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Genetic Manipulation of Telomerase in
HIV-Specific CD8 + T Cells: Enhanced
Antiviral Functions Accompany the Increased
Proliferative Potential and Telomere Length
Stabilization
Mirabelle Dagarag, Tandik Evazyan, Nagesh Rao and Rita
B. Effros
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 173:6303-6311; ;
doi: 10.4049/jimmunol.173.10.6303
http://www.jimmunol.org/content/173/10/6303
The Journal of Immunology
Genetic Manipulation of Telomerase in HIV-Specific CD8ⴙ T
Cells: Enhanced Antiviral Functions Accompany the Increased
Proliferative Potential and Telomere Length Stabilization1
Mirabelle Dagarag,* Tandik Evazyan,* Nagesh Rao,* and Rita B. Effros2*†
A
growing body of evidence from both humans and macaque cell depletion studies suggests that the CD8⫹ T
lymphocyte is a crucial effector cell type responsible for
controlling viremia during HIV-1 infection (1–5). The antiviral
effects of CD8⫹ T cells are presumed to be mediated through multiple mechanisms, including lysis of HIV-1-infected cells via release of perforin and granzyme proteases, production of such cytokines as IFN-␥ and TNF-␣ that exert pleiotropic antiviral and
immunological effects, and secretion of soluble factors that suppress HIV-1 replication (6, 7). High frequencies of HIV-1-specific
CD8⫹ T cells and vigorous immune responses against HIV-1 have
been observed during both the acute and chronic phases of the
infection, yet CD8⫹ T cells ultimately fail to prevent the progression to AIDS in seropositive individuals. Even the currently most
successful treatment strategy, highly active antiretroviral therapy
does not eradicate the virus, allowing HIV-specific CD8⫹ T cells
to continue being stimulated for many years. Indeed, it is likely
that the persistence of suboptimal (i.e., low perforin) HIV-specific
CD8⫹ T cell responses despite prolonged therapeutic viral suppression is associated with continuous proliferation. Thus, the in-
*Department of Pathology and Laboratory Medicine and †AIDS Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
Received for publication March 17, 2004. Accepted for publication September
8, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The work was supported by National Institutes of Health Grants AI47665 (to
R.B.E.) and AG 05920 (to M.D.). R.B.E. holds the Elizabeth and Thomas Plott Endowed Chair in Gerontology.
2
Address correspondence and reprint requests to Dr. Rita B. Effros, Department of
Pathology and Laboratory Medicine, David Geffen School of Medicine, 10833 Le Conte
Avenue, Los Angeles, CA 90095-1732. E-mail address: [email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
trinsic cellular program of replicative senescence, which poses a
strict limit on proliferation of normal T cells and is associated with
shortened telomeres, altered function, and changes in gene expression, has the potential to play a role in HIV disease progression.
It is becoming increasingly clear that, in addition to viral
strategies that evade immune recognition, CD8⫹ T cell defects
that develop due to chronic cell turnover also contribute to the
failure of the immune system to control HIV-1 (8, 9). Indeed,
telomere length studies have demonstrated aberrantly short
telomeres in the CD8⫹ T lymphocyte compartment of HIV⫹
patients, observations suggestive of a history of prolonged and
continuous cellular expansion that could potentially lead to the
premature exhaustion of protective antiviral responses and replicative senescence (10, 11). Consistent with this probable link
between shortened telomere status and cellular dysfunction are
reports documenting maintenance of T cell telomere length in
HIV-infected long-term survivors and a more rapid disease progression in older HIV-infected persons (12, 13).
Telomeres are synthesized and maintained by telomerase, a ribonucleoprotein consisting of a catalytic subunit, human telomerase (hTERT),3 and an RNA component, human telomerase RNA
(14). Most human somatic cells have no endogenous telomerase
activity, so that the telomere shortening that accompanies each cell
division is likely to be the major contributing factor to their finite
replicative life span (15). By contrast, lymphocytes express telomerase during development, and transiently following activation, a
unique property believed to increase their replicative potential and
allow for the extensive clonal expansion necessary for an effective
immune response (16). Recent studies using a dominant-negative
mutant of hTERT clearly demonstrate that endogenous hTERT
3
Abbreviations used in this paper: hTERT, human telomerase; B-LCL, B lymphoblastoid cell; PD, population doubling; TRF, telomere restriction fragment.
0022-1767/04/$02.00
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A large proportion of the CD8ⴙ T cell pool in persons chronically infected with HIV consists of cells that show features of
replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and
shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human
telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8ⴙ T cells, particularly in the
context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8ⴙ lymphocytes show both an enhanced
and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-␥
and TNF-␣ in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28
expression, the signature change of replicative senescence in cell culture, was retarded in those CD8ⴙ T cell cultures that had high
levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving
force behind several aspects of CD8ⴙ T cell dysfunction associated with replicative senescence. We also demonstrate reduced
accumulation of the p16INK4a and p21WAF1 cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8ⴙ T cells. Given the key role of CD8ⴙ
T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements
associated with replicative senescence may lead to novel types of immunotherapy. The Journal of Immunology, 2004, 173:
6303– 6311.
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hTERT TRANSDUCTION OF CD8 T CELLS ENHANCES ANTI-HIV FUNCTION
Materials and Methods
Subjects
This study included cultures derived from peripheral blood samples of
eight HIV-1-seropositive HLA-A2⫹ participants in the University of California Multicenter AIDS Cohort Study. The study was approved by the
University of California Institutional Review Board, and all subjects gave
informed consent.
Peptides
Synthetic peptides used in this study represented the following HIV-1derived HLA-A*0201-restricted epitopes: Gag p17 epitope SLYNTVATL
(SL9), the RT epitope ILKEPVGHV (IV9), and the Env epitope
LWVTVYYGV (LV9). Peptides were obtained from the University of California Peptide Synthesis Facility.
