Skip to main content

Main menu

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons

User menu

  • Subscribe
  • My alerts
  • Log in
  • Log out

Search

  • Advanced search
The Journal of Immunology
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons
  • Subscribe
  • My alerts
  • Log in
  • Log out
The Journal of Immunology

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Follow The Journal of Immunology on Twitter
  • Follow The Journal of Immunology on RSS

Virus-Induced CD8+ T Cell Clonal Expansion Is Associated with Telomerase Up-Regulation and Telomere Length Preservation: A Mechanism for Rescue from Replicative Senescence

Mala K. Maini, Maria Vieira D. Soares, Christian F. Zilch, Arne N. Akbar and Peter C. L. Beverley
J Immunol April 15, 1999, 162 (8) 4521-4526;
Mala K. Maini
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Maria Vieira D. Soares
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Christian F. Zilch
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Arne N. Akbar
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Peter C. L. Beverley
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

In acute infectious mononucleosis (AIM), very large clones of Ag-specific CD8+ effector T cells are generated. Many clones persist as memory cells, although the clone size is greatly reduced. It would be expected that the large number of cell divisions occurring during clonal expansion would lead to shortening of telomeres, predisposing to replicative senescence. Instead, we show that clonally expanded CD8+ T cells in AIM have paradoxical preservation of telomere length in association with marked up-regulation of telomerase. We postulate that this allows a proportion of responding T cells to enter the memory pool with a preserved capacity to continue dividing so that long-term immunological memory can be maintained.

In many acute viral infections, there is a massive increase in circulating T cells, followed by a rapid loss of most of these cells by apoptosis. Recent data have highlighted the large Ag-specific component to this CD8+ T cell proliferation, both in primary and secondary responses (1, 2, 3). Initial clonal burst size has been suggested to determine the duration of memory (4) and correlates with the size of the memory pool for a given epitope (2). In acute infectious mononucleosis (AIM)3, there is a striking CD8 lymphocytosis consisting of multiple, very large clonal expansions (5). Generation of such large clones would involve at least 28 population doublings in the acute phase (6), raising the possibility that cells enter memory with reduced replicative capacity.

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.

Materials and Methods

Isolation of PBMC and T cell subsets

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 1× Tris acetate. The gel was depurinated briefly, alkaline denatured, neutralized, and the DNA transferred in 20× 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 32PγATP 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 33PγATP 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.

Results

CD8+ T cells in AIM have paradoxically long telomeres compared with CD4+ T cells

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. 4⇓A) 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. 1⇓C). 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.

  FIGURE 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 1.

Telomere length in CD8+ compared with CD4+ T cells in AIM donors and controls. Digested genomic DNA from CD4+ and CD8+ purified T cells from AIM patients was electrophoresed through a 0.7% agarose gel, transferred to a nylon membrane, and hybridized with a 32PγATP end-labeled (TTAGGG)3 probe. A, TRF blot of CD4+ and CD8+ T cells (>95% pure) from two AIM donors sampled within 1 wk of symptom onset. The mean TRF length was calculated as described (7), marking the area above the background and adjusting the intensity of signals for molecular size calculated from a curve fitted to the DNA m.w. markers. B, Comparison of mean TRF length (kb) in CD4+ and CD8+ T cells from eight AIM patients (each represented by a different pair of symbols). CD8+ T cells had longer telomere lengths than CD4+ T cells, with a mean ± SD difference of 0.69 ± 0.17 kb. This difference was statistically significant both with a paired t test (p < 0.0001) and with the Wilcoxon matched-pairs signed-ranks test (p = 0.01), since telomere lengths could not be assumed to follow a Gaussian distribution. C, Comparison of the difference between CD8+ and CD4+ telomere lengths in eight patients with AIM, five of these patients on disease resolution (Follow-up) and five healthy donors with no history of AIM (Controls). Results are presented as the TRF length (kb) for CD8+ T cells minus that for CD4+ T cells from the same sample (TRF[CD8 − CD4] kb). The mean (TRF[CD8 − CD4] kb), represented by the horizontal bar for each group, was 0.69 kb (± SD 0.17 kb) for the AIM patients compared with −0.05 ± 0.23 kb for follow-ups and −0.19 ± 0.39 kb for controls. Only the AIM patients consistently have longer telomeres in the CD8 than the CD4 fraction, and this difference was significant compared with the follow-ups (p < 0.0001) and healthy controls (p = 0.0002). The mean (TRF[CD8 − CD4] kb) did not differ between AIM follow-up samples and those from healthy controls (p = 0.49).

