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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1011-1019
Normal T-Cell Telomerase Activity and Upregulation in Human
Immunodeficiency Virus-1 Infection
By
Katja C. Wolthers,
Sigrid A. Otto,
G. Bea A. Wisman,
Sylvain Fleury,
Peter Reiss,
Reinier W. ten Kate,
Ate G.J. van der Zee, and
Frank Miedema
From the Department of Clinical Viro-Immunology, CLB, Sanquin Blood
Supply Foundation, Amsterdam; the Laboratory for
Experimental and Clinical Immunology and the Department of Human
Retrovirology, Academic Medical Centre, University of Amsterdam,
Amsterdam; the Department of Gynaecology and Obstetrics, Academic
Hospital Groningen, University of Groningen, Groningen; the National
AIDS Therapy Evaluation Centre, Academic Medical Centre, University of
Amsterdam, Amsterdam; the Department of Internal Medicine, Kennemer
Gasthuis, Haarlem, The Netherlands; and the Laboratorie
d'Immunopathologie du SIDA, Division des maladies infectieuses,
Department de medecine interne, Hopital de Beaumont, Lausanne,
Switzerland.
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ABSTRACT |
In human immunodeficiency virus (HIV)-1 infection, decrease of
telomere length is mainly found in CD8+ T cells and not
in CD4+ T cells. Telomerase, a ribonucleoprotein enzyme
that can synthesize telomeric sequence onto chromosomal ends, can
compensate for telomere loss. Here, we investigated if telomerase
activity could explain differential telomere loss of CD4+
and CD8+ T cells in HIV-1 infection. Telomerase activity
was higher in CD8+ than in CD4+ T cells
from HIV-infected patients, but still in the same range as in healthy
controls, and upregulation after stimulation was comparable to normal.
Telomerase activity in lymph node CD4+ and
CD8+ T cells from HIV-infected patients was in the same
range as that in CD4+ and CD8+ T cells from
peripheral blood (PB) and was normal in unseparated bone marrow cells.
Thus, our study did not provide evidence for compartmentalized
elongation of telomeres in HIV infection. In patients treated with
reverse transcriptase inhibitors, telomerase activity was
inhibited, but this did not lead to accelerated loss of telomere length
in vivo. Thus, differential telomere loss in CD4+ and
CD8+ T cells in HIV-1 infection cannot be explained by
telomerase activity.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN IMMUNODEFICIENCY virus (HIV-1)
infection is characterized by a decrease in CD4+ T-cell
numbers, an increase in CD8+ T-cell numbers, and
progressive immune dysfunction eventually leading to acquired
immunodeficiency syndrome (AIDS). It has been proposed that progression
to AIDS is related to exhaustion of CD4+ T-cell renewal
capacity due to persistently enhanced CD4+ T-cell
turnover.1 However, longitudinal analysis of
CD4+ T-cell telomere length showed that telomere loss was
not accelerated in the course of HIV-1 infection, suggesting that
turnover of CD4+ T cells might not be increased in HIV
infection.2 Telomeres are the extreme ends of chromosomes
consisting of TTAGGG repeats, which shorten progressively during cell
division in vitro and with aging.3-5 Telomerase, a
ribonucleoprotein enzyme that synthesizes telomeric DNA repeats onto
chromosomal ends, can compensate for telomere shortening.6
In germline cells and immortalized cells, telomerase activity is high
and telomeres do not shorten.4,7 Initially, telomerase
activity was only found in neoplastic somatic cells.8 More
recently, several reports showed that hematopoietic cells have low
telomerase activity, which can be upregulated by cell
activation.9-12 However, after activation in vitro,
increase of telomerase activity was transient and did not prevent
telomere shortening in long-term culture.13,14 It has been
suggested that telomerase provides partial compensation for telomere
loss during cell division.12 Therefore, changes in telomere
length might be caused by proliferation, but could also be related to altered telomerase activity. We and others previously addressed the
question whether normal telomere length in
CD4+ T cells from HIV-infected individuals could be due to
increased telomerase activity.2,15 Here, we compared
telomerase activity in lymphocyte subsets from HIV-infected individuals
with controls and studied upregulation, inhibition by antiviral therapy
with reverse transcriptase inhibitors, and telomerase activity in
tissues from HIV-infected individuals.
