|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Department of Clinical Immunology and
Department of Haematology, Royal Free and University College Medical
School, London; Division of Immunology, Birmingham University Medical
School, Birmingham; Division of Immunology, Institute of Food Research,
Norwich Research Park, Norwich; and Department of Rheumatology, The
Medical School, University of Birmingham, Birmingham, United
Kingdom.
Acute infectious mononucleosis (AIM) induced by Epstein-Barr virus
(EBV) infection is characterized by extensive expansion of
antigen-specific CD8+ T cells. One potential consequence of
this considerable proliferative activity is telomere shortening, which
predisposes the EBV-specific cells to replicative senescence. To
investigate this, a method was developed that enables the simultaneous
identification of EBV specificity of the CD8+ T cells,
using major histocompatibility complex (MHC) class I/peptide complexes,
together with telomere length, which is determined by fluorescence in
situ hybridization. Despite the considerable expansion,
CD8+ EBV-specific T cells in patients with AIM maintain
their telomere length relative to CD8+ T cells in normal
individuals and relative to CD4+ T cells within the
patients themselves and this is associated with the induction of the
enzyme telomerase. In 4 patients who were studied up to 12 months after
resolution of AIM, telomere lengths of EBV-specific CD8+ T
cells were unchanged in 3 but shortened in one individual, who was
studied only 5 months after initial onset of infection. Substantial
telomere shortening in EBV-specific CD8+ T cells was
observed in 3 patients who were studied between 15 months and 14 years
after recovery from AIM. Thus, although telomerase activation may
preserve the replicative potential of EBV-specific cells in AIM
and after initial stages of disease resolution, the capacity of these
cells to up-regulate this enzyme after restimulation by the persisting
virus may dictate the extent of telomere maintenance in the memory
CD8+ T-cell pool over time.
(Blood. 2001;97:700-707) Primary infection with Epstein-Barr virus (EBV)
leads to a syndrome known as acute infectious mononucleosis (AIM)
characterized by a massive expansion of CD8+ T cells, which
controls the spread of virus.1-3 This increase in
CD8+ T-cell numbers is not unique to EBV infections;
different viruses, including varicella zoster and influenza in
humans4 and lymphocytic choriomeningitis virus (LCMV) in
mice,5,6 all induce similar changes. Disease resolution is
associated with a dramatic reduction in cell numbers to preinfection
levels and this homeostasis is achieved by the induction of apoptosis
in the expanded CD8+ T-cell pool.3,4,7,8
However, for a memory CD8+ T-cell population to persist
after the original immune challenge, some specific cells have to be
retained and this involves mechanisms that prevent apoptosis and enable
them to survive in vivo.5,9
The use of tetrameric major histocompatibility complex (MHC) class
I/peptide complexes (tetramers) to identify antigen-specific CD8+ T cells has shown that in patients with acute EBV
infection10 and also in mice with LCMV
infections11 up to 50% of CD8+ T cells may be
specific for a single viral epitope.2,10 It is estimated
that the number of divisions required to reach such expansions would
require the precursor populations to undergo at least 28 population
doublings.12 This level of expansion would result in
telomere shortening and be sufficient to induce replicative
senescence.13 Telomeres are repeating hexameric sequences
of DNA found at the ends of chromosomes, which are responsible for maintaining chromosomal stability and integrity.14
With each cell division, between 50 and100 base pairs (bp) of telomeric DNA are lost due to the inability of DNA polymerase to fully replicate the ends of chromosomes. Critically short telomeres trigger cell cycle
arrest and replicative senescence.15 This raises the
possibility that virus-specific CD8+ T cells, which escape
apoptosis and enter the memory pool, may have reduced replicative
capacity,16,17 thus reducing the ability of these
cells to proliferate on re-encounter with virus.
To assess the impact of telomere loss in antigen-specific
CD8+ T cells during acute EBV infection, a method that
combines the use of MHC class I tetramers with telomere analysis is
required. Current techniques that are most commonly used for telomere
length measurement would require the isolation of large numbers of
tetramer-positive cells for Southern blot analysis of telomeric
restriction fragments (TRFs).18 The labor-intensive
isolation steps required for this18 and the substantial
reduction of tetramer-binding cells in blood samples after resolution
of the infection10 would preclude the routine analysis of
large numbers of samples by this method. Another protocol for measuring
telomere length involves fluorescence in situ hybridization (FISH)
using fluorescencelabeled telomere probes on fixed cell
suspensions, which are then analyzed for staining by flow cytometry
(flow-FISH).19-21 However, in previous studies this method
has only been used to measure telomere length in isolated subpopulations of cells, therefore once again requiring the
purification of tetramer-positive CD8+ T cells.