CD8+ T cells do not maintain longer telomeres than CD4+ T cells on follow-up

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.

  FIGURE 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 2.

Longitudinal analysis of TRF length following AIM. Mean TRF length was analyzed as described for Fig. 1⇑ for CD4+ and CD8+ T cells from sequential samples following AIM (run in adjacent lanes on a single gel). Time 0 represents sampling at the time of recruitment, which was within 10 days of onset of acute symptomatic disease. As shown in two representative patients, there was more rapid telomere shortening in CD8+ than CD4+ T cells on follow-up, so that by 1–3 mo after AIM, there was no longer any difference between mean TRF length in CD4+ and CD8+ T cells.

Up-regulation of telomerase activity is found in association with telomere length maintenance

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. 3⇓A). 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. 3⇓B). 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).

  FIGURE 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 3.

Up-regulation of telomerase activity in AIM T cells. Telomerase activity was measured using the TRAP assay, as described in the text. The cell extracts were from the same CD4+ and CD8+ purified T cells used for telomere length measurement (Fig. 1⇑B). A, The TRAP assay was performed using cell extracts from 5000 cells from a healthy control and an AIM donor, with two separate extracts from the AIM CD4+ purified (4+) and CD8+ purified (8+) T cells. PCR amplification was conducted using only 25 cycles of 30 sec at 94°C and 30 sec at 59°C in this experiment. Relative telomerase activity was calculated as a percentage of that from 1250 cells of the 293 transformed cell line, correcting for cell numbers used. For the negative control, cell extract was replaced by lysis buffer. Heat inactivation to destroy the RNA component of telomerase was performed by heating cell extracts to 85°C for 10 min (samples labeled 4+ -HI and 8+ -HI on gel). The arrow indicates the 36-bp internal control standard. B, Telomerase activity was measured as described in A, but using serial dilutions, shown here with extracts from 5000 and 500 cells and with 28 cycles of PCR. The CD4+ and CD8+ purified T cells were from a different AIM patient from A, taken in acute disease (Time 0) and after 10 wk. The two faint bands in the heat-inactivated (HI) track had the typical migration of primer-dimers (15).

Telomere length and telomerase levels in an acute oligoclonal TCRBV restricted expansion

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. 4⇓A), 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. 4⇓B). 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).

  FIGURE 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 4.

Telomere length in a phenotypically characterized oligoclonal expansion. A, The proportion of CD4+, CD8+, and BV22+CD8+ T cells in this AIM donor positive for CD45RO (60%, 90%, and 93%, respectively) and HLA-DR (17%, 82%, and 95%), and the proportion negative for CD28 (1%, 35%, and 53%), as assessed by triple staining of PBMC with directly conjugated mAbs and FACS analysis. B, Genomic DNA was extracted from CD4+ T cells, BV22+CD8+ purified T cells and from the remaining CD8+ T cells. The mean TRF length (calculated as described in Fig. 1⇑) was increased in the oligoclonal highly activated BV22 expansion (9.3 kb), compared with the rest of the CD8+ T cells (8.7 kb) and the CD4+ fraction (7.8 kb).

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).

