SIV-infected nonhuman primates Blood, March 15, 2003; 101(6): 2294 - 2299. [Abstract] [Full Text] [PDF]
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N. Manel, S. Kinet, J.-L. Battini, F. J. Kim, N. Taylor, and M. Sitbon The HTLV receptor is an early T-cell activation marker whose expression requires de novo protein synthesis Blood, March 1, 2003; 101(5): 1913 - 1918. [Abstract] [Full Text] [PDF]
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S. Jaleco, S. Kinet, J. Hassan, V. Dardalhon, L. Swainson, D. Reen, N. Taylor, and L. Al-Harthi IL-7 and CD4+ T-cell proliferation Blood, December 15, 2002; 100(13): 4676 - 4677. [Full Text] [PDF]
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 D. D. Scripture-Adams, D. G. Brooks, Y. D. Korin, and J. A. Zack Interleukin-7 Induces Expression of Latent Human Immunodeficiency Virus Type 1 with Minimal Effects on T-Cell Phenotype J. Virol., November 13, 2002; 76(24): 13077 - 13082. [Abstract] [Full Text] [PDF]
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E. Davila, M. G. Velez, C. J. Heppelmann, and E. Celis Creating space: an antigen-independent, CpG-induced peripheral expansion of naive and memory T lymphocytes in a full T-cell compartment Blood, September 18, 2002; 100(7): 2537 - 2545. [Abstract] [Full Text] [PDF]
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S. Kinet, F. Bernard, C. Mongellaz, M. Perreau, F. D. Goldman, and N. Taylor gp120-mediated induction of the MAPK cascade is dependent on the activation state of CD4+ lymphocytes Blood, September 18, 2002; 100(7): 2546 - 2553. [Abstract] [Full Text] [PDF]
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A. Suarez, L. Mozo, and C. Gutierrez Generation of CD4+CD45RA+ Effector T Cells by Stimulation in the Presence of Cyclic Adenosine 5'-Monophosphate- Elevating Agents J. Immunol., August 1, 2002; 169(3): 1159 - 1167. [Abstract] [Full Text] [PDF]
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T. J. Fry and C. L. Mackall Interleukin-7: from bench to clinic Blood, May 13, 2002; 99(11): 3892 - 3904. [Full Text] [PDF]
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C. M. Steffens, E. Z. Managlia, A. Landay, and L. Al-Harthi Interleukin-7-treated naive T cells can be productively infected by T-cell-adapted and primary isolates of human immunodeficiency virus 1 Blood, May 1, 2002; 99(9): 3310 - 3318. [Abstract] [Full Text] [PDF]
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Y. Okamoto, D. C. Douek, R. D. McFarland, and R. A. Koup Effects of exogenous interleukin-7 on human thymus function Blood, April 15, 2002; 99(8): 2851 - 2858. [Abstract] [Full Text] [PDF]
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M. Maurice, E. Verhoeyen, P. Salmon, D. Trono, S. J. Russell, and F.-L. Cosset Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide Blood, April 1, 2002; 99(7): 2342 - 2350. [Abstract] [Full Text] [PDF]
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S. Kimmig, G. K. Przybylski, C. A. Schmidt, K. Laurisch, B. Mowes, A. Radbruch, and A. Thiel Two Subsets of Naive T Helper Cells with Distinct T Cell Receptor Excision Circle Content in Human Adult Peripheral Blood J. Exp. Med., March 18, 2002; 195(6): 789 - 794. [Abstract] [Full Text] [PDF]
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O. Alpdogan, C. Schmaltz, S. J. Muriglan, B. J. Kappel, M.-A. Perales, J. A. Rotolo, J. A. Halm, B. E. Rich, and M. R. M. van den Brink Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease Blood, October 1, 2001; 98(7): 2256 - 2265. [Abstract] [Full Text] [PDF]
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J. Hassan and D. J. Reen Human Recent Thymic Emigrants-Identification, Expansion, And Survival Characteristics J. Immunol., August 15, 2001; 167(4): 1970 - 1976. [Abstract] [Full Text] [PDF]
