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*
Imperial Cancer Research Fund Tumour Immunology Unit and
Departments of Oncology and Sexually Transmitted Diseases, University College London Medical School, London, United Kingdom;
Department of Clinical Immunology, Royal Free Hospital Medical School, London, United Kingdom; and
§
The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, United Kingdom.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Telomeres are terminal chromosome structures consisting of hexameric repeats that progressively shorten with each successive round of cell division due to the inability of DNA polymerase to replicate the terminal portion (7). The mean telomere length of a phenotypically defined population of cells is therefore thought to indicate its mitotic history and residual replicative potential (8). Consistent with this, memory and effector T cells have been shown to have shorter telomeres than naive cells and a corresponding reduction in replicative capacity in vitro (9, 10). Critically shortened telomeres might therefore be expected to limit the replicative capacity of Ag-driven clones in chronic infection following AIM. This would impair the ability of memory cells to respond to reactivation of virus. However, the activity of the enzyme telomerase, which is capable of adding telomeric repeats back on to chromosome ends, needs to be taken into account (11). Low levels of telomerase activity have been detected in lymphocytes, and up-regulation of activity has been demonstrated on in vitro activation and in vivo in selected subpopulations, such as thymic and bone marrow progenitor T cells (12, 13) and germinal center B cells (14). Here, we have investigated telomere length and telomerase activity in AIM T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight patients (age range 18–24 yr) diagnosed with AIM on the basis of characteristic clinical and laboratory features were studied. Patients were first sampled within 10 days of symptom onset and followed for up to 1 yr. Controls were age-matched healthy volunteers who had not had an illness suggestive of infectious mononucleosis. Local ethical committee approval for the study was obtained.
PBMC were isolated from heparinized blood samples by Ficoll-Hypaque density centrifugation, and the nonadherent fraction was recovered after a 1-h plastic adherence step at 37°C. T cell subsets were isolated by positive selection using directly conjugated anti-CD4 or anti-CD8 MiniMacs magnetic beads (Militenyi Biotech, Bisley, Surrey, U.K.). Separated fractions were always >95% pure, as assessed by mAb staining and FACS analysis.
Phenotyping and purification procedure for TCRBV expansion
Expression of activation markers by CD4+ and CD8+ subsets was assessed by flow cytometric analysis following triple staining of nonadherent PBMC with directly conjugated Abs to CD3, CD4, or CD8, and CD45RO or HLA-DR (all from Sigma, Poole, U.K.) or CD28 (Becton Dickinson, San Jose, CA). Expression of these markers by the BV expansion was analyzed by gating on CD8+ T cells that had also been stained with Abs to BV22 (Immunotech, Marseille, France) and CD45RO or HLA-DR or CD28.
BV22+CD8+ T cells were purified by incubating CD4-depleted T cells with anti-BV22 mAb for 30 min on ice, followed by goat anti-mouse Ig MiniMacs beads and fractionation on a MiniMacs column. The resultant population was 87% BV22+ 97% CD8+ T cells. The negative fraction was then used to perform a further positive selection for all remaining CD8+ T cells, using directly conjugated anti-CD8 MiniMacs beads (purity 98%). This selection procedure ensured that CD4+ T cells (purity 99% in this experiment), CD8+BV22+ T cells, and remaining CD8+ T cells had all been through a positive selection step before telomere length and telomerase analysis.
Terminal restriction fragment (TRF) length measurement
Genomic DNA was extracted from 1–3 million purified
CD4+/CD8+ T cells and 2 µg digested with
MspI and RsaI (Pharmacia Biotech, Herts, U.K.).
Digested DNA was electrophoresed through a 0.7% agarose gel in 1x
Tris acetate. The gel was depurinated briefly, alkaline
denatured, neutralized, and the DNA transferred in 20x SSC to a nylon
membrane (Hybond N+; Amersham Life Science, Amersham, U.K.),
which was fixed by baking at 80°C. Membranes were prehybridized in
Rapid Hyb Buffer (Amersham), hybridized at 42°C for 1 h with the
oligonucleotide (TTAGGG)3, which had been end-labeled with
32P
ATP using polynucleotide kinase (Pharmacia), and
washed. TRF length was calculated from the phosphor imager (using
ImageQuant) or by densitometry of autoradiographs using Molecular
Analyst software (Bio-Rad, Hemel Hempstead, U.K.). The mean TRF
length was calculated as described (7).