Discussion

The cellular basis of T cell memory is poorly defined, but appears to involve persistence of many of the same clones involved in the primary response (24 and M.K.M., manuscript in preparation). There is evidence that much of the memory pool is a cycling rather than long-lived resting population, necessitating repeated rounds of division following the initial clonal expansion (9, 25). In a chronic infection such as with EBV, there is undoubtedly continuing antigenic stimulation due to the persistence of the virus, with maintenance of a high frequency of CTL (3, 26). The symptomatic form of the acute infection, infectious mononucleosis, is characterized by extensive oligoclonal expansions, followed by an efficient cellular down-regulation to restore homeostasis. The role of apoptosis in limiting the acute response (and rescue from this allowing survival of some activated T cells) is well described (27, 28). However, the potential constraints on continued division of such expansions and the possibility of specifically avoiding replicative senescence have not been addressed in this context. We have examined the phenomenon of rescue from replicative senescence in CD8+ T cells, analyzed directly ex vivo from AIM patients, in whom the phenotype and extent of clonal expansion had also been defined.

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.

Acknowledgments

We thank K. C. Wolthers (Department of Clinical Viro-Immunology, University of Amsterdam) for advice, A. Copas (Department of Sexually Transmitted Diseases, University College London Medical School) for statistical advice, University College London Hospital Department of Haematology for their cooperation, and local general practitioners and patients who participated in the study.

Footnotes

  • ↵1 This work is supported in part by a Medical Research Council Clinical Training Fellowship (to M.K.M) and Grants G-9319116 MA and G-9218555 MA (to A.N.A.). M.V.D.S. is supported by Fundação para a Ciência e Tecnologia, Portugal (Grant PRAXIS XXI/BD/9254/96).

  • ↵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: peter.beverly{at}jenner.ac.uk

  • ↵3 Abbreviations used in this paper: AIM, acute infectious mononucleosis; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment.

  • Received September 30, 1998.
  • Accepted January 28, 1999.
  • Copyright © 1999 by The American Association of Immunologists