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MATERIALS AND METHODS |
Patients.
For telomerase activity in CD4+ and CD8+ T-cell
subsets, sequential cryopreserved samples were used from HIV-infected
individuals participating in the Amsterdam cohort study on HIV
infection in homosexual men. Patient clinical and laboratory
characteristics are described in Table 1. To study telomerase activity
during treatment, eight HIV-infected cohort participants treated with azidothymidine (AZT) monotherapy (600 mg/d) or a
combination of AZT (600 mg/d) and dideoxyinosine (DDI)
(400 mg/d) or dideoxycytidine (DDC) (2.25 mg/d) were
selected, of which frozen blood samples were used before and after the
start of treatment. Patient clinical and laboratory characteristics are
described in Table 2. As controls, frozen blood samples from healthy
HIV homosexual cohort participants and fresh blood
samples from laboratory workers were used. Lymphoid tissue and
peripheral blood (PB) were obtained from four HIV-infected individuals
with CD4+ T-cell counts >400 cells/µL who
did not receive treatment at the time of lymph node biopsy. Bone marrow
was obtained from four AIDS patients and two HIV
individuals by aspiration for diagnostic reasons.
Cell separation and culture.
Frozen PB mononuclear cells (PBMC) from cohort participants were used
either to analyze telomerase activity directly or to purify
CD4+ and CD8+ T cells by magnetic separation
over columns (MiniMACS; Miltenyi Biotec Inc, Sunnyvale, CA) as
described previously.2 For separation in
CD45RA+ and CD45RO+ T cells,
MiniMACS multisort kit was used for purification of CD4+ T
cells according to the manufacturer's instructions. In brief, after
purification, cells were released from the magnetic beads and the cells
were further separated in CD45RA+ and CD45RO+
fractions by positive selection over columns after a 15-minute incubation with 20 µL magnetic microbeads conjugated with CD45RA, respectively, CD45RO monoclonal antibodies (MoAbs) per 107
cells. Purity was checked by fluorescence-activated cell sorting (FACS)
staining. To analyze upregulation of telomerase activity, PBMC were
stimulated for 4 days with CD3 MoAb (CLB T3/4.E 1:1,000 final dilution
of ascites) in Iscove's modified Dulbecco's medium (IMDM)
complemented with 10% fetal calf serum, 2-mercaptoethanol, and
antibiotics. After stimulation, PBMC were purified for CD4+
and CD8+ T cells by magnetic separation over columns
(MiniMACS; Miltenyi Biotec Inc) and used for extract preparation.
Cells from tissues.
Lymphocytes from lymph nodes and bone marrow were purified on a Ficoll
Hypaque (Pharmacia Biotech, Uppsala, Sweden) gradient. Lymphocytes from lymph nodes were separated in CD4+ and
CD8+ T cells by sorting on a FACSVantage (Becton Dickinson,
San Jose, CA) with a combination of CD3 and CD4 MoAbs or CD3 and CD8
MoAbs (Pharmingen, Hamburg, Germany). Cells were frozen until further use.
Telomeric repeat amplification protocol (TRAP) assay.