Bulk stimulation of fresh PBMCs
To initiate the cultures, freshly isolated PBMCs (4 ⫻ 106) obtained by
density gradient centrifugation over Ficoll-Hypaque (Pharmacia Biotech,
Uppsala, Sweden) were stimulated with 1 ⫻ 106 autologous peptide-pulsed
PBMCs. Pulsing was performed by incubating PBMCs with each peptide
(10 ␮g/ml) for 90 min, followed by two washes. Lymphocytes were plated
in a T25 flask with 15 ⫻ 106 irradiated feeder and cultured in Yssel’s
medium (Gemini Bio-Products, Calabasas, CA) supplemented with L-glutamine and penicillin and streptomycin (both from Mediatech, Herndon,
VA). rIL-2 (Roche, Basel, Switzerland) was added on day 4 and twice per
week thereafter. After 10 –14 days, the cells were tested for specificity in
a standard 51Cr release CTL assay using the HLA-A2⫹ T2 cell line pulsed
with SL9, IV9, or LV9 peptides.
Purification and establishment of CTL-enriched polyclonal
CD8⫹ cell lines
CD8⫹ T cells from bulk PBMC cultures recognizing the SL9, IV9, or LV9
peptides were purified by magnetic cell sorting (Miltenyi Biotec, Auburn,
CA) and plated at 4 ⫻ 106 cells/well in a 12-well plate in Yssel’s medium
supplemented with 150 IU/ml IL-2. Cells were restimulated every 3– 4 wk
with irradiated peptide-pulsed autologous EBV-transformed B lymphoblastoid cells (B-LCLs) to enrich for CTLs. Viable cells were counted by
trypan blue exclusion every 1–2 wk. Population doublings (PDs) of the cell
lines were determined from the point of transduction and calculated according to the formula: PDs ⫽ log2(final cell concentration/initial cell
concentration).
Cumulative PDs of a cell line is the summation of all prior doublings in
response to previous restimulations.
Retrovirus and gene transduction method
The amphotrophic PA317 packaging cell lines containing the stably transfected pBABE retroviral vector with and without hTERT cDNA were provided by Geron (Menlo Park, CA). Supernatants containing retrovirus were
pooled from plates at 40 – 60% confluence, filtered through a 0.45-␮m filter, and mixed with DMEM. CD8⫹ T cells were plated on RetroNectin
(Takara Shuzo, Shiga, Japan)-coated 24-well plates (Falcon) 2 days after
stimulation and transduced with fresh retroviral supernatant containing
hTERT or empty vector for 12 h. Infection was repeated a second time for
optimal gene transfer. Transduced cells were then selected in pretitrated
concentrations of puromycin (Sigma-Aldrich, St. Louis, MO) for 5 days.
The efficiency of transduction varied from 2 to 8%.
Telomere length assay
Measurement of telomere restriction fragment length (TRF) was performed, as previously described (29). Briefly, genomic DNA was isolated
from 2 ⫻ 106 CD8⫹ T cells using DNAzol (Molecular Research Center,
Cincinnati, OH). Each DNA sample was digested with RsaI and HinfI
(New England Biolabs, Beverly, MA) and electrophoresed through a 0.5%
agarose gel. The gel was then dried for 1 h at 60°C, denatured, and subsequently hybridized overnight to a [␥-32P]ATP end-labeled telomeric oligoprobe (TTAGGG)4. Autoradiography was performed for at least 3 days.
Mean TRF lengths were measured from phosphor imager (Packard Instrument, Downers Grove, IL) scans using ImageQuant software.
Flow cytometric analysis
mAbs used for surface staining were all purchased from BD Biosciences
(San Diego, CA). To surface stain cells, Abs were added for 15 min at
room temperature, after which the cells were washed in PBS, fixed in 2%
paraformaldehyde, and stored at 4°C until acquisition on a FACSCalibur.
For staining with HLA-A*0201 peptide-MHC tetramers, PE-labeled tetramer containing the SL9 peptide and allophycocyanin-labeled tetramer
containing the IV9 peptide were obtained from the National Institute of
Allergy and Infectious Diseases AIDS Reagent Program Tetramer Core
Facility. LV9-bound Cy5-labeled tetramer was purchased from ProImmune
(Oxford, U.K.). Pretitrated amounts of the appropriate tetramer were used
to stain 5 ⫻ 105 lymphocytes for 30 min at 4°C, after which cells were
washed and fixed in 2% paraformaldehyde and stored at 4°C until flow
cytometry analysis.
Intracellular staining for cytokines
Analyses of intracellular IFN-␥ and TNF-␣ productions were performed by
stimulating 2 ⫻ 105 CD8⫹ T cells with peptide-pulsed or unpulsed autologous B-LCLs for 6 –12 h in the presence of 1 ␮g/ml monensin (BD Biosciences). Cells were then washed, surface stained with anti-CD8 and antiCD3 at 4°C for 20 min, fixed, and permeabilized with Cytofix/Cytoperm
buffer (BD Biosciences). Intracellular staining was performed for 15 min
using pretitrated concentrations of anti-IFN-␥ or anti-TNF-␣ mAbs (BD
Biosciences). Cells were then washed and stored in 2% paraformaldehyde
at 4°C until flow cytometry analysis was performed.
Chromium release assay
Standard chromium release assays were performed using hTERT- and vector-transduced CD8⫹ T lymphocytes as effector cells. Briefly, autologous
B-LCLs were labeled with 100 ␮Ci of Na251CrO4 in the presence or absence of synthetic peptides at 10 ␮g/ml for 1.5 h. After three washes, cells
were plated in a 96-well U-bottom plate at 10,000/well. Effector cells were
then added at the indicated ratios for a 4-h incubation, after which supernatants were harvested and radioactivity measured with a gamma counter.
Spontaneous release was determined in the absence of effector cells, and
maximal release was determined in the presence of 2% Triton X-100 (Sigma-Aldrich). All wells were run in triplicates. The percent specific lysis
was calculated according to the formula: ((experimental release ⫺ spontaneous release)/(maximum release ⫺ spontaneous release)) ⫻ 100.
Western blot analysis
Frozen pellets of hTERT- and vector-transduced cells were lysed for 30
min on ice in modified radioimmunoprecipitation buffer (50 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM
EDTA, and 0.5% Nonidet P-40) supplemented with proteinase inhibitors.
Samples were then centrifuged for 20 min at 4°C, and aliquots of the
supernatants were stored at ⫺80°C until further use. Total protein concentration was determined using the Lowry method (Sigma-Aldrich). Proteins
were separated on 7.5 or 15% SDS-PAGE (Bio-Rad, Richmond, CA),
transferred to polyvinylidene difluoride membrane (Amersham, Arlington
Heights, IL), and blocked overnight in PBS containing 10% dried milk and
0.1% Tween 20. Membranes were then probed with Abs against p16INK4a
(BD Pharmingen, San Diego, CA), p21WAF1 (Oncogene Science, Carpenteria, CA), and actin (Calbiochem, La Jolla, CA). After washing, proteins
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plays a key role in the in vitro longevity of T lymphocytes (17).