In this study we describe a rapid way to identify simultaneously the
telomere length and surface tetramer staining of individual EBV-specific CD8+ T cells by 2-color FISH and flow
cytometry (2-color flow-FISH). Using this technique we demonstrate that
despite the massive expansion of the EBV-specific CD8+ T
cells during acute infection, they preserve their telomere length and
that this is associated with up-regulation of the enzyme telomerase.
Isolation of peripheral blood mononuclear cells
Cell staining for flow cytometric analysis
Assessment of the mean TRF length using Southern blot Genomic DNA was extracted from frozen samples using the WIZARD Genomic DNA purification Kit (Promega Ltd, Southampton, United Kingdom) and digested for 6 hours at 37°C with restriction enzymes MspI and RsaI (both from Pharmacia Biotech, Herts, United Kingdom). The digests were then electrophoresed in an 0.7% agarose gel for 48 hours at 20 V. The gel was then subjected to depurination, denaturation in an alkaline solution followed by neutralization. The DNA was transferred onto a nitrocellulose membrane (Hybond N+, Amersham Life Science, Amersham, United Kingdom) by Southern blotting and fixed by baking at 80°C. Prior to hybridization with the telomeric probe, the membranes were prehybridized with Rapid Hyb Buffer (Amersham Life Science). Hybridization was performed at 42°C for 1 hour with the telomeric specific probe (TTAGGG)3, which had previously been labeled with 32 P [ATP] using polynucleotide kinase (Pharmacia
Biotech). Autoradiographs were obtained by exposing the autoradiography
film (Hyperfilm MP, Amersham Life Science) 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 MultiAnalyst software (BioRad, Herts, United Kingdom). The mean TRF length was calculated as described.18
Assessment of telomere length using flow-FISH Telomere length was determined using the flow-FISH technique as described recently.19 Briefly, PBMCs were washed in phosphate-buffered BSA (PBSA) (0.2% bovine serum albumin [BSA]) followed by permeabilzation using Ortho PermeaFix (Ortho Diagnostic Systems, Bucks, United Kingdom) for 30 minutes in the dark. Samples were then washed twice in PBSA followed by a wash in 1 mL hybridization buffer (70% deionized formamide [BDH, Poole, United Kingdom], 20 mM Tris pH 7, 1% BSA [both from Sigma Chemical]). Samples were then resuspended in 200-µL hybridization buffer and incubated with 0.3 µg/mL of the PNA telomeric (C3TA2)3 or the PNA control probe (alphoid sequences of the X chromosome, CCCATAACTAAACAC) both conjugated to fluorescein isothiocyanate (FITC), described previously.19-21 PNA probes were obtained from Perseptive Biosystems (Framingham, MA). Samples were incubated for 20 minutes in the dark followed by heating at 80°C for 10 minutes. Cells were rapidly cooled and further hybridized for 2 hours at room temperature in the dark. Samples were washed twice in posthybridization buffer (70% formamide [BDH], 10 mM Tris, 0.1% BSA, 0.1% Tween 20 [all from Sigma Chemical]) followed by 2 further washes in PBSA. Samples were then analyzed by flow cytometry (FACSCalibur cytometer, Becton Dickinson) using Cell Quest Software (Becton Dickinson). Polyfluorescent beads (Dako Fluospheres, Dako, Denmark) were used at the beginning of each experiment to standardize the cytometer. Following the last wash, samples were incubated with propidium iodide (5 µg/mL; Sigma Chemical) and RNAase (50 ng/mL; Sigma Chemical) for 15 minutes for the exclusion of doublets as determined by FL3-area versus FL3-width.Assessment of telomere length in specific populations using 2-color flow-FISH Cells were first stained with biotin-labeled anti-CD4 or CD8-biotin (Immunotech) followed by streptavidin-Cy5 (Southern Biotech Associates, Inc, supplied by Euro-Path, Cornwall, United Kingdom) or with A2-GLC-Cy5/B8-RAK-Cy5. These cells were then washed, fixed, and permeabilized using Ortho Permeafix (Ortho Diagnostic Systems, Raritan, NJ) and processed for flow-FISH as described above. Analysis was performed using the CellQuest software as described above.Measurement of telomerase activity A modified version of the telomeric repeat amplification protocol (TRAP) was used (Oncor Inc, Gaithersburg, MD) as previously described.22 Extracts from varying cell numbers were used for telomeric elongation, using a 33P[ATP] (Amersham
Life Science) end-labeled primer. These samples were used for
polymerase chain reaction (PCR) amplification (Perkin-Elmer Cetus,
Norwalk, CT), using 25 to 28 cycles of 30 seconds at 94°C and
30 seconds at 59°C. The PCR products were run on a 12%
polyacrylamide gel (Amersham Life Science), which was vacuum dried
before exposure to autoradiography film (Hyperfilm MP, Amersham Life
Science). 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. Telomerase activity was calculated according to the
manufacturer's instructions using the optical density of the telomeric
repeat bands, on samples and heat-inactivated controls, divided by the strength of the internal PCR control band (which also served to indicate the absence of Taq inhibitors).