For detection of telomerase activity, a modification of the TRAP
assay8 was used. PBMC or purified lymphocytes (1 × 106 to 2 × 106) were washed once with
phosphate-buffered saline (PBS) and once with ice-cold wash buffer (10 mmol/L HEPES-KOH [pH, 7.5], 1.5 mmol/L MgCl2, 10 mmol/L
KCl, 1 mmol/L dithiothreitol), and resuspended in 100 µL/1 × 106 cells of ice-cold lysis buffer (0.5%
CHAPS, 10 mmol/L Tris/HCl [pH, 7.5], 1 mmol/L
MgCl2, 1 mmol/L EGTA, 10% glycerol, 5 mmol/L  -mercaptoethanol, 0.1 mmol/L phenylmethylsulfonylamide), incubated on ice for 25 minutes, centrifuged at 12,000g for 20 minutes, and the supernatant was quick-frozen in liquid nitrogen and stored at
 70°C. Cell extracts (10 to 15 µL), corresponding to
100,000 to 150,000 cells, were diluted by end-point dilution. Three
subsequent extract dilutions were incubated for 30 minutes at room
temperature (RT) in a 50-µL reaction mixture containing
50 µmol/L each of 2'deoxynucleoside triphosphate
(dNTP), 0.1 µg of TS primer, 0.2 µL
[ -32P]deoxycytidine triphosphate (dCTP)
(10 µCi/µL, 3,000 Ci/mmol), 3 U Taq polymerase (Promega, Leiden,
The Netherlands), 20 mmol/L Tris/HCl (pH, 8.3), 1.5 mmol/L
MgCl2, 63 mmol/L KCl, 1 mmol/L EGTA, and 0.1 mg/mL bovine
serum albumin (BSA). After 30 minutes, 0.1 µg of CX
primer was added, and polymerase chain reaction (PCR) was performed as
follows: 90 seconds 90°C, and 34 cycles 30 seconds 94°C, 30 seconds 50°C, 90 seconds 72°C. The PCR product was
electrophoresed on a 12.5% nondenaturing polyacrylamide gel in
0.5× TBE. Gels were fixed in 50% ethanol (vol/vol), 50 mmol/L
NaCl, and 40 mmol/L NaAc, and dried. Films (Kodak, Rochester, NY) were
exposed overnight (O/N). Telomerase activity was
expressed as the percentage of telomerase activity in a
telomerase-positive immortalized cell line.9 The minimum
number of cell equivalents of the lung carcinoma cell line
GLC4 needed for specific telomerase activity, usually derived from 13 cells, was taken as 100%. The cell equivalent of the
last positive signal in the diluted extracts of the sample was
expressed as a percentage of this. For example, the last positive signal in a sample of 10 cell equivalents represents 130% activity, and in a sample of 10,000 cell equivalents, it represents 0.13% activity. A sample was considered positive when a fragment of the
typical 6-bp ladder was still present. When samples were repeatedly measured, results could differ a factor of 10. To confirm that the PCR
products were the product of telomerase activity, samples were treated
with 0.5 µg RNase at 37°C for 15 minutes, which resulted in
abolishment of the specific signal. A modification of the Internal Telomerase Assay Standard (M-ITAS)16 was used to exclude
inhibitors of Taq polymerase in the samples, which could lead to
artificially low telomerase activity in the sample.
Nonradioisotopic and semiquantitative TRAP assay.
Because the original TRAP assay as described above is limited in
quantitating the telomerase activity, samples were reanalyzed in a TRAP
assay using fluorescence-labeled primers and an automatic sequencer
where indicated.17,18 Telomerase products were represented by fluorescent curves, and the peak height, peak area, and size (bp)
were calculated automatically by the Fragment Manager computer program
(ALF, Pharmacia Biotech). To obtain semiquantitative
levels of telomerase activity, the M-ITAS (5 attogram) was included in the TRAP buffer. As a standard, the GLC4 cell line was
used. Peaks representing telomerase activity in GLC4
equivalents were summed, then relatively expressed to telomerase
activity of 100 GLC4 cell equivalents (set at 100%) and
normalized to the M-ITAS signal. For the samples (10,000 to 100,000 cell equivalents), the peaks representing the telomerase activity were
summed, normalized to the M-ITAS, and correlated to the
GLC4 cell number. For example, if a sample of 10,000 cell
equivalents is comparable to 10 GLC4 cells, the activity is
0.1%. After heat inactivation (10 minutes, 85°C) telomerase
activity peaks disappeared, while the M-ITAS was still detected,
indicating that the peaks are representing telomerase activity. The
assay is highly reproducible (variation <20%).
Determination of telomeric restriction fragment (TRF) length.
TRF length was analyzed in patients treated with nucleoside analogs.
DNA was isolated from 1 × 106 PBMC by the Qiagen
Blood and Body Fluid Protocol according to manufacturer's instructions
(Qiagen, Hilden, Germany). Genomic DNA (5 µg) was digested with 40 U
of HinfI and Rsa I (GIBCO Life Technologies, Breda, The
Netherlands). The digested DNA was electrophoresed on 0.6% agarose
gels (50 mA, 24 hours). Gels were then denatured in 0.25 N HCl and
neutralized in 0.4 N NaOH/0.6 mol/L NaCl, and blotted to Genescreen
plus (DuPont NEN, Brussels, Belgium) in 0.5 N NaOH/1.5 mol/L NaCl.