Telomerase assay
A modified version of the telomeric repeat amplification
protocol (TRAP) was used (Oncor, Gaithersburg, MD) (15). Extracts from
varying cell numbers were used for telomeric elongation at 30° for 30
min, using a 33PATP end-labeled primer. The same sample
was used for PCR amplification, using 25–28 cycles of 30 sec at 94°C
and 30 sec at 59°C. PCR product (equivalent to 500-5000 cells) was
run on a 12% polyacrylamide gel. Telomerase activity was quantified by
comparison between samples of the number and strength of telomeric
repeat bands divided by the strength of the internal PCR control band
(which also served to indicate the absence of TAQ inhibitors). The
positive control was cell extract from the immortalized 293 cell line;
negative controls were conducted with a heat-inactivated sample for
each cell extract (to inactivate the RNA template), and in one tube,
lysis buffer was used in place of cell extract.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At a time when most circulating CD8+ T cells had a
phenotype previously associated with shortened telomeres
(CD45RO+ CD28-) and contained oligoclonal
expansions generated by repeated cell division, there was a paradoxical
maintenance of telomere length. In all eight AIM patients analyzed,
mean TRF length was longer in the CD8+ than the
CD4+ fraction (Fig. 1
,
A and B), with a mean ± SD difference in
TRF length of 0.69 ± 0.17 kb, p < 0.0001 (paired
t test), p = 0.01 (Wilcoxon matched-pairs
signed-ranks test). At this time, the CD4+ T cells
contained many fewer cells with an activated phenotype than the
CD8+ T cells (illustrated for one patient in Fig. 4A) and had no clonal expansions detectable by a very
sensitive heteroduplex method (16 and M.K.M, unpublished data).
They would therefore be expected to have longer telomeres than the
CD8+ T cells. In five healthy age-matched controls and in
five AIM patients sampled after disease resolution, mean TRF length was
not longer in CD8+ than in CD4+ T cells (Fig. 1C). Similarly, in a previous study of nine healthy controls
(17), there was no significant difference between mean TRF length in
CD4+ and CD8+ T cell fractions. The finding
that the CD8+ T cells have longer telomeres than
CD4+ T cells in AIM strongly suggests differential
telomeric replacement counteracting the expected shortening.
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The difference in CD8+ and CD4+ telomere
length was not maintained on disease resolution (Fig. 1
C).
To examine this normalization in more detail, telomere lengths were
measured sequentially for CD4+ and CD8+ subsets
from patients sampled at least three times. All comparisons of TRF
between subsets and time points for an individual were made from
samples run in parallel on the same gel to minimize technical
variations in measurement. As shown for two patients (Fig. 2), from 3 mo after AIM onwards, when
lymphocytosis had resolved and large TCRBV expansions were no longer
detectable with mAbs, there was no difference in mean telomere length
between CD4+ and CD8+ T cells. Samples from
five of the AIM patients during convalescence showed more telomeric
shortening in CD8+ (range of loss of 0.8–1.7 kb) than
CD4+ (0–0.8 kb) T cells, over 3–11 mo follow-up. This
suggests that telomere length is only maintained in the first month of
the infection when CD8 cellular proliferation is most intense;
unopposed telomeric loss in this early phase would leave little reserve
for the subsequent shortening seen. Longitudinal sampling allowed the
patients to serve as their own internal controls, strengthening the
significance of the CD4/CD8 differences observed only in acute disease.
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There was an up-regulation of telomerase activity in both CD4 and
CD8 purified fractions from AIM samples. Fig. 3 shows telomerase activity quantified by
serial dilutions and densitometry of the ladder generated by enzyme
activity divided by the internal PCR control band. The relative
telomerase activity in AIM CD4+ T cells was estimated to be
between 35 and 43% that of the positive control 293 cells in different
donors, while that in CD8+ T cells was even higher, with
relative activities of 45–70% (Fig. 3, A and
B). Telomerase activity was undetectable or minimal in the
CD4+ and CD8+ T cells of healthy age-matched
controls (Fig. 3A). We could detect telomerase activity on
serial dilution from an extract equivalent to 50 CD8+ AIM T
cells, whereas telomerase activity was never detected in extracts from
<1000 cells from healthy age-matched controls by us or others (12).