Characterization of 2-color flow-FISH method The first aim of the study was to develop a method for the analysis of telomere length in small subsets of cells without the need for their prior isolation. To do this, we adapted a technique that has been previously described,20,21 but we have included an initial surface staining step. The technique described by Rufer and colleagues19 involves heating cells in suspension for 10 minutes at 80°C to enable dissociation of DNA strands, which allows access to the FITC-labeled telomeric probe. This heating step did not substantially alter the forward scatter (FSC) and side scatter (SSC) characteristics of normal human T cells (Figure 1A,B). Heated cells were stained with propidium iodide and analyzed on the flow cytometer. For single cells, the area and width were set using the gain adjustments for equal pulse heights. Doublets were excluded by their wider pulse area than the single cells of comparable DNA content. Using this method to exclude cellular aggregates from the analysis, single cells were then gated by their FSC and SSC characteristics. We only analyzed cells located within the R1 gate shown in Figure 1B, which only contains nonblastoid cells in the G0 or early G1 phase of the cell cycle (F.J.P. and A.N.A., unpublished observations, 2000). Complications that arise from the increased FISH signal in cycling cells compared to resting cells can therefore be ruled out.21 To characterize the telomere length in CD8+ T-cell subsets within a mixed population, surface labeling was performed first using biotinylated anti-CD8 antibody followed by addition of a Cy5-coupled streptavidin second layer, chosen because Cy5 is heat stable. The PBMCs were stained for CD8, then fixed, and percentage positivity determined by flow cytometry before and after heating for 10 minutes at 80°C (Figure 1C). Although heating induced a reduction in intensity of CD8 staining, the percentage of CD8+ cells remained relatively unchanged (75.3% before versus 69.5% after heating). We next investigated if we could detect telomeres by flow-FISH in surface-labeled cells. Cells were first labeled with anti-CD8 biotin and Cy5-labeled streptavidin. These cells were then fixed, permeabilized, and heated to 80°C as above. The cells were then stained with the FITC-labeled X-chromosome control probe (Figure 1D) or the FITC-labeled telomeric probe (Figure 1E) followed by flow cytometric analysis. The percentage of CD8+ T cells detected in cultures with the control or telomeric probe was 68% and 65%, respectively (Figure 1D,E). Although the control probe showed a low level of staining, all the CD8+ T cells were stained with the telomeric probe. These results demonstrated the feasibility of detecting telomere length in subsets of cells in a mixed population by 2-color flow-FISH.
Analysis of telomere length by flow-FISH in healthy individuals We compared telomere length changes obtained using the flow-FISH method with those obtained by measuring TRFs by Southern blot analysis. PBMCs isolated from 14 different healthy individuals of different ages were analyzed for telomere length using both methods (Figure 2A). We found a significant correlation between flow-FISH and TRF analysis (P = .018), confirming previous results.19,20 Using the flow-FISH method we also confirmed that isolated CD45RA+ T cells have longer telomeres than CD45RO+ T cells (Figure 2B). We next compared the telomere lengths of mononuclear cell populations from 4 cord blood and 4 adult samples (individuals over 60 years old) using the flow-FISH method (representative experiment in Figure 2C). We found that the older adults showed significantly decreased telomeres as measured by flow-FISH (average median fluorescence intensity [MFI] 5.9 compared to 11.3 in the cord samples) as assessed by the Mann-Whitney test (P < .029). When TRF analysis was performed on the same samples, the cord samples also showed longer telomeres compared to the older individuals as assessed by the Mann-Whitney test (P = .0029; data not shown). Collectively these results indicate that the flow-FISH method of telomere analysis gives the same results as TRF analysis in human lymphocyte populations confirming previous reports.19-21
Analysis of telomere length in MHC class I tetramer-binding cells We next investigated if we could perform telomere staining in A2-GLC tetramer-positive cells, which was used to identify EBV-specific CD8+ T cells. The specificity of the A2-GLC tetramer staining has been described previously.10 A representative labeling profile is shown in Figure 3A. In this study A2-GLC tetramer-positive cells composed between 2% and 11% of the CD8 pool of AIM patients. To identify telomere staining the tetramer was conjugated to Cy5 instead of PE because PE is degraded by heating. After staining with the Cy5-conjugated A2-GLC tetramer, the cells were fixed, permeabilized, and then heated to enable staining with the telomeric probe. Using this method we were able to detect telomere labeling in tetramer-specific cells (Figure 3B). We also showed that the telomere length detected by flow-FISH in A2-GLC-positive cells was almost identical when whole mononuclear cell populations were labeled (MFI 16) or when CD8+ T cells were first isolated from the mononuclear cell population before labeling (MFI 16.5).