Blots were washed twice in 2× saline-saturated citrate
(SSC) and cross-linked (UV Stratalinker; Stratagene,
Leusden, The Netherlands). The telomeric probe
(TTAGGG)5 was radiolabeled with -32P-dCTP
using terminal transferase (Boehringer Mannheim, Almere, The
Netherlands). Hybridization was at 65°C in 0.5 mol/L
Na2HPO4/7% sodium dodecyl sulfate (SDS) (pH,
7.2). Blots were washed in buffer with decreasing salt concentration
starting with 3× SSC/0.5% SDS onto a final concentration of
0.1× SSC/0.5% SDS (15 minutes at 65°C). Blots were exposed
to Phosphor-Imager screens (Fuji, Kanagawa, Japan) for 4 hours or O/N,
and mean telomere length was analyzed by Phosphor-Imager software
(TINA, Raytest Company, Straubenhardt, Germany), which calculates the
integrated signal of the area above the background. The mean value in
kilobases was calculated using the molecular weight marker
Lamda/HindIII.
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RESULTS |
Telomerase activity in blood T lymphocytes from HIV-infected
individuals.
In normal human T cells, low to undetectable levels of telomerase
activity are found.9-12 In healthy controls, telomerase activity in extracts from PBMC (n = 8, data not shown) and purified CD4+ and CD8+ T cells (n = 3, Table 1 and Fig 1A) was
less than 1% of activity in the GLC4 cell line. To assess
telomerase activity changes during HIV-1 infection, we analyzed
telomerase activity in purified CD4+ and CD8+ T
cells from seven HIV-infected individuals in the first year after
seroconversion (SC) and 3 to 9 years after seroconversion (Table 1, Fig
1A). The upper four patients remained asymptomatic during follow-up,
while the lower three patients progressed to AIDS 3 to 5 years after
SC. In the HIV-infected individuals both early and late in infection,
telomerase activity in CD4+ T cells and CD8+ T
cells was in the same range as that of healthy controls (Mann-Whitney U
test, P > .4). During the course of infection, there was no consistent change of telomerase activity in CD4+ or
CD8+ T cells (Wilcoxon matched-pairs signed-ranks test,
P > .6). However, in HIV-infected individuals, telomerase
activity in CD8+ T cells was significantly higher than
telomerase activity in CD4+ T cells (Wilcoxon matched-pairs
signed-ranks test, P = .016). Furthermore, telomerase activity
was analyzed in purified CD45RA+ (naive) and
CD45RO+ (memory) CD4+ T-cell subsets obtained
from fresh blood samples of seven asymptomatic HIV-infected individuals
(CD4+ T-cell counts >300 cells/µL and viral load <4
log copies/mL). These data confirmed that in HIV-1 infection telomerase
activity in CD4+ T-cell subsets was in the same range as in
healthy controls (Fig 1B and 1C).
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| Fig 1.
Telomerase activity in CD4+ and
CD8+ T cells. Telomerase activity was measured by the
TRAP assay and is expressed as a percentage of the activity detected in
the lung carcinoma cell line GLC4. (A) Telomerase activity
in the original TRAP assay. Telomerase activity in 130, 13, and 1.3 GLC4 cell equivalents is shown at the left. Specific
telomerase activity could still be measured in 130 GLC4
cell equivalents. Addition of RNAse is indicated (+). Abolishment of
the signal confirmed specific telomerase activity. No cell extracts
were added to the PCR controls a (lysis buffer only) and b (PCR mix).