Thus, the observation that telomerase activity of CD8⫹ T cells
declines progressively with repeated antigenic stimulation in cell
culture and is undetectable by the fourth encounter with Ag may
explain the presence of high proportions of memory CD8⫹ T cells
with shortened telomeres in aged persons and individuals chronically infected with HIV-1 (18).
Based on the central role of telomere shortening in the replicative senescence program, genetic manipulation of hTERT has been
used to prevent senescence and increase proliferative potential in a
variety of cell types (19 –23). Additionally, constitutive expression
of hTERT has also been increasingly recognized to prevent certain
aspects of senescence-associated cellular dysfunction, such as response to oxidative stress (23–28). In the present study, we apply
this genetic strategy to CD8⫹ T cells derived from persons infected
with HIV to evaluate the long-term effects of hTERT on the phenotypic, cell cycle, cytokine, and functional characteristics of virus-specific lymphocytes. Our results suggest that, in addition to
increasing proliferative potential and stabilizing telomere length,
gene transduction with hTERT enhances several key CD8⫹ T cell
functions involved in the control of HIV-1.
The Journal of Immunology
were detected with HRP-conjugated secondary Abs (Amersham) and developed with ECL reagents (ECLplus) (Amersham), according to the manufacturer’s instructions. Densitometry on ECL films was performed using
an Amersham Biosciences densitometer (Amersham Biosciences, Sunnyvale, CA).
Inhibition of HIV-1 replication by CD8⫹ T cells
The ability of hTERT-transduced CD8⫹ T cells to suppress viral replication was determined, as described previously (30). Briefly, T1 cells were
acutely infected with HIV-1 IIIB strain at a multiplicity of infection of
0.01. The infected T1 cells were then mixed with telomerase- or vectortransduced CD8⫹ T cells in 24-well flat-bottom plates (Falcon) at a ratio of
5 ⫻ 105 T1 cells to 2.5 ⫻ 105 CD8⫹ lymphocytes in a final volume of 2
ml. At the indicated time points, 1 ml of medium was removed for measurement of HIV-1 replication by p24 Ag ELISA and replaced with fresh
medium.
Results
Expression of hTERT retards telomere shortening
pressing cells maintained a normal karyotype, and required antigenic stimulation and IL-2 for continued proliferation (data not
shown). The hTERT⫹ CD8⫹ T cells maintained high levels of
telomerase activity throughout the culture period (data not shown),
suggesting that the increased proliferative potential might be due
to effects of the exogenous hTERT gene on telomere length. To
test this possibility, cryopreserved lymphocytes collected at different PDs were evaluated in parallel for telomere length. Results
from a representative experiment are provided in Fig. 1B, which
shows the pattern of TRF length in hTERT- and vector-transduced
cells from donor 1. These data demonstrate that during the initial
period following transduction, there was an increase in telomere
length in both cultures, an observation consistent with the welldocumented high levels of endogenous telomerase activity induced
by antigenic restimulation (18). However, consistent with our previous demonstration that the level of endogenous telomerase activity induced in memory T cells declines with repeated antigenic
stimulation, the vector-transduced culture underwent progressive
telomere shortening, reaching a mean TRF of 2.4 kb at senescence.
By contrast, the rate of telomere shortening was retarded in the
hTERT-expressing cells, and by 45 PDs, the mean TRF had become stabilized at ⬃3.0 kb.
hTERT prevents up-regulation of cell cycle inhibitors
Replicative senescence in other cell types has been correlated with
elevated levels of both p16INK4A (p16) and p21WAF1 (p21) cyclindependent kinase inhibitors, suggesting an important role for both
FIGURE 1. Extension of replicative life span and telomere length maintenance in hTERT-transduced polyclonal CD8⫹ T cells from HIV-infected
individuals. A, In vitro growth curves of representative (donors 1 and 4) CD8⫹ T cell lines transduced with retrovirus containing hTERT or vector only.
Long-term cultures were maintained by periodic restimulations with SL9-, IV9-, and LV9-pulsed irradiated autologous B-LCLs. B, Samples for telomere
length analysis were collected throughout the life span of the cell lines and measured by TRF assay. The data shown are for donor 1. Mean TRF lengths
are indicated.
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We recently reported that CTL-enriched polyclonal CD8⫹ T cell
lines from HLA-A2⫹ HIV-1-infected individuals transduced with
hTERT cDNA exhibited significantly increased replicative life
spans (31). We continued to expand some of these cell lines by
periodic restimulations with HIV peptides, and at the time of this
submission, hTERT-expressing CD8⫹ T cells from donor 1 have
undergone 66 PDs, which is nearly three times the number at
which the control cultures reached senescence (Fig. 1A). Similarly,
hTERT-transduced cells from donor 4 continued to show robust
proliferation well beyond the PD level at which the control cells
ceased dividing. Despite the dramatic increase in proliferative activity, there was no evidence of transformation; the hTERT-ex-
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hTERT TRANSDUCTION OF CD8 T CELLS ENHANCES ANTI-HIV FUNCTION
CD8⫹ T lymphocytes (vector transduced) with increasing numbers
of cell divisions, and that expression of hTERT delayed the accumulation of both proteins. As early as 4 PDs after gene transfer, the
vector-transduced cells expressed 1.5 times more p16, and 1.6
times more p21 than the hTERT-transduced cells. By 21 PDs, just
a few PDs before the vector-transduced cells senesced, the levels
of p16 and p21 were 4.7 and 7.9 times greater than those of the
hTERT-transduced cells, respectively. hTERT- and vector-transduced cell lines analyzed from two other donors varied with respect to the degree of change in p16 and p21 expression, but in all
cases, the level of one or both senescence-associated proteins was
significantly reduced in the cells constitutively expressing hTERT.
hTERT retards the loss of CD28 expression
proteins in mediating the senescence program (32–34). We therefore compared the levels of p16 and p21 proteins in the hTERTand vector-transduced CD8⫹ T cell lines. Data from Western blot
analysis of the cell lines from donor 1 are shown in Fig. 2, in which
we compared the levels of expression of p16 and p21 in early (PD
4) and later passage (PD 21) quiescent hTERT- and vector-transduced cells. The results show that p16 and p21 accumulate in
FIGURE 3. Ectopic hTERT expression prolongs cell surface expression of CD28. hTERT- and vector-transduced CD8⫹ T cells from HIV-infected
donors were examined for CD28 expression at the indicated PDs. Cells were stained with anti-CD28 and anti-CD8 mAbs and analyzed by flow cytometry.