References

  1. ↵
    Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8: 167
    OpenUrlCrossRefPubMed
  2. ↵
    Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. D. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8: 177
    OpenUrlCrossRefPubMed
  3. ↵
    Callan, M. F., L. Tan, N. Annels, G. S. Ogg, J. D. Wilson, C. A. O’Callaghan, N. Steven, A. J. McMichael, A. B. Rickinson. 1998. Direct visualization of antigen-specific CD8(+) T cells during the primary immune response to epstein-barr virus in vivo. J. Exp. Med. 187: 1395
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Hou, S., L. Hyland, K. W. Ryan, A. Portner, P. C. Doherty. 1994. Virus-specific CD8+ T-cell memory determined by clonal burst size. Nature 369: 652
    OpenUrlCrossRefPubMed
  5. ↵
    Callan, M. F., N. Steven, P. Krausa, J. D. Wilson, P. A. Moss, G. M. Gillespie, J. I. Bell, A. B. Rickinson, A. J. McMichael. 1996. Large clonal expansions of CD8+ T cells in acute infectious mononucleosis. Nat. Med. 2: 906
    OpenUrlCrossRefPubMed
  6. ↵
    Effros, R. B., G. Pawelec. 1997. Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion?. Immunol. Today 18: 450
    OpenUrlCrossRefPubMed
  7. ↵
    Harley, C. B., A. B. Futcher, C. W. Greider. 1990. Telomeres shorten during ageing of human fibroblasts. Nature 345: 458
    OpenUrlCrossRefPubMed
  8. ↵
    Allsopp, R. C., H. Vaziri, C. Patterson, S. Goldstein, E. V. Younglai, A. B. Futcher, C. W. Greider, C. B. Harley. 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89: 10114
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Weng, N. P., B. L. Levine, C. H. June, R. J. Hodes. 1995. Human naive and memory T lymphocytes differ in telomeric length and replicative potential. Proc. Natl. Acad. Sci. USA 92: 11091
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Monteiro, J., F. Batliwalla, H. Ostrer, P. K. Gregersen. 1996. Shortened telomeres in clonally expanded CD28−CD8+ T cells imply a replicative history that is distinct from their CD28+ CD8+ counterparts. J. Immunol. 156: 3587
    OpenUrlAbstract
  11. ↵
    Blackburn, E. H.. 1991. Structure and function of telomeres. Nature 350: 569
    OpenUrlCrossRefPubMed
  12. ↵
    Hiyama, K., Y. Hirai, S. Kyoizumi, M. Akiyama, E. Hiyama, M. A. Piatyszek, J. W. Shay, S. Ishioka, M. Yamakido. 1995. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J. Immunol. 155: 3711
    OpenUrlAbstract
  13. ↵
    Weng, N. P., B. L. Levine, C. H. June, R. J. Hodes. 1996. Regulated expression of telomerase activity in human T lymphocyte development and activation. J. Exp. Med. 183: 2471
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Hu, B. T., S. C. Lee, E. Marin, D. H. Ryan, R. A. Insel. 1997. Telomerase is up-regulated in human germinal center B cells in vivo and can be re-expressed in memory B cells activated in vitro. J. Immunol. 159: 1068
    OpenUrlAbstract
  15. ↵
    Holt, S. E., J. C. Norton, W. E. Wright, J. W. Shay. 1996. Comparison of the telomeric repeat amplification protocol (TRAP) to the new TRAP-eze telomerase detection kit. Methods Cell Sci. 18: 237
  16. ↵
    Wack, A., D. Montagna, P. Dellabona, G. Casorati. 1996. An improved PCR-heteroduplex method permits high-sensitivity detection of clonal expansions in complex T cell populations. J. Immunol. Methods 196: 181
    OpenUrlCrossRefPubMed
  17. ↵
    Wolthers, K. C., G. Bea, A. Wisman, S. A. Otto, A. M. de Roda Husman, N. Schaft, F. de Wolf, J. Goudsmit, R. A. Coutinho, A. G. van der Zee, L. Meyaard, F. Miedema. 1996. T cell telomere length in HIV-1 infection: no evidence for increased CD4+ T cell turnover. Science 274: 1543
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Weng, N., L. D. Palmer, B. L. Levine, H. C. Lane, C. H. June, R. J. Hodes. 1997. Tales of tails: regulation of telomere length and telomerase activity during lymphocyte development, differentiation, activation, and aging. Immunol. Rev. 160: 43
    OpenUrlCrossRefPubMed
  19. ↵
    Bodnar, A. G., N. W. Kim, R. B. Effros, C. P. Chiu. 1996. Mechanism of telomerase induction during T cell activation. Exp. Cell. Res. 228: 58
    OpenUrlCrossRefPubMed
  20. ↵
    van Steensel, B., T. de Lange. 1997. Control of telomere length by the human telomeric protein TRF1. Nature 385: 740
    OpenUrlCrossRefPubMed
  21. ↵
    Zakian, V. A.. 1995. Telomeres: beginning to understand the end. Science 270: 1601
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Lundblad, V., W. E. Wright. 1996. Telomeres and telomerase: a simple picture becomes complex. Cell 87: 369
    OpenUrlCrossRefPubMed
  23. ↵
    Strahl, C., E. H. Blackburn. 1996. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. Mol. Cell. Biol. 16: 53
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Silins, S. L., S. M. Cross, S. L. Elliott, S. J. Pye, S. R. Burrows, J. M. Burrows, D. J. Moss, V. P. Argaet, I. S. Misko. 1996. Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis. J. Exp. Med. 184: 1815
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Michie, C. A., A. McLean, C. Alcock, P. C. Beverley. 1992. Lifespan of human lymphocyte subsets defined by CD45 isoforms. Nature 360: 264
    OpenUrlCrossRefPubMed
  26. ↵
    Merkenschlager, M., P. C. Beverley. 1989. Evidence for differential expression of CD45 isoforms by precursors for memory-dependent and independent cytotoxic responses: human CD8 memory CTLp selectively express CD45RO (UCHL1). Int. Immunol. 1: 450
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Lenardo, M. J.. 1996. Fas and the art of lymphocyte maintenance. J. Exp. Med 183: 721
    OpenUrlFREE Full Text
  28. ↵
    Akbar, A. N., M. Salmon. 1997. Cellular environments and apoptosis: tissue microenvironments control activated T-cell death. Immunol. Today 18: 72
    OpenUrlCrossRefPubMed
  29. ↵
    Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner, W. E. Wright. 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Pawelec, G., A. Rehbein, K. Haehnel, A. Merl, M. Adibzadeh. 1997. Human T cell clones in long-term culture as a model of immunosenescence. Immunol. Rev. 160: 31
    OpenUrlCrossRefPubMed
  31. ↵
    Effros, R. B., R. Allsopp, C. P. Chiu, M. A. Hausner, K. Hirji, L. Wang, C. B. Harley, B. Villeponteau, M. D. West, J. V. Giorgi. 1996. Shortened telomeres in the expanded CD28−CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis. AIDS 10: F17
    OpenUrlCrossRefPubMed
  32. ↵
    Soares, M. V. D., N. J. Borthwick, M. K. Maini, G. Janossy, M. Salmon, A. N. Akbar. 1998. IL-7 dependent extrathymic expansion of CD45RA+ T cells enables preservation of a naive repertoire. J. Immunol. 161: 5909
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Hathcock, K. S., N. Weng, R. Merica, M. K. Jenkins, R. Hodes. 1998. Cutting Edge: Antigen-dependent regulation of telomerase activity in murine T cells. J. Immunol. 160: 5702
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Weng, N. P., L. Granger, R. J. Hodes. 1997. Telomere lengthening and telomerase activation during human B cell differentiation. Proc. Natl. Acad. Sci. USA 94: 10827
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Kelsoe, G.. 1996. Life and death in germinal centers (redux). Immunity 4: 107
    OpenUrlPubMed
PreviousNext
Back to top