Cell extracts equivalent to 105, 104, and
103 purified CD4+ and CD8+ T
cells from one healthy control and one representative HIV-infected
person early (0.1 year after seroconversion) and late (7.5 years after
seroconversion) in infection were analyzed. Addition of the M-ITAS
resulted in specific bands (arrow) indicating that Taq polymerase was
not limiting. (B) Telomerase activity in freshly isolated purified
naive (CD4RA) and memory (CD4RO) CD4+ T cells from one
HIV-infected individual and one healthy control performed as described
in (A), but without M-ITAS. (C) Telomerase activity in freshly isolated
naive (CD4RA) and memory (CD4RO) CD4+ T cells from six
healthy controls and seven HIV-infected individuals. Bars with error
bars indicate the mean telomerase activity ± the standard deviation
of the population. (D) Representative fluorescence curves showing
telomerase activity and M-ITAS (at 145 bp). Lanes 1 and 2, healthy
control CD4+ and CD8+ T cells (100,000 cell
equivalents); lanes 3 and 4, HIV+ CD4+ T
cells early and late (100,000, respectively, 75,000 cell equivalents);
lanes 5 and 6, HIV+ CD8+ T cells early and
late (identical [id]); lane 7, GLC4 (100 cell equivalents); and lane 8, lysis buffer.
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The original method to quantitate telomerase activity in the TRAP
assay9 has been modified17,18 so that
telomerase activity of the sample can be accurately correlated to the
amount of GLC4 cells equaling this activity. Telomerase
activity in T cells from patient 1024 and control 2 (Fig 1A) was
reanalyzed by this semiquantitative method (Fig 1D). Telomerase
activity in T cells from the healthy control was 0.001% (Fig 1D, lanes
1 and 2). In CD4+ T cells from the patient, activity was
now 0.01%, which was 10-fold higher than in the control (Fig 1D, lanes
3 and 4). As before, the highest telomerase activity was measured in
CD8+ T cells from the HIV-infected individual early in
infection (Fig 1D, lane 5, activity 0.08%). In addition, reanalysis by
the semiquantitative method showed a twofold difference in telomerase
activity between CD45RA+ and CD45RO+
CD4+ T cells from a healthy control (0.01% and 0.02%,
respectively), while in the HIV-infected individual, telomerase
activity was 0.01% for both subsets (data not shown). Furthermore, the
amplification of the internal standard (M-ITAS, Fig 1D, right peaks)
confirmed that there were no Taq inhibitors present in the cell
extracts, which could lead to artificially low telomerase activity in
the samples.
Thus, telomerase activity in T cells from HIV-infected individuals is
low compared with activity in the GLC4 cell line, and CD4+ T cells differ less than 10-fold in activity compared
with healthy controls.
Induction of telomerase activity in blood lymphocytes.
In HIV-1 infection, T-cell dysfunction is already found before
CD4+ T-cell numbers decline.19 Therefore, the
induction of telomerase activity after cell activation might be
impaired in HIV-1 infection. However, if during activation telomerase
activity is upregulated to a higher extent, proliferation-induced
telomere loss might be compensated. To determine if activation of T
cells from HIV-infected individuals induces telomerase activity as
described for T cells from healthy controls,9 PBMC were
cultured in vitro with CD3 MoAbs. After 4 days of culture, telomerase
activity was measured in extracts equivalent to 500, 100, and 50 purified CD4+ and CD8+ T cells
(Fig 2A) or 10,000 cell equivalents (Fig
2B). Cell stimulation could upregulate telomerase activity in
lymphocytes from healthy controls, resulting in telomerase activity on
the order of 2% to 30% (Fig 2A and B). In the HIV-infected
individual, telomerase upregulation was comparable to the healthy
controls in CD8+ T cells and CD4+ T cells early
in infection, while CD4+ T cells late in infection showed
less activity compared with the healthy control (Fig 2A and B).
Differences in upregulation of telomerase activity in lymphocytes early
and late in infection were found between HIV-infected individuals (Fig
2C), but activity did not exceed the level of activity in healthy
controls after stimulation. Furthermore, these differences were not
related to T-cell function, which was declining in three of four
patients.
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| Fig 2.