Comparison of telomere length in T-cell subsets of normal and EBV patients Using the 2-color flow-FISH method, we investigated telomere length in CD4+ and CD8+ T cells in 9 healthy controls and compared these to the CD4+, CD8+, and A2-GLC tetramer-positive cells of 12 AIM patients (Figure 4). In these studies, control samples were always processed at the same time as AIM patient samples. Reproducible results were also obtained when the same samples were run on separate occasions. In addition, telomere length in different T-cell subsets were compared within the same patients themselves, allowing them to act as their own controls. Although most CD8+ T cells from these patients expressed HLA class II, indicating that they were activated (Figure 6A) and contained oligoclonal expansions generated by repeated cell division,23,24 they had significantly longer telomeres than CD8+ T cells from normal individuals (Figure 4; P = .008). The A2-GLC tetramer-positive cells in AIM patients were also activated, as shown by elevated class II expression (Figure 6A). Like the total CD8+ T-cell pool, these expanded A2-GLC tetramer-positive cells also had significantly longer telomeres than CD8+ T cells from normals (P < .016). It was of interest that the mean telomere length of CD4+ T cells from AIM patients was also longer than that of normals (P < .01), suggesting that this subset was involved in the cellular response in AIM and that mechanisms responsible for telomere maintenance were also engaged in this population.
We next investigated the relative differences in telomere lengths
between CD4+ and CD8+ populations within each
individual AIM patient and in the controls (Figure
5). We subtracted the median telomere
length of CD8+ T cells from CD4+ T cells.
Although values above zero reflect longer telomeres in CD4 compared to
CD8 cells, values below zero indicate longer telomeres in the CD8
subset. We found that, on average, CD4+ T cells had longer
telomeres than the CD8+ T cells in healthy controls,
whereas in AIM patients, the CD8+ T cells had longer
telomeres than the CD4+ cells (Figure 5). When the
differences between CD4 and CD8 subsets in controls and AIM patients
were compared, there was a significant difference between the 2 groups
(P < .013; Mann-Whitney test). This indicates that
despite the greater proliferative activity within the CD8 relative to
the CD4 subset in AIM,1,12 the CD8+ cells had
preserved their telomere length relative to the CD4+ cells
in the same individuals. These results confirm our previous observations using Southern blot analysis for telomere
length.12
Telomere length in EBV-specific CD8+ T cells in patients before and after resolution of AIM The absolute numbers of both total CD8+ and A2-GLC tetramer-positive CD8+ T cells decrease substantially after resolution of AIM, as described previously.3,10 Although MHC class II is not expressed in nonactivated CD8+ T cells,25 the majority of the CD8+ T-cell pool and A2-GLC tetramer-positive CD8+ T cells expressed high levels of class II during AIM suggesting intense activation (Figure 6A). In this analysis we gated on nonblastoid cells (gate RI in Figure 1) as described above. MHC class II expression was substantially reduced on total CD8+ and A2-GLC tetramer-positive T cells in the follow-up samples (Figure 6A). Nevertheless, the expression of MHC class II remained elevated on some tetramer-positive cells suggesting continued activation on a proportion of these cells after resolution of the acute infection. Although there also was increased MHC class II expression in CD4+ T cells during AIM, this level of activation was much lower than in the CD8+ T cells as reported previously.25
Using the 2-color flow-FISH technology we analyzed telomere length of tetramer-positive cells during and after resolution of AIM in the same patients. PBMCs from 7 AIM patients and the same individuals between 5 months and 14 years after the resolution of the infection were investigated (Figure 6B). The acute and follow-up samples were cryopreserved immediately after isolation, thawed, and analyzed simultaneously. We found that telomere lengths were identical in the acute and follow-up samples in 3 patients tested 8 to 12 months after AIM (patients 1-3). In patient 4, who was tested after 5 months, however, telomere shortening was observed. Patients who were tested at longer times after AIM, ranging from 15 months (patient 5) to 14 years (patients 6 and 7), all showed decreased telomere lengths in the A2-GLC tetramer-positive cells (Figure 6B). These results suggest that although telomere length is maintained in the EBV-specific cells during the acute phase of infection and up to 1 year after resolution in the majority of patients, telomere shortening occurs after longer periods of time in vivo. There may be individual variation in the capacity to maintain telomere shortening of EBV-specific cells after disease resolution (eg, patient 4) and we are currently investigating the impact of telomere length in EBV-specific CD8+ T cells and subsequent functional responses to the virus by post-AIM patients in vitro. Telomerase activity in EBV-specific CD8+ T cells during acute infection and after follow-up One crucial mechanism for the maintenance of telomere length involves the induction of the enzyme telomerase, which can extend telomeres in cells.17,26,27 We therefore investigated if the telomere maintenance that is seen in cells from AIM patients was related to their up-regulation of telomerase activity. We first isolated CD8+ T cells from AIM patients by negative selection. We next enriched for tetramer-binding cells within the CD8+ fraction by positive selection (see "Patients, materials, and methods"). The CD8+ T-cell, the tetramer-enriched, and the CD8-depleted cell fractions were all analyzed for telomerase activity (Figure 7). Both the CD8+ and the tetramer-enriched fractions showed high telomerase activity during the acute phase of infection. Low but detectable levels of telomerase activity were found in the CD8-depleted subset, which confirms our previous observations that telomerase is activated in CD4+ T cells in AIM.12