Stainings were performed in duplicates.
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FIGURE 2. Reduced accumulation of the cell cycle inhibitors p21WAF1,
p16INK4a in CD8⫹ T lymphocytes stably expressing hTERT. Ten micrograms of lysates from CD8⫹ T cells collected at the indicated numbers of
PDs were analyzed for the expression levels of p21 and p16 by Western
blot. Densitometric ratios of p21 and p16 to actin at early (䡺) and later (f)
passage cells are represented by bar graphs.
A progressive decline in the proportion of CD8⫹ T cells that express the CD28 costimulatory molecule has been extensively documented during aging and in disease states, and also in cell culture
with increasing PDs (35). To investigate whether hTERT transduction has an effect on CD28 expression, we performed a longitudinal analysis of the CD28 expression profiles in the polyclonal
CD8⫹ T cell lines using anti-CD28 mAb and flow cytometry (Fig.
3). In the two CD8⫹ T cell cultures that had high proportions of
CD28⫹ cells at the time of transduction (donors 1 and 3), constitutive expression of hTERT was associated with a retardation in
the loss of cell surface CD28. Indeed, at a time point when all of
the cells in their corresponding control cultures no longer expressed CD28, 35% of the hTERT-transduced cells from donor 1
and 50% of the hTERT-expressing lymphocytes from donor 3
were still CD28⫹. Moreover, in contrast to the control cells in
which complete loss of CD28 is associated with cell cycle arrest
and replicative senescence, the hTERT-transduced lines with expanded life span remained capable of further proliferation even
when they became totally CD28 negative. In the case of the cell
lines from donor 5 in which gene transfer was performed when the
culture already contained a high proportion (60%) of cells that
were CD28 negative, no effect of hTERT on the rate of loss of
CD28 expression was observed. These data suggest that hTERT
gene transfer is capable of retarding the loss of CD28 expression
in CD8⫹ T cells from HIV-1-infected persons as long as there are
sufficient numbers of CD28⫹ lymphocytes at the time of
transduction.
The Journal of Immunology
FIGURE 4. Constitutive expression of telomerase enhances antiviral
suppressive capacity of CD8⫹ T cells. CD8⫹ T lymphocytes were cocultured for 8 days with T1 cells acutely infected with HIV-1 IIIB. Inhibition
assay supernatants were collected every 2 days and assayed for p24 production. Percentage of inhibition was calculated against an untreated control. Error bars of triplicate coculture wells are shown.
6307
peptides at multiple time points after transduction to determine the
long-term kinetics of this HIV-1-suppressive activity. hTERT- and
vector-transduced CD8⫹ lymphocytes at several stages of their in
vitro proliferative life spans were cocultured with the acutely HIV1-infected HLA-A2 cell line T1 cells. Supernatants from these
cultures were collected at 2-day intervals and were then assayed by
ELISA for p24 levels. Inhibition data from one of three donors
analyzed (Fig. 4) show that the hTERT⫹ cells had superior ability
to inhibit viral replication at every time point tested. Indeed, the
hTERT-transduced cultures maintained levels of inhibition that
ranged from 68 to 82%, even as late as 50 PDs, in comparison with
the more modest inhibitory levels of 35– 45% of the vector-transduced cultures just before their senescence at 22 PDs. These results, together with data from other cultures tested, indicate that the
ability to inhibit HIV-1 replication is a stable feature of HIV-1specific CD8⫹ T cells, regardless of proliferative history. However, in all donors tested, constitutive expression of hTERT enhanced antiviral suppressive activity, consistent with our previous
data on an HIV-specific CTL clone.
hTERT retards the loss of IFN-␥ and TNF-␣ production by
HIV-1-specific CD8⫹ T cells
CD8⫹ T lymphocytes produce soluble factors that have been
shown to inhibit HIV-1 replication in vitro and have been hypothesized to contribute to control of the virus in vivo (36, 37). Previously, we reported that an hTERT-transduced IV9-specific CTL
clone showed markedly enhanced HIV-1-suppressive activity in
vitro at a time point 8 wk after gene transduction (31). In this
study, we evaluated uncloned CD8⫹ cell lines that have been enriched for virus-specific CTLs through repeated stimulation with
We compared the capacity of hTERT- and vector-transduced HIV1-specific CD8⫹ T cells to produce IFN-␥ and TNF-␣ in response
to stimulation with viral peptides. Cells at multiple stages of their
in vitro proliferative life span were restimulated with peptidepulsed APCs and evaluated 6 h later by flow cytometry for intracellular cytokines (Fig. 5A). The data shown, which is representative of cultures from three donors examined, demonstrate that
hTERT-transduced lymphocytes have a prolonged capacity to
FIGURE 5. hTERT-transduced HIV-specific CD8⫹ T cells from HIV-1-seropositive individuals show prolonged capacity for production of IFN-␥ and
TNF-␣ in response to stimulation. CD8⫹ T cells were stimulated for 6 h with autologous B-LCLs that were unpulsed or pulsed with SL9, IV9, and LV9
peptides in the presence of brefeldin A. Next, cells were surface stained with a mAb against CD8, fixed, and intracellularly stained with anti-IFN-␥ or
anti-TNF-␣ mAbs. A, Flow cytometric data at multiple time points during their in vitro proliferative life span. B, To demonstrate that the progressive decline
in cytokine-producing cells is not due to loss of the Ag-specific CD8⫹ T cells from the cultures, hTERT- and vector-transduced CD8⫹ T lymphocytes were
also stained with tetramers at two different time points during the culture life span.