TRAP assays with more PCR cycles, to enhance detection of low-level
activity, supported this estimate of telomerase activity being at least
10-fold higher in CD4+ and CD8+ T cells from
AIM patients than healthy age-matched controls. In two AIM patients, in
whom we examined convalescent samples at 6–10 wk, telomerase activity
in CD4+ and CD8+ T cells had decreased by 2- to
4-fold (Fig. 3B). This was consistent with the telomeric
shortening observed and with in vitro data showing that repeated
restimulation of T cells results in eventual telomeric shortening,
partly attributed to attenuated levels of telomerase (18, 19).
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To further investigate the unexpected finding of telomere length
preservation in the context of CD8 clonal expansion, an oligoclonal
TCRBV expansion was examined. A BV22 expansion, accounting for 10% of
an AIM patient’s circulating CD8+ T cells, was separated
from the remaining CD8+ T cells (which contained other
smaller clonal expansions as demonstrated by the heteroduplex assay
(data not shown)). This BV22 expansion had a higher proportion of
HLA-DR+ CD28- staining (as a measure of
activation status) than the rest of the CD8+ T cells (Fig. 4A), and heteroduplex analysis
showed one large and two smaller clonal TCR expansions within it,
accounting for most of the BV22 CD8+ T cells (data not
shown). The oligoclonally expanded population was shown to have a
longer mean telomere length (9.3 kb) than the rest of the
CD8+ T cells (8.7 kb) (Fig. 4B). This provided
clear confirmation that cells that have undergone repeated rounds of
division can maintain long telomeres in the acute stage of this
disease. In another AIM patient, mean telomere length of the
CD8+ T cells was found to shorten by 0.3 kb following
removal of a BV14 expansion accounting for 13% of the total
CD8+ T cells, providing further evidence for the presence
of relatively long telomeres in clonally expanded CD8+ T
cells. Preliminary data in two AIM patients do not show telomeric
shortening of the CD8+CD28- subset (data not
shown). At the point of sampling, many of the CD8+ T cells
were acutely activated and cycling, and therefore differed from the
relatively resting CD8+CD28- subset in healthy
individuals, 8 out of 10 of whom showed telomeric shortening relative
to the CD28+ CD8+ subset (10).
Telomerase levels in the oligoclonal expansion were much higher than in healthy controls, but slightly less than in the remaining CD8+ T cells (36% and 55% of the positive cell line control, respectively), perhaps because they had already reached their peak in this population. Telomerase activity is only measured at a single time point and can undergo rapid fluctuations, whereas telomere length is a cumulative measure. Lack of a direct quantitative relationship between telomerase levels and telomere length has been observed previously and suggests the involvement of other regulatory factors (20, 21, 22, 23).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mean and spread of telomere lengths of a population of lymphocytes represents the composite of the opposing effects of telomere shortening of 50–100 base pairs per cell division and the counteracting effects of any telomeric replacement. Although there is wide variability between individuals in absolute telomere lengths, the differences between CD4 and CD8 subsets within a sample are minimal and highly conserved in healthy controls (17). Our unexpected finding of telomere maintenance in a population of circulating CD8+ T cells that were known to be of activated/memory phenotype and to have undergone extensive clonal expansion, suggests that counteractive telomere-lengthening mechanisms can operate in vivo. One such mechanism is the activity of the ribonucleoprotein enzyme telomerase, which uses its own RNA template to synthesize telomeric repeats. The recent induction of telomerase activity in two human somatic cell types by transfection of the telomerase catalytic subunit has clearly demonstrated its association with telomere preservation and rescue from senescence (29). Telomerase activity has been demonstrated at very low levels in circulating lymphocytes of healthy individuals with the highly sensitive PCR-based TRAP technique, with the amount detected being inversely related to donor age (12). We demonstrate for the first time that human peripheral CD4+ and CD8+ T cells can up-regulate telomerase activity in vivo. The level of telomerase activity in AIM CD8+ T cells approximating that in a carcinoma cell line is consistent with the marked degree of lymphoproliferation seen in this condition. Telomere length maintenance would be compatible with the elevated telomerase levels seen, and has been observed to occur in vitro in the presence of elevated telomerase (18 and M.V.D.S. et al., unpublished data). Some initial change in telomere length of the CD4+ T cells cannot be excluded, especially since telomerase is also elevated in this fraction. However, telomere length was consistently longer in the CD8+ than CD4+ T cells in AIM, a difference not observed in healthy controls. The converse would be expected if telomeric shortening had occurred unopposed in AIM, since the generation of the extensive large clones is restricted to the CD8+ fraction and requires multiple divisions.