We next investigated if the telomerase activity in the CD8+ T cells from AIM patients was maintained after resolution of the infection. We therefore investigated telomerase activity in isolated CD8+ and tetramer-enriched cells from the same patient after resolution of AIM. We found that on follow-up a marked reduction of telomerase activity in both the CD8+ and tetramer-positive cells compared to the same cells in AIM (Figure 7). There is, however, low but detectable telomerase activity in both these subsets suggestive of a residual level of activation in these cells. In 5 patients studied to date, CD8+ T cells that were isolated during AIM all had high telomerase activity relative to CD8+ T cells from the same patient after follow-up (data not shown). This suggests that the ability to maintain telomeres of EBV-specific CD8+ T cells is not due to the ability to up-regulate telomerase during the acute phase of disease. However, because telomerase activity decreases after AIM, telomere maintenance or shortening of EBV-specific CD8+ T cells will depend on the capacity of these cells to up-regulate telomerase on subsequent challenge with virus after longer periods in vivo (Figure 6B). Longitudinal studies are now in progress to address this point.
We have modified a method for detecting telomere length by flow cytometry to enable us to investigate the relative replicative senescence of EBV-specific CD8+ T cells in patients during AIM and after resolution of the infection. We show that despite the massive activation and proliferation of EBV-specific CD8+ T cells during AIM,2,3 these cells maintain their telomeres relative to the CD8+ T cells from normal individuals and also their own CD4+ T cells. However, our results suggest that the CD4+ T cells pool in these patients have also increased their telomeres suggesting that they have also been activated and have a role in the acute phase of this infection. We only analyzed cells within the nonblastoid gate, which excludes the possibility that any differences between the subsets analyzed arose from the increased FISH signal resulting from telomere replication in cycling cells.21 Although the cells from AIM patients that fell in this gate were not blasts, the majority expressed HLA-DR suggesting that they had been previously activated and were likely to be recently activated postmitotic cells rather than resting cells. The increased telomere length of CD8+ relative to CD4+ T cells in AIM patients detected by flow-FISH analysis confirms our previous results using DNA analysis by Southern blot.12 The maintenance of telomere length of EBV-specific CD8+ T cells in AIM is associated with induction of telomerase activity and suggests that the CD8+ T cells that escape apoptosis during resolution of AIM enter the memory pool with relatively preserved replicative capacity. Using the 2-color flow-FISH technology, we were able to analyze the relative changes of telomere length of the EBV-specific CD8+ T cells during the acute phase and in the same patients after the infection resolved. We found that in 3 of 4 individuals, telomere length remained virtually identical up to 1 year after disease resolution. In patients who were studied after longer periods after AIM, there was consistent telomere shortening. The ability to maintain telomere length is not related to the capacity of different individuals to up-regulate telomerase activity in the acute phase of disease because all the patients we have studied during AIM have up-regulated telomerase activity in the CD8+ T-cell population. However, because EBV causes a persistent infection1 there will be episodes of viral replication followed by some reactivation and expansion of specific CD8+ T cells. It is likely, therefore, that the ability of EBV-specific CD8+ T cells from different individuals to up-regulate telomerase activity on subsequent challenge and also the extent of reactivation of the cells by virus over time determines residual telomere length. Previous studies have shown that although initial antigenic challenge of T cells leads to highly increased telomerase activity, subsequent challenges induce progressively less enzymic activity.27 The observation that EBV epitope-specific primary responses may be dominated by just a few highly expanded clonotypes and that with persistent viral challenge, these dominant T-cell clonotypes may be lost and replaced by others in memory provides indirect support for the loss of reactive clones by telomere shortening.28 The 2-color flow-FISH technology described here will enable further detailed investigation of the heterogeneity of telomere length changes in patients at multiple time points after acute infection. This information is clearly crucial for the clarification of the role of replicative senescence in the regulation of CD8+ T-cell memory. The identification of signals that may regulate telomerase