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hTERT-expressing CD8⫹ T cells show enhanced inhibition of
HIV-1 replication in vitro
6308
hTERT TRANSDUCTION OF CD8 T CELLS ENHANCES ANTI-HIV FUNCTION
elaborate both IFN-␥ and TNF-␣ in response to Ag-specific stimulation. At PD 24, which was the last time point we were able to
examine the vector-transduced culture before it senesced, only
3–5% of the lymphocytes responded to stimulation by producing
cytokines, whereas 15 and 19% of the cells in the hTERT⫹ culture
still actively produced IFN-␥ and TNF-␣, respectively. The decline in cytokine production was not due to altered kinetics of
cytokine expression in later passage cells, as no IFN-␥ or TNF-␣
production was detected by intracellular cytokine staining performed at the 8-, 10-, and 12-h time points after stimulation (data
not shown). Moreover, the decline in cytokine production by the
vector-transduced cells was not due to loss of Ag-specific lymphocytes, as tetramer staining demonstrated that 19.1% were Gag
(SL9)⫹, 16.1% were Pol (IV9)⫹, and 7.8% were Env (LV9)⫹ at
the time point when the cytokines ceased being produced (Fig.
5B). Indeed, our analysis indicates that despite the prolongation of
Ag-induced cytokine responses in the hTERT cultures during the
period between PD 6 and 24, there was nevertheless a decrease in
the percentage of cytokine-producing cells (40 to 11% for IFN-␥,
and 42 to 17% for TNF-␣). This functional decline occurred in
parallel with an increase in tetramer-binding cells from 24 to 43%
during the same time frame. Similar enrichment of tetramer-binding cells accompanied the progressive decline of Ag-induced
IFN-␥ and TNF-␣ production in the hTERT cultures of two other
donors examined. Taken together, these data demonstrate that
hTERT transduction delays, but does not prevent the progressive
loss of Ag-induced cytokine production by HIV-specific CD8⫹ T
cells. Importantly, because the levels of exogenous telomerase activity (assayed by the telomerase repeat amplification protocol assay) (31) were maintained throughout the entire culture period, our
data demonstrate a dissociation between hTERT effects on proliferation and cytokine function.
hTERT does not prevent the loss of cytolytic function of
HIV-1-specific CD8⫹ T cells
Finally, we examined the effect of ectopic hTERT expression on
the cytolytic effector function of HIV-1-specific CD8⫹ T cells.
Before transduction, chromium release assays on in vitro expanded
PBMCs from our subjects demonstrated robust SL9-, IV9-, or
LV9-specific CTL responses (data not shown). The same high
level of cytotoxicity was observed in the purified CD8⫹ T cells
from the same individuals after transduction with hTERT cDNA or
vector only, showing that CD8 T⫹ lymphocytes are cytolytic when
tested in the absence of other cell types, and that hTERT does not
alter the specificity of the cells as shown in the representative
experiment in Fig. 6A. However, with increasing PDs, cytotoxic
activity of HIV-1-specific CTLs in both hTERT-expressing and
control cultures from all of our donors steadily declined with parallel kinetics to the point of being undetectable. At PD 25, the
noncytolytic hTERT⫹ culture from our representative donor still
contained substantial proportions of HIV-1-specific cells, as indicated by tetramer binding (Fig. 6B). However, the high proportion
of perforin-expressing cells (55%) present in this culture at PD 4,
a time point of high lytic activity, was almost completely absent
from the same culture at PD 25, providing an explanation for the
failure of CTL function (Fig. 6C). Altogether, these findings suggest that the loss of HIV-1-specific CTL activity with increasing
culture age is primarily due to impaired expression of perforin, and
that ectopic expression of hTERT does not maintain CTL function.
Discussion
The extensive analysis of long-term cultures of virus-specific
CD8⫹ T cells established from multiple HIV-1-infected donors
described in the present study has provided novel insights into the
effects of ectopic telomerase expression on a variety of functional,
cell cycle, and genetic characteristics of these cells. The most striking consequence of constitutively expressing telomerase was the
enhanced viral suppressive activity of the CD8⫹ T cells. The effects of the soluble inhibitory factors released by CD8⫹ T cells on
suppressing HIV-1 replication have been well established in vitro,
and have been suggested to play an important role in HIV-1 host
defense (36 –38). Thus, augmentation of the suppressive activity
through telomerase transduction may lead to new forms of immunotherapy, particularly during the later stages of disease when
CD8⫹ T cell cytolytic activity has been reported to wane (reviewed in Ref. 8).
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FIGURE 6. hTERT does not affect cytolytic activity of bulk HIV-specific CD8⫹ T cells. A, At the indicated PDs, hTERT- and vector-transduced CD8⫹
T cells were tested in a chromium release assay for the ability to kill peptide-pulsed autologous B-LCL targets. Results obtained at an E:T ratio of 20:1
target are shown; similar results were seen at lower E:T ratios. B, Tetramer staining of hTERT-transduced culture at PD 25. C, Perforin staining of
hTERT-transduced culture at PDs 4 and 25.
The Journal of Immunology
cells to prevent accumulation of random oxidative DNA damage,
and by this mechanism prevents the increase of p16 and p21 expression (45, 46). Current studies are directed at distinguishing
between these two possibilities.
Replicative senescence in vitro is also associated with complete
loss of CD28 cell surface and gene expression (47). The prolonged
retention of CD28 on hTERT-transduced cell lines, therefore, suggests that loss of telomere length is somehow involved in the
down-regulation of CD28 expression. The fact that stable expression of telomerase delays the loss of CD28 expression is a novel
observation, and is consistent with the recently described association between loss of telomerase inducibility in CD8⫹ lymphocytes and the loss of cell surface CD28 expression (18). Taken
together, these findings suggest that in CD8⫹ T cells, there is an
underlying relationship between CD28 expression, telomere
length, and telomerase activity. Even so, the maintenance of telomere length in the cells transduced with hTERT was ultimately
unable to prevent the eventual progressive decline in CD28 expression. Based on our finding that the prolonged expression of
CD28 was observed only in CD8⫹ cultures that were predominantly CD28⫹ at the time of hTERT introduction, it is possible that
the efficacy of this gene manipulation technique is dependent on
the age of the culture. Indeed, just as there was a lag between the
introduction of hTERT and telomere length stabilization, it is possible that gene transduction at an earlier time point during the
culture life span may be more efficient in retarding/preventing the
CD28 expression changes. The use of lentivirus vectors, which can
infect cells that are quiescent, and can therefore introduce hTERT
at the earliest stage of the culture life span, may clarify this issue.