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 V. Dardalhon, S. Jaleco, S. Kinet, B. Herpers, M. Steinberg, C. Ferrand, D. Froger, C. Leveau, P. Tiberghien, P. Charneau, et al. IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells PNAS, July 19, 2001; (2001) 161272698. [Abstract] [Full Text] [PDF]
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L. Vivien, C. Benoist, and D. Mathis T lymphocytes need IL-7 but not IL-4 or IL-6 to survive in vivo Int. Immunol., June 1, 2001; 13(6): 763 - 768. [Abstract] [Full Text] [PDF]
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T. J. Fry, B. L. Christensen, K. L. Komschlies, R. E. Gress, and C. L. Mackall Interleukin-7 restores immunity in athymic T-cell-depleted hosts Blood, March 15, 2001; 97(6): 1525 - 1533. [Abstract] [Full Text] [PDF]
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L. A. Geiselhart, C. A. Humphries, T. A. Gregorio, S. Mou, J. Subleski, and K. L. Komschlies IL-7 Administration Alters the CD4:CD8 Ratio, Increases T Cell Numbers, and Increases T Cell Function in the Absence of Activation J. Immunol., March 1, 2001; 166(5): 3019 - 3027. [Abstract] [Full Text] [PDF]
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K. Weinberg, B. R. Blazar, J. E. Wagner, E. Agura, B. J. Hill, M. Smogorzewska, R. A. Koup, M. R. Betts, R. H. Collins, and D. C. Douek Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation Blood, March 1, 2001; 97(5): 1458 - 1466. [Abstract] [Full Text] [PDF]
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S. Gozalo-Sanmillan, J. M. McNally, M. Y. Lin, C. A. Chambers, and L. J. Berg Cutting Edge: Two Distinct Mechanisms Lead to Impaired T Cell Homeostasis in Janus Kinase 3- and CTLA-4-Deficient Mice J. Immunol., January 15, 2001; 166(2): 727 - 730. [Abstract] [Full Text] [PDF]
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E. C. Tsark, M. A. Dao, X. Wang, K. Weinberg, and J. A. Nolta IL-7 Enhances the Responsiveness of Human T Cells That Develop in the Bone Marrow of Athymic Mice J. Immunol., January 1, 2001; 166(1): 170 - 181. [Abstract] [Full Text] [PDF]
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G. Lombardi, P. J. Dunne, D. Scheel-Toellner, T. Sanyal, D. Pilling, L. S. Taams, P. Life, J. M. Lord, M. Salmon, and A. N. Akbar Type 1 IFN Maintains the Survival of Anergic CD4+ T Cells J. Immunol., October 1, 2000; 165(7): 3782 - 3789. [Abstract] [Full Text] [PDF]
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V. Dardalhon, S. Jaleco, C. Rebouissou, C. Ferrand, N. Skander, L. Swainson, P. Tiberghien, H. Spits, N. Noraz, and N. Taylor Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector Blood, August 1, 2000; 96(3): 885 - 893. [Abstract] [Full Text] [PDF]
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N. C. Matthews, M. Wadhwa, C. Bird, F. E. Borras, and C. V. Navarrete Sustained Expression of CD154 (CD40L) and Proinflammatory Cytokine Production by Alloantigen-Stimulated Umbilical Cord Blood T Cells J. Immunol., June 15, 2000; 164(12): 6206 - 6212. [Abstract] [Full Text] [PDF]
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D. Unutmaz, V. N. KewalRamani, S. Marmon, and D. R. Littman Cytokine Signals Are Sufficient for HIV-1 Infection of Resting Human T Lymphocytes J. Exp. Med., June 7, 1999; 189(11): 1735 - 1746. [Abstract] [Full Text] [PDF]
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M. K. Maini, M. V. D. Soares, C. F. Zilch, A. N. Akbar, and P. C. L. Beverley Virus-Induced CD8+ T Cell Clonal Expansion Is Associated with Telomerase Up-Regulation and Telomere Length Preservation: A Mechanism for Rescue from Replicative Senescence J. Immunol., April 15, 1999; 162(8): 4521 - 4526. [Abstract] [Full Text] [PDF]
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V. Dardalhon, S. Jaleco, S. Kinet, B. Herpers, M. Steinberg, C. Ferrand, D. Froger, C. Leveau, P. Tiberghien, P. Charneau, et al. IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells PNAS, July 31, 2001; 98(16): 9277 - 9282. [Abstract] [Full Text] [PDF]
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