In this issue

The Journal of Immunology: 162 (8)
The Journal of Immunology
Vol. 162, Issue 8
15 Apr 1999
  • Table of Contents
Print
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word about The Journal of Immunology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Virus-Induced CD8+ T Cell Clonal Expansion Is Associated with Telomerase Up-Regulation and Telomere Length Preservation: A Mechanism for Rescue from Replicative Senescence
(Your Name) has forwarded a page to you from The Journal of Immunology
(Your Name) thought you would like to see this page from the The Journal of Immunology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Virus-Induced CD8+ T Cell Clonal Expansion Is Associated with Telomerase Up-Regulation and Telomere Length Preservation: A Mechanism for Rescue from Replicative Senescence
Mala K. Maini, Maria Vieira D. Soares, Christian F. Zilch, Arne N. Akbar, Peter C. L. Beverley
The Journal of Immunology April 15, 1999, 162 (8) 4521-4526;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Virus-Induced CD8+ T Cell Clonal Expansion Is Associated with Telomerase Up-Regulation and Telomere Length Preservation: A Mechanism for Rescue from Replicative Senescence
Mala K. Maini, Maria Vieira D. Soares, Christian F. Zilch, Arne N. Akbar, Peter C. L. Beverley
The Journal of Immunology April 15, 1999, 162 (8) 4521-4526;
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
  • Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
  • Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
Show more CELLULAR IMMUNOLOGY AND IMMUNE REGULATION

Similar Articles

Navigate

  • Home
  • Current Issue
  • Next in The JI
  • Archive
  • Brief Reviews
  • Pillars of Immunology
  • Translating Immunology

For Authors

  • Submit a Manuscript
  • Instructions for Authors
  • About the Journal
  • Journal Policies
  • Editors

General Information

  • Advertisers
  • Subscribers
  • Rights and Permissions
  • Accessibility Statement
  • FAR 889
  • Privacy Policy
  • Disclaimer

Journal Services

  • Email Alerts
  • RSS Feeds
  • ImmunoCasts
  • Twitter

Copyright © 2022 by The American Association of Immunologists, Inc.

Print ISSN 0022-1767        Online ISSN 1550-6606