It is not possible to distinguish between actual physical shortening of telomeres of individual Ag-specific cells over time as opposed to the effects of changes in the cell composition of the subsets examined. Some of the apparent shortening on follow-up could be due to a reduction in the number of circulating Ag-specific cells. During recovery from the acute infection, the majority of cells with long telomeres could be lost by apoptosis, and the smaller number entering memory would no longer bias the telomere length measurement. Decreased telomerase activity with an ongoing high rate of cell division may also contribute to the situation we have observed in convalescence of equal CD4+ and CD8+ T cell telomere length.
Since the CD4+ and CD8+ populations are heterogeneous with respect to phenotype, the elevated TRF length and telomerase activity cannot definitely be attributed to a particular subset. At the stage of first sampling in AIM, many more CD8+ T cells were found to have acquired the CD45RO+, HLA-DR+ phenotype than the CD28- phenotype, suggesting that not all activated clonally expanding cells were CD28-. Direct visualization of EBV-specific CD8+ T cells in AIM using HLA-peptide tetramers (3) has confirmed that acutely Ag-activated cells are highly variable in their CD28 phenotype. CD28 subsets might therefore be expected to be heterogeneous with respect to telomere length in the setting of the dramatic T cell activation seen in the acute phase of EBV infection. The CD8+ T cells in AIM patients, many of which are highly activated and cycling, differ from the relatively resting population of CD8+ T cells, which show shortening of the CD28- subset in 8 out of 10 healthy individuals as reported previously (10). In addition, the clonally expanded CD8+ T cells examined in a healthy individual (10) differed from those seen in AIM in that they were all CD28- and CD57+, suggesting terminal differentiation. The presence of shortened telomeres in such long-term expansions but not in the Ag-specific oligoclonal expansions in acute EBV infection is consistent with our data and that from in vitro work (18) showing attenuation of compensatory telomerase production over time.
It is clear that, at least in vitro, normal T cells do undergo replicative senescence after a finite number of population doublings, found to average 23 for bulk cultures of CD8+ T cells activated by mitogen or irradiated allogeneic B lymphoblastoid cell lines (6, 30). Estimates of the number of population doublings involved in successive rounds of Ag-driven clonal expansion (6), and based on the size of the EBV-related clonal expansions we and others have documented, indicate that replicative senescence of specific responding clones could be a limitation in antiviral responses. In HIV infection, telomeric shortening in the expanded CD8+CD28- fraction has been postulated to contribute to clonal exhaustion and, hence, loss of viral control (31). This may be exacerbated by early and prolonged treatment with reverse transcriptase inhibitors, which have been shown to inhibit telomerase activity in immortalized cell lines (23). By contrast, we find EBV infection, which is characterized by an effective long-term memory response, to be associated with increased telomerase activity and preserved telomere length in the responding cells, which may protect against replicative senescence.
It is possible that EBV has an effect on telomerase activity, either directly or indirectly, for example by inducing changes in the cytokine environment that can alter telomerase activity (32). However, existing data suggest the telomerase up-regulation is more likely to be mediated by Ag-dependent activation, which has now been shown to induce telomerase activity in a transgenic mouse model (33). It remains to be seen whether such changes occur in other acute viral infections in humans. It would be instructive to be able to analyze changes in telomere length over the course of AIM in the Ag-specific population. With the use of EBV-specific HLA-peptide tetramers to sort these cells, such an approach should be possible in the future.
An equivalent paradoxical increase in telomere length in association with raised telomerase levels has recently been demonstrated in germinal center B cells, which also undergo extensive clonal expansion and selection (34). Germinal center B cells, like the T cells in AIM, are prone to apoptosis due to low Bcl-2 expression (35). Thus, extensive clonal expansion of cells prone to apoptosis and rescue of some of these cells with lengthened telomeres appear to be parallel mechanisms regulating entry into the memory pool in B and T cells. These data raise important considerations for the effective induction and maintenance of immunological memory following virus infection, vaccination, and immunotherapy with CTL.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. P. C. L. Beverley, The Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, U.K. E-mail address:
3 Abbreviations used in this paper: AIM, acute infectious mononucleosis; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment.
Received for publication September 30, 1998. Accepted for publication January 28, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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