activity is undoubtedly of importance because these may be manipulated to enhance immunity after vaccination, especially in the aged. It has
been shown previously that both T-cell receptor and costimulation via
CD28 are required for optimal telomerase induction in antigen-specific T cells.27 However, CD28 expression by CD8+ T
cells in humans is decreased considerably after activation both in
vitro and in vivo and most of the CD8+ T cells that are
generated during acute viral infection in humans and simians are
CD28 Although there are many similarities in mechanisms that may
regulate the CD8+ T-cell pool in humans and mice, there are
also some striking differences. Similarities include the ability of
cytokines such as those that signal via the These observations have important implications for the maintenance of CD8 memory in different species. In mice, memory CD8+ T cells have been shown to undergo slow but continuous cycling, induced by cytokines such as IL-15 and this may enable the maintenance of the memory pool.33,43 Although this has not yet been studied in detail, the maintenance of CD8 memory in humans by continuous bystander proliferation by cytokines would potentially lead to excessive telomere shortening and loss of the cells through replicative senescence. The fact that we can see telomere shortening in patients after long-term follow-up and that certain clones of EBV-specific T-cell activities are lost with time,28 provides indirect support for this possibility. Also, accelerated shortening of telomeres in leukocytes in human bone marrow transplant recipients may be due to extensive expansion of these cells to repopulate the host.44 Other additional mechanisms that enable maintenance of memory CD8+ T cells without inducing proliferation may therefore have to operate in humans. One such mechanism may be the maintenance of cells by type I IFNs, which prevent apoptosis of activated T cells without inducing proliferation.45 This would enable survival without overt proliferative activity. This would not preclude the possibility of episodes of activation and proliferation in memory CD8+ T cells, which is induced by antigen or cytokines, which are then interspersed with periods of proliferative quiescence induced by the type I IFNs.46 In conclusion, the use of MHC class I tetramer technology together with the ability to analyze changes in telomere length in these cells by 2-color flow-FISH now permits the study ofreplicative senescence in memory CD8+ T cells in humans. This will allow the investigation of changes within cells that are reactive to different epitopes of viruses in response to vaccination and will clarify if replicative senescence may be the reason why immunity to some vaccines offer lifelong protection, whereas others are only effective for a limited period. This technology will also enable us to investigate antigen-specific CD8+ T-cell responses in other viral infections such human immunodeficiency virus, and will enable us to determine reasons for the compromised antigen-specific T-cell responses in the elderly.47
We would like to thank Dr Paul Travers and Professor P. C. L. Beverley for discussions. We would also like to thank Dr Caroline Sabin for advice on statistical analysis.
Submitted July 28, 2000; accepted October 3, 2000.
Supported by grant 77/SAG1002 from the BBSRC SAGE initiative to F.J.P. and grant BD/9254/96 from Programa PRAXIS XXI to M.V.D.S.
F.J.P., M.V.D.S., and N.A. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Arne N. Akbar, Department of Clinical Immunology, Royal Free and University College Medical School, Pond St, Hampstead, London NW3 2QG, United Kingdom; e-mail: akbar{at}rfhsm.ac.uk.
1. Rickinson AB, Moss JM. Human cytotoxic T lymphocytes responses to Epstein-Barr virus infection. Ann Rev Immunol. 1997;15:405-431[CrossRef][Medline] [Order article via Infotrieve]. 2. Callan MC, Steven J, Krausa P, et al. Large clonal expansions of CD8+ T cells in acute infectious mononucleosis. Nat Med. 1996;2:906-911[CrossRef][Medline] [Order article via Infotrieve].
3.
Silins SL, Cross SM, Elliott SL, et al.
Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.
J Exp Med.
1996;184:1815-1824
4.
Akbar AN, Borthwick NJ, Salmon M, et al.
The significance of low bcl-2 expression by CD45RO+ T cells in normal individuals and patients with acute viral infections: the role of apoptosis in T cell memory.
J Exp Med.
1993;178:427-438 5. Ahmed R, Gray D. Immunological memory and protective immunity: understanding their relation. Science. 1996;272:54-60[Abstract].
6.
Gallimore A, Glithero A, Godkin A, et al.
Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I peptide complexes.
J Exp Med.
1998;187:1383-1393 7. Razvi ES, Welsh RM. Apoptosis in viral-infections. Ad Virus Res. 1995;45:1-60[Medline] [Order article via Infotrieve].
8.
Grayson JM, Zajac AJ, Altman JD, Ahmed R.
Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells.
J Immunol.
2000;164:3950-3954 9. Akbar AN, Salmon M. Cellular environments and apoptosis: tissue microenvironments control activated T-cell death. Immunol Today. 1997;18:72-76[CrossRef][Medline] [Order article via Infotrieve].
10.
Callan MF, Tan L, Annels N, et al.
Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo.
J Exp Med.
1998;187:1395-1402 11. Murali-Krishna K, Altman JD, Suresh M, et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity. 1998;8:177-187[CrossRef][Medline] [Order article via Infotrieve].
12.
Maini MK, Soares MVD, Zilch CF, Akbar AN, Beverley PCL.
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.