Upregulation of telomerase activity in CD4+
and CD8+ T cells after in vitro stimulation. PBMC were
cultured in vitro for 4 days with CD3 MoAbs and telomerase activity was
analyzed in extracts prepared from purified CD4+ and
CD8+ T cells. (A) Telomerase activity in 500, 100, and 50 cell equivalents from one healthy control (HIV-) and one representative
HIV-positive individual (HIV+) early and late (3 years
after seroconversion, 1 year before AIDS) in infection is shown as
described in Fig 1A. (B) Representative fluorescence
curves showing telomerase activity in activated CD4+ and
CD8+ T cells and M-ITAS. Lanes 1 and 2, CD4+ and CD8+ T cells from a second healthy
control (10,000 cell equivalents); lanes 3 and 4, CD4+
and CD8+ T cells from HIV+ early in
infection (id); lanes 5 and 6, CD4+ and
CD8+ T cells from HIV+ late in infection
(id); lanes 7 and 8, GLC4 (100 cell equivalents) and lysis
buffer. (C) Telomerase activity in CD4+ and
CD8+ T cells after activation in vitro in four
HIV-infected individuals early and late in infection as analyzed with
the original TRAP assay.
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Telomerase activity after treatment with nucleoside analogs.
Telomerase is a reverse transcriptase, which uses its own RNA template.
It has been described that reverse transcriptase inhibitors inhibit
telomerase activity in ciliates and in cultured cell lines in vitro,
which caused telomere shortening.20,21 In the group of
HIV-infected individuals initially studied, we could not find any
relation between treatment with nucleoside analogs and loss of telomere
length.2 To address the question if telomerase activity is
inhibited by treatment with nucleoside analogs in vivo, we analyzed
telomerase activity in PBMC from eight individuals before and around 2 years after the start of treatment with AZT monotherapy or a
combination of AZT and DDI or DDC (Table
2). Using the semiquantitative analysis, telomerase in PBMC from these HIV-infected individuals before therapy generated a specific banding pattern, which equalled an activity of 0.001% to 0.01%
(Fig 3A, lane 1 and Fig 3B). During
therapy, the specific banding pattern vanished in two patients (Fig 3A,
lane 2, Fig 3B). In three patients, telomerase activity disappeared
(Fig 3B). This was not due to Taq inhibitors in the samples or an assay
artefact, because the M-ITAS was amplified as expected. No specific
differences were found in telomerase activity between patients with a
different treatment regimens or different laboratory parameters,
although patient groups may have been too small to allow for a good
comparison. To see if diminished telomerase activity due to treatment
would lead to accelerated loss of telomere length, telomere loss in PBMC was measured over the same time span. However, telomere loss over
the follow-up period was within the normal range (<100
bp/yr2) in these treated HIV-infected individuals (Fig 3C).
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| Fig 3.
Telomerase activity in PBMC from HIV-infected individuals
before and after start of treatment with nucleoside analogs. (A)
Representative fluorescence curves showing telomerase activity in
100,000 cell equivalents from patient 2 (Table 2). M-ITAS at 145 bp.
Lane 1, before therapy; lane 2, during therapy; lanes 3 and 4, GLC4 (100 cell equivalents) and lysis buffer. (B)
Telomerase activity in eight HIV-infected individuals before and after
the start of treatment with nucleoside analogs. Telomerase activity was
analyzed in PBMC with the semiquantitative method and expressed as
percentage of activity in the GLC4 cell line. Lines connect
telomerase activity before and during treatment from the same patient.
Bars with error bars indicate the mean telomerase activity ± the
standard deviation of the population. (C) Telomere length of PBMC from
the eight HIV-infected patients at the same time points as in (B).
Telomere length was analyzed by Southern blot. Telomere length is
expressed as change of telomere length in kilobases over time.
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Telomerase activity in lymphoid tissues.
In T-cell progenitors and tonsil T cells, telomerase activity is
reported to be higher than in PB T cells.12 Therefore, high
turnover of T cells could lead to increased telomerase activity in
other compartments than PB. Telomerase activity in CD4+ and
CD8+ T cells from lymph nodes was compared with that in PB
lymphocytes obtained from four HIV-infected individuals. One
representative patient is shown in Fig 4A.
Telomerase activity was comparable in blood and lymph node
CD4+ and CD8+ T cells (Fig 4A and B).
Telomerase activity in lymphoid tissue CD4+ and
CD8+ T cells from these patients was similar to that in a
HIV individual (data not shown). In addition,
telomerase activity in total bone marrow cells obtained from four AIDS
patients and two HIV controls was comparable, on the
order of 1%, which is in agreement with previous
reports9,10 (Fig 4C, two patients shown). Extracts from
lymph node and bone marrow cells tested in the semiquantitative method
did show amplification of the M-ITAS, indicating that there are no Taq
inhibitors present in extracts from these tissue cells (data not
shown), and telomerase activity measured by this method was on the
order of 0.01% to 0.1%.