In HIV-1 disease, shortened telomeres in the CD8⫹ population
have been suggested to play a role in the decline of virus-specific
responses, leading to progression to AIDS (48, 49). Interestingly,
a large-scale aging study on the relationship between telomere
length and infection has recently demonstrated that in persons over
60, the mortality rate from infectious disease was 8 times higher
for individuals in the bottom 25% of the lymphocyte telomere
length distribution than for persons in the top 75% (50). Consistent
with these findings and with the notion that HIV disease constitutes accelerated immunological aging (49), the present longitudinal analysis of the functional behavior of virus-specific CD8⫹ T
cells derived from HIV-infected persons revealed specific impairments in antiviral activity associated with telomere shortening and
senescence. With increasing number of PDs, the vector-transduced
cell lines showed diminished numbers of HIV-1-specific lymphocytes that produced the effector cytokines IFN-␥ and TNF-␣ in
response to antigenic stimulation. In fact, even before the actual
end stage of replicative senescence, there was almost a complete
absence of cells capable of producing these cytokines, despite the
presence of a substantial number of tetramer-positive cells. This
finding is reminiscent of clinical studies of patients with late-stage
disease, who had fewer cells that secreted IFN-␥ when stimulated
with HIV-1 (51, 52). Cultures that were transduced with hTERT
showed a prolonged retention of significant numbers of Ag-reactive cells that produced IFN-␥ and TNF-␣ in response to stimulation. Because of the pleiotropic antiviral effects of both IFN-␥ and
TNF-␣, our findings suggest that ectopic expression of telomerase
in virus-specific CD8⫹ T might be useful as a tool to increase the
functional longevity of these cells.
The current study on bulk populations of CD8⫹ T cells yielded
results on cytotoxicity that differ from our previous findings on an
IV9-specific CD8⫹ T cell clone, although in both cases the T cells
were derived from HIV-1-infected donors (31). In the present set
of experiments, hTERT was unable to prevent the impaired cytotoxic capacity of the bulk-cultured senescent HIV-1-specific CTLs.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The present study underscores the notion that the proliferative
block is only one facet of the replicative senescence cellular program, which is now known to also involve a variety of fundamental changes in gene expression and functional characteristics (15).
Constitutive expression of telomerase, which retards/prevents replicative senescence, would therefore be predicted to influence not
only the rate of telomere shortening and proliferative capacity, but
also functional traits as well. Indeed, our results show that stable
hTERT expression in HIV-specific CD8⫹ T cells derived from
HIV-infected individuals, in addition to increasing longevity and
stabilizing telomere length, also prevents the increase in cell cycle
inhibitors, prolongs the period of secretion of cytokines associated
with antiviral immune responses, and delays the loss of CD28
expression resulting from chronic Ag stimulation in vitro. These
observations are consistent with reports on telomerase manipulation in other cell types, in which hTERT has been increasingly
documented to affect multiple cellular functions. For example,
constitutive hTERT expression enhances the ability of endothelial
cells to form microvascular structures, and restores the functional
capacity of senescent fibroblasts in a dermal reconstitution model
(22, 25). Ectopic expression of hTERT also enhances the differentiation potential and bone-forming ability of human bone marrow stromal cells (26, 27). Similarly, exogenous telomerase prevents chromosomal instability and spontaneous immortalization of
Li Fraumeni Syndrome cells (39). Finally, our own previous study
showed that hTERT transduction of a selected HIV-specific CTL
clone enhanced cytotoxicity and antiviral suppressive activity (31).
The results of the present study showing pleiotropic and possibly
beneficial effects of constitutive telomerase expression in HIVspecific CD8⫹ T cells derived from persons chronically infected
with HIV, therefore, suggest that the functional decline associated
with chronic immune stimulation may be amenable to therapeutic
strategies that modulate the process of replicative senescence.
Telomere length in normal somatic cells, including CD8⫹ T
cells, shortens with increasing PDs in cell culture (18, 40). Due to
chronic activation by virus, this process of telomere shortening is
actually accelerated in the CD8⫹ T cells from HIV-infected individuals, resulting in CD8⫹ lymphocytes with abnormally short
telomere lengths (10). In the present study, we show that following
an initial decrease in telomere length in the hTERT-expressing
CD8⫹ T cell cultures, the rate of loss of telomeric sequences was
retarded compared with the vector control, and telomere length
was ultimately stabilized. This stabilization of telomeres is consistent with previous observations in hTERT-immortalized melanoma-specific CTLs (21).
A longstanding theory regarding the mechanism by which short
telomeres signal senescence is that they generate a DNA damage
signal that activates p53, which, in turn, is associated with induction in expression of the cell cycle inhibitory protein p21 (41, 42).
Similarly, up-regulation of p16 expression can be induced by DNA
damage (43). Indeed, in senescing cultures of fibroblasts, keratinocytes, and T lymphocytes, levels of p16 and p21 proteins or their
corresponding mRNAs are significantly higher than in younger
and more proliferation-competent cells (32–34, 44). Our observation of a diminished accumulation of p16 and/or p21 in the
hTERT-transduced CD8⫹ T cell cultures offers further support for
the notion that short telomeres are sensed as DNA damage, and
also provides insight into the mechanism by which stable expression of telomerase is able to confer life span extension. The simplest scenario is that the telomerase stabilizes telomere length, and
in that way, the expression of p16 and/or p21 is maintained at a
low level, allowing the cells to remain in cycle. Alternatively,
based on the ability of telomerase to heal damaged chromosomes
in lower eukaryotes, it is possible that telomerase is acting in T
6309
6310
hTERT TRANSDUCTION OF CD8 T CELLS ENHANCES ANTI-HIV FUNCTION
Acknowledgments
We thank Drs. Kenneth Dorshkind and Otto Yang for reviewing the manuscript. We are grateful to Geron for supplying the hTERT and control
constructs used in this study.
References
1. Borrow, P., H. Lewicki, B. H. Hahn, G. M. Shaw, and M. B. A. Oldstone. 1994.
Virus-specific CD8⫹ CTL activity associated with control of viremia in primary
HIV infection. J. Virol. 68:6103.
2. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrew, G. MacLeod, W. Borkowsky,
C. F. Farthing, and D. Ho. 1994. Temporal association of cellular immune responses with the initial control of viremia in primary HIV infection. J. Virol.
68:4650.
3. Ogg, G., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard,
J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, et al. 1998. Quantitation
of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103.
4. Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon,
M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, et al. 1999.
Control of viremia in simian immunodeficiency virus infection by CD8⫹ lymphocytes. Science 283:857.
5. Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard,
C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, et al. 1999. Dramatic rise in
plasma viremia after CD8⫹ T cell depletion in simian immunodeficiency virusinfected macaques. J. Exp. Med. 189:991.
6. Yang, O. O., and B. D. Walker. 1997. CD8⫹ cells in human immunodeficiency
virus type I pathogenesis: cytolytic and noncytolytic inhibition of viral replication. Adv. Immunol. 66:273.
7. Bailer, R. T., A. Holloway, J. Sun, J. B. Margolick, M. Martin, J. Kostman, and
L. J. Montaner. 1999. IL-13 and IFN-␥ secretion by activated T cells in HIV-1
infection associated with viral suppression and a lack of disease progression.
J. Immunol. 162:7534.
8. Lieberman, J., S. Premlata, N. Manjunath, and J. Andersson. 2001. Dressed to
kill? A review of why antiviral CD8 lymphocytes fail to prevent progressive
immune deficiency in HIV-1 infection. Blood 98:1667.
9. Klenerman, P., Y. Wu, and R. Phillips. 2002. HIV: current opinion in escapology.
Curr. Opin. Microbiol. 5:408.
10. Effros, R. B., R. Allsopp, C. P. Chiu, M. A. Hausner, K. Hirji, L. Wang,
C. B. Harley, B. Villeponteau, M. D. West, and 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.
11. Palmer, L. D., N. Weng, B. L. Levine, C. H. June, H. C. Lane, and R. J. Hodes.
1997. Telomere length, telomerase activity, and replicative potential in HIV infection: analysis of CD4⫹ and CD8⫹ T cells from HIV-discordant monozygotic
twins. J. Exp. Med. 185:1381.
12. Tucker, V., J. Jenkins, J. Gilmour, H. Savoie, P. Easterbrook, F. Gotch, and
M. J. Browning. 2000. T-cell telomere length maintained in HIV-infected longterm survivors. HIV Med. 1:116.
13. Phillips, A. N., C. A. Lee, A. Webster, G. Janossy, A. Timms, M. Bofill, and
P. B. Kernoff. 1991. More rapid progression to AIDS in older HIV-infected
people: the role of CD4⫹ T cell counts. J. Acquired Immune Defic. Syndr. 4:970.
14. Blackburn, E. 1992. Telomerases. Annu. Rev. Biochem. 61:113.
15. Campisi, J. 2001. Cellular senescence as a tumor-suppressor mechanism. Trends
Cell Biol. 11:S27.
16. Weng, N. P., B. L. Levine, C. H. June, and R. Hodes. 1996. Regulated expression
of telomerase activity in human T lymphocyte development and activation.
J. Exp. Med. 183:2471.
17. Roth, A., H. Yssel, J. Pene, E. A. Chavez, M. Schertzer, P. Lansdorp, H. Spits,
and R. Luiten. 2003. Telomerase levels control the life span of human lymphocytes. Blood 102:849.
18. Valenzuela, H. F., and R. B. Effros. 2002. Divergent telomerase and CD28 expression patterns in human CD4 and CD8 T cells following repeated encounters
with the same antigenic stimulus. Clin. Immunol. 105:117.
19. Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C. P. Chiu, G. B. Morin,
C. B. Harley, J. W. Shay, S. Lichtsteiner, and W. E. Wright. 1998. Extension of
life-span by introduction of telomerase into normal human cells. Science
279:349.
20. Vaziri, H., and S. Benchimol. 1998. Reconstitution of telomerase activity in
normal human cells leads to elongation of telomeres and extended replicative life
span. Curr. Biol. 8:279.
21. Hooijberg, E., J. J. Ruizendaal, P. J. Snijders, E. W. Kueter, J. M. Walboomers,
and H. Spits. 2000. Immortalization of human CD8⫹ T cell clones by ectopic
expression of telomerase reverse transcriptase. J. Immunol. 165:4239.
22. Yang, J., E. Chang, A. M. Cherry, C. D. Bangs, Y. Oei, A. Bodnar, A. Bronstein,
C. P. Chiu, and G. S. Herron. 1999. Human endothelial cell life extension by
telomerase expression. J. Biol. Chem. 274:26141.
23. Luiten, R., J. Pene, H. Yssel, and H. Spits. 2003. Ectopic hTERT expression
extends the life span of human CD4⫹ helper and regulatory T-cell clones and
confers resistance to oxidative stress-induced apoptosis. Blood 101:4512.
24. Yang, J., U. Nagavarapu, K. Relloma, M. D. Sjaastad, W. C. Moss, A. Passaniti,
and G. S. Herron. 2001. Telomerized human microvasculature is functional in
vivo. Nat. Biotechnol. 19:219.
25. Funk, W. D., C. K. Wang, D. N. Shelton, C. B. Harley, G. D. Pagon, and
W. K. Hoeffler. 2000. Telomerase expression restores dermal integrity to in vitroaged fibroblasts in a reonstituted skin model. Exp. Cell Res. 258:270.
26. Shi, S., S. Gronthos, S. Chen, A. Reddi, C. M. Counter, P. G. Robey, and
C. Wang. 2002. Bone formation by human postnatal bone marrow stromal stem
cells is enhanced by telomerase expression. Nat. Biotechnol. 20:587.
27. Simonsen, J. L., C. Rosada, N. Serakinci, J. Justesen, K. Stenderup, S. Rattan,
T. G. Jensen, and M. Kassem. 2002. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marow
stromal cells. Nat. Biotechnol. 20:592.
28. Elmore, L. W., and S. E. Holt. 2000. Telomerase and telomere stability: a new
class of tumor suppressor? Mol. Carcinog. 28:1.
29. Valenzuela, H. F., and R. B. Effros. 2000. Telomeres and replicative senescence.
In Methods in Molecular Medicine: Aging Methods and Protocols, Vol. 38. Humana Press, Totowa.
30. Yang, O. O., S. A. Kalams, A. Trocha, H. Cao, A. Luster, R. P. Johnson, and
B. D. Walker. 1997. Suppression of human immunodeficiency virus type 1 replication by CD8⫹ T cells: evidence for HLA class I-restricted triggering of cytolytic and non-cytolytic mechanisms. J. Virol. 71:3120.
31. Dagarag, M., H. Ng, R. Lubong, R. B. Effros, and O. O. Yang. 2003. Differential
impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotoxic lymphocytes. J. Virol. 77:3077.
32. Alcorta, D. A., Y. Xiong, D. Phelps, G. Hannon, D. Beach, and J. C. Barrett.
1996. Involvement of the cyclin kinase inhibitor p16(INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl. Acad. Sci. USA 93:13742.
33. Erickson, S., O. Sangfelt, M. Heyman, J. Castro, S. Einhorn, and D. Grander.
1998. Involvement of the INK4 proteins p16 and p15 in T-lymphocyte senescence. Oncogene 17:595.
34. Pawelec, G. 1999. T cell immunosenescence in vitro and in vivo. Exp. Gerontol.
34:419.
35. Effros, R. B., N. Boucher, V. Porter, X. Zhu, C. Spaulding, R. L. Walford,
M. Kronenberg, D. Cohen, and F. Schachter. 1994. 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. 29:601.
36. Garzino-Demo, A., R. B. Moss, J. B. Margolick, F. Cleghorn, A. Sill,
W. A. Blattner, F. Cocchi, D. J. Carlo, A. L. DeVico, and R. C. Gallo. 1999.
Spontaneous and antigen-induced production of HIV-inhibitory ␤-chemokines
are associated with AIDS-free status. Proc. Natl. Acad. Sci. USA 96:11986.
37. Scarlatti, G., E. Tresoldi, A. Bjorndal, R. Fredriksson, C. Colognesi, H. K. Deng,
M. S. Malnati, A. Plebani, A. G. Siccardi, D. R. Littman, et al. 1997. In vivo
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
By contrast, the IV9-specific CTL clone showed maintenance of
cytotoxic potential even at senescence, and, further enhancement
by hTERT expression. The most likely explanation for the different outcomes is that the clone was originally screened and selected
based on vigorous cytotoxic activity, and may therefore not necessarily be representative of the majority of CD8⫹ T cells in HIV1-infected persons. It is also possible that intrinsic differences between the particular HIV-infected donors from whom the bulk
cultures and T cell clones were derived may contribute to the disparate cytotoxicity results.
In conclusion, in the present study, we have shown that transduction of the gene for the catalytic component of telomerase,
hTERT, in CD8⫹ T cells from HIV-1-infected persons delayed or
partially inhibited a number of the alterations in function and phenotype that developed as a consequence of in vitro aging and subsequent entry into replicative senescence. Our results suggest that
telomere shortening is a major driving force behind the CD8⫹
cellular dysfunction observed in late-stage HIV-1 disease, and that
the senescence-associated changes may ultimately lead to loss of
control over the virus. The study thus supports the notion that
replicative senescence and premature immunological aging might
play a role in the functional immune deficiency induced by HIV-1
(10, 49). Our results are highly relevant to the ongoing efforts to
develop therapeutic vaccines aimed at stimulating CD8⫹ T cell
responses to alleviate symptoms after HIV-1 infection, as well as
to current immunotherapy protocols based on adoptive transfer of
in vitro expanded virus-specific CD8⫹ T cells. The ramifications
and functional consequences of replicative senescence may significantly alter the effectiveness of such approaches for fighting
HIV-1. For this reason, the demonstrated efficacy of ectopic
hTERT expression in modulating a variety of senescence-associated changes in virus-specific CD8⫹ T cells from HIV-1-infected
persons represents a potential new strategy for HIV-1 therapy that
might result in prolonged protection by HIV-1-specific CD8⫹ T
cells.
The Journal of Immunology
38.
39.
40.
41.
42.
43.
44.
evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat. Med. 3:1259.
Levy, J. A. 2003. The search for the CD8⫹ cell anti-HIV factor (CAF). Trends
Immunol. 24:628.
Elmore, L. W., K. C. Turner, L. S. Gollahon, M. R. Landon, C. K. Jackson-Cook,
and S. E. Holt. 2002. Telomerase protects cancer-prone human cells from chromosomal instability and spontaneous immortalization. Cancer Biol. Ther. 1:391.
Harley, C., A. B. Futcher, and C. W. Greider. 1990. Telomeres shorten during
ageing of human fibroblasts. Nature 345:458.
De Lange, T. 1994. Activation of telomerase in a human tumor. Proc. Natl. Acad.
Sci. USA 91:2882.
Wynford-Thomas, D., J. A. Bond, F. S. Wylie, and C. J. Jones. 1995. Does
telomere shortening drive selection for p53 mutation in human cancer? Mol.
Carcinog. 12:119.
Robles, S. J., and G. R. Adami. 1998. Agents that cause DNA double strand
breaks lead to p16INK4a enrichment and the premature senescence of normal
fibroblasts. Oncogene 16:1113.
Harley, C. B. 1997. Human ageing and telomeres. Ciba Found. Symp. 211:129.
6311
45. Harrington, L., and C. W. Greider. 1991. Telomerase primer specificity and chromosome healing. Nature 353:451.
46. Yu, G., and E. Blackburn. 1991. Developmentally programmed healing of chromosomes by telomerase in Tetrahymena. Cell 67:823.
47. Effros, R. B. 2000. Costimulatory mechanisms in the elderly. Vaccine 18:1661.
48. Effros, R. B., and G. Pawelec. 1997. Replicative senescence of T cells: does the
Hayflick Limit lead to immune exhaustion? Immunol. Today 18:450.
49. Appay, V., and S. L. Rowland-Jones. 2002. Premature ageing of the immune
system: the cause of AIDS? Trends Immunol. 23:580.
50. Cawthon, R. M., K. R. Smith, E. O’Brien, A. Sivatchenko, and R. A. Kerber.
2003. Association between telomere length in blood and mortality in people aged
60 years or older. Lancet 361:393.
51. Goepfert, P. A., A. Bansal, B. H. Edwards, G. D. Ritter, Jr., I. Tellez,
S. A. McPherson, S. Sabbaj, and M. J. Mulligan. 2000. A significant number of
human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce ␥ interferon. J. Virol. 74:10249.
52. Shankar, P., M. Russo, B. Harnisch, M. Patterson, P. Skolnik, and J. Lieberman.
2000. Impaired function of circulating HIV-specific CD8⫹ T cells in chronic
human immunodeficiency virus infection. Blood 96:3094.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017