1999;162:4521-4526 13. Effros RB, Pawelec G. Replicative senescence of T cells: does the Hayflick limit lead to immune exhaustion? Immunol Today. 1997;18:450-454[CrossRef][Medline] [Order article via Infotrieve]. 14. Blackburn EH. Structure and function of telomeres. Nature. 1991;350:569-573[CrossRef][Medline] [Order article via Infotrieve]. 15. Hayflick L. Mortality and immortality at the cellular level: a review. Biochem-Moscow. 1997;62:1180-1190.
16.
Effros RB, Allsopp R, Chiu CP, et al.
Shortened telomeres in the expanded CD28 17. Weng NP, Hathcock KS, Hodes RJ. Regulation of telomere length and telomerase in T and B cells: a mechanism for maintaining replicative potential. Immunity. 1998;9:151-157[CrossRef][Medline] [Order article via Infotrieve]. 18. Harley CB, Futcher AB, Greider CW. Telomeres shorten during aging of human fibroblasts. Nature. 1990;345:458-460[CrossRef][Medline] [Order article via Infotrieve]. 19. Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotech. 1998;16:743-747[CrossRef][Medline] [Order article via Infotrieve].
20.
Rufer N, Brummendorf TH, Kolvraa S, et al.
Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.
J Exp Med.
1999;190:157-167
21.
Hultdin M, Gronlund E, Norrback KF, Eriksson Lindstrom E, Just T, Roos G.
Telomere analysis by fluorescence in situ hybridization and flow cytometry.
Nucleic Acids Res.
1998;26:3651-3656 22. Holt SE, Norton JC, Wright WE, Shay JW. Comparison of the telomeric repeat amplification protocol (TRAP) to the new TRAP-eze telomerase detection kit. Meth Cell Sci. 1996;18:237-248. 23. Maini MK, Wedderburn LR, Hall FC, Wack A, Casorati G, Beverley PCL. Comparison of two techniques for the molecular tracking of specific T-cell responses; CD4+ human T-cell clones persist in a stable hierarchy but at a lower frequency than clones in the CD8 population. Immunology. 1998;94:529-535[CrossRef][Medline] [Order article via Infotrieve]. 24. Maini MK, Casorati G, Dellabona P, Wack A, Beverley PCL. T-cell clonality in immune responses. Immunol Today. 1999;20:262-266[CrossRef][Medline] [Order article via Infotrieve].
25.
Akbar AN, Savill J, Gombert W, et al.
The specific recognition by macrophages of CD8+CD45RO+ T cells undergoing apoptosis
26.
Hodes RJ.
Telomere length, aging, and somatic cell turnover.
J Exp Med.
1999;190:153-156
27.
Hathcock KS, Weng NP, Merica R, Jenkins MK, Hodes R.
Cutting edge: antigen-dependent regulation of telomerase activity in murine T cells.
J Immunol.
1998;160:5702-5706 28. Annels N, Callan MFC, Tan L, Rickinson RB. Changing patterns of dominant T cell receptor usage with maturation of an Epstein-Barr virus-specific cytotoxic T cell response. J Immunol. 2000;9:4831-4841. 29. Borthwick NJ, Bofill M, Hassan I, et al. Factors that influence activated CD8+ T-cell apoptosis in patients with acute herpesvirus infections: loss of costimulatory molecules CD28, CD5 and CD6 but relative maintenance of Bax and Bcl-X expression. Immunology. 1996;88:508-515[Medline] [Order article via Infotrieve].
30.
Borthwick NJ, Lowdell M, Salmon M, Akbar AN.
Loss of CD28 expression on CD8+ T cells is induced by IL-2R 31. Effros RB, Boucher N, Porter V, et al. Decline in CD28+ T cells in centenarians and in long-term T cell cultures: a possible cause for both in vivo and in vitro immunosenescence. Exp Gerontol. 1994;29:601-609[CrossRef][Medline] [Order article via Infotrieve].
32.
Monteiro J, Batliwalla F, Ostrer H, Gregersen PK.
Shortened telomeres in clonally expanded CD28
33.
Ku CC, Murakami M, Sakamato A, Kappler J, Marrack P.
Control of homeostasis of CD8+ memory T cells by opposing cytokines.
Science.
2000;288:675-678
34.
Marrack P, Kappler J, Mitchell T.
Type I interferons keep activated T cells alive.
J Exp Med.
1999;189:521-529
35.
Vella AT, Dow S, Potter TA, Kappler J, Marrack P.
Cytokine-induced survival of activated T cells in vitro and in vivo.
Proc Natl Acad Sci U S A.
1998;95:3810-3815
36.
Pilling D, Akbar AN, Girdlestone J, et al.
Interferon
37.
Scheel-Toellner D, Pilling D, Akbar AN, et al.
Inhibition of T cell apoptosis by IFN-
38.
Akbar AN, Borthwick NJ, Wickremasinghe RG, et al.