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| Fig 4.
Telomerase activity in lymph nodes and bone marrow from
HIV-infected patients. (A) Telomerase activity in extracts equivalent
to 105, 104, and 103 purified
CD4+ and CD8+ T cells isolated from PB (CD4
and CD8) and lymph node tissue (LN, CD4 and CD8) shown for one
representative HIV-infected individual. (B) Telomerase activity in
CD4+ and CD8+ T cells isolated from PB and
LN from four HIV-infected individuals, as analyzed by the original TRAP
assay. (C) Telomerase activity in extracts equivalent to
104, 103, and 102 total bone marrow
cells from two HIV-infected patients after AIDS diagnosis and two
HIV controls. Total bone marrow cell samples contained
approximately 40% to 45% CD3+ cells, 4%
CD19+ cells, and 2% CD34+ cells. In bone
marrow samples from the AIDS patients, the percentage of
CD4+ cells was decreased (6.5% compared with 15% in
HIV samples) and the percentage CD8+ cells
was increased (30% compared with 18%).
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DISCUSSION |
It has been found that telomere length in HIV-infected
individuals is decreased in CD8+ T cells, but
normal or even increased in CD4+ T cells in HIV-1
infected individuals with CD4+ T-cell numbers
>200 cells/µL.2,15 Telomerase activity could play a role in compensating proliferation-induced telomere loss in
HIV-1 infection, which could explain why high
CD4+ T-cell turnover in CD4+ T cells
is not reflected in shorter telomeres or accelerated telomere loss. We
showed here that this is unlikely to be the case.
First, telomerase activity in T cells from HIV-infected individuals was
low compared with activity in the GLC4 cell line and in the
same range as that in healthy controls. The original TRAP assay is
limited in quantification of telomerase activity because telomerase
activity is quantitated by end-point dilution, while in the
semiquantitative method, telomerase activity of the sample can be more
accurately related to telomerase activity in a specific amount of
GLC4 cells. Because of these differences in quantification, one cannot compare the percentages of activities in the different assays. However, when telomerase activity in different samples were
compared, the semiquantitative method confirmed that telomerase activity was low in quiescent T cells from HIV-infected individuals and
could be upregulated after stimulation (Figs 1 and 2). Now, a
difference of 10-fold in telomerase activity between CD4+ T
cells from the healthy control and the HIV-infected individual could be
shown, and differences of less than 10-fold in telomerase activity
between samples could be analyzed (Fig 1). However, the relevance of
10-fold differences in telomerase activity in quiescent cells as
calculated semiquantitatively by these methods is not yet clear. A
large variation already exists between telomerase activity in T cells
from healthy controls. Fluctuations of 10-fold were observed in
telomerase activity of CD4+ T cells during the course of
infection (Table 1), but no relation to disease progression could be
found in this patient group, while telomere length is stable during the
course of infection in these patients.2 In addition, a
10-fold difference in telomerase activity was found between samples of
different timepoints from a healthy control (data not shown).
Furthermore, increases of 10-fold to 100-fold in telomerase activity
during progression, as shown in the CD8+ T cells from
patients 232 and 39, did not prevent these cells from losing telomere
length.2 This indicates that differences in low telomerase
activity levels as found in a population of quiescent T cells by the
TRAP assay do not correlate with telomere length changes.
Secondly, higher levels of telomerase activity could be measured after
stimulation in vitro. These levels of telomerase activity are shown to
compensate telomere shortening of cells for a limited amount of
population doublings.13,14 In HIV-infected patients, telomerase activity was upregulated after stimulation in vitro, but did
not exceed the normal level of activation. Moreover, decreased upregulation of telomerase activity late in infection did not lead to
accelerated loss of CD4+ T-cell telomere length, while
normal upregulation late in infection did not prevent CD8+
T cells from losing telomere length (Table 12).
Thirdly, we could find no indication for localized or compartmentalized
elongation of telomeres, as telomