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

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.

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.

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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.

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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).

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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).

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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).