Interleukin-2 receptor common 39. Garcia S, DiSanto J, Stockinger B. Following the development of a CD4 T cell response in vivo: from activation to memory formation. Immunity. 1999;11:163-171[CrossRef][Medline] [Order article via Infotrieve]. 40. Salmon M, Scheel-Toellner D, Huissoon AP, et al. Inhibition of T cell apoptosis in the rheumatoid synovium. J Clin Invest. 1997;99:439-446[Medline] [Order article via Infotrieve]. 41. Kipling D, Cooke HJ. Hypervariable ultra-long telomeres in mice. Nature. 1990;347:400-402[CrossRef][Medline] [Order article via Infotrieve]. 42. Engwerda CR, Handwerger BS, Fox BS. Aged T-cells are hyporesponsive to costimulation mediated by CD28. J Immunol. 1994;152:3740-3747[Abstract]. 43. Tough DF, Sun SQ, Zhang XH, Sprent J. Stimulation of naive and memory T cells by cytokines. Immunol Rev. 1999;170:39-47[CrossRef][Medline] [Order article via Infotrieve]. 44. Wynn RF, Cross MA, Hatton C, et al. Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants. Lancet. 1998;351:178-181[CrossRef][Medline] [Order article via Infotrieve].
45.
Akbar AN, Salmon M, Savill J, Janossy G.
A possible role for Bcl-2 in regulating T cell memory
46.
Akbar AN, Lord JM, Salmon M.
IFN- 47. Hodes RJ. Aging and the immune system. Immunol Rev. 1997;160:5-8[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. M. Henson, O. Franzese, R. Macaulay, V. Libri, R. I. Azevedo, S. Kiani-Alikhan, F. J. Plunkett, J. E. Masters, S. Jackson, S. J. Griffiths, et al. KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative dysfunction of highly differentiated CD8+ T cells Blood, June 25, 2009; 113(26): 6619 - 6628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Aubert and P. M. Lansdorp Telomeres and Aging Physiol Rev, April 1, 2008; 88(2): 557 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. J. Plunkett, O. Franzese, H. M. Finney, J. M. Fletcher, L. L. Belaramani, M. Salmon, I. Dokal, D. Webster, A. D. G. Lawson, and A. N. Akbar The Loss of Telomerase Activity in Highly Differentiated CD8+CD28-CD27- T Cells Is Associated with Decreased Akt (Ser473) Phosphorylation J. Immunol., June 15, 2007; 178(12): 7710 - 7719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
M. V. D. Soares, F. J. Plunkett, C. S. Verbeke, J. E. Cook, J. M. Faint, L. L. Belaramani, J. M. Fletcher, N. Hammerschmitt, M. Rustin, W. Bergler, et al. Integration of apoptosis and telomere erosion in virus-specific CD8+ T cells from blood and tonsils during primary infection Blood, January 1, 2004; 103(1): 162 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Rufer, A. Zippelius, P. Batard, M. J. Pittet, I. Kurth, P. Corthesy, J.-C. Cerottini, S. Leyvraz, E. Roosnek, M. Nabholz, et al. Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions Blood, September 1, 2003; 102(5): 1779 - 1787. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Hathcock, S. M. Kaech, R. Ahmed, and R. J. Hodes Induction of Telomerase Activity and Maintenance of Telomere Length in Virus-Specific Effector and Memory CD8+ T Cells J. Immunol., January 1, 2003; 170(1): 147 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Dunne, J. M. Faint, N. H. Gudgeon, J. M. Fletcher, F. J. Plunkett, M. V. D. Soares, A. D. Hislop, N. E. Annels, A. B. Rickinson, M. Salmon, et al. Epstein-Barr virus-specific CD8+ T cells that re-express CD45RA are apoptosis-resistant memory cells that retain replicative potential Blood, July 18, 2002; 100(3): 933 - 940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N.-p. Weng Interplay between telomere length and telomerase in human leukocyte differentiation and aging J. Leukoc. Biol., December 1, 2001; 70(6): 861 - 867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Engelhardt, J. Finke, N. Rufer, T. H. Brummendorf, B. Chapuis, C. Helg, P. M. Lansdorp, and E. Roosnek Does telomere shortening count? Blood, August 1, 2001; 98(3): 888 - 890. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
; follow-ups, 
. For individual patients, each
acute and follow-up sample was assessed at the same time.
, and
tetramer-enriched T-cell fractions from the patient during AIM and 12 months after follow-up (A). The TRAP-eze protocol, in which telomerase
activity is amplified by PCR, gives rise to a ladder of products with
6-bp increments, starting at 50 nucleotides. Heat-inactivated (HI) cell
extracts provided a telomerase negative control for each sample. The
negative control (
a mechanism for T cell clearance during resolution of viral-infections.
J Exp Med.
1994;180:1943-1947
mediates stromal cell rescue of T cells from apoptosis.
Eur J Immunol.
1999;29:1041-1050
.
Eur J Immunol.
1999;29:2603-2612
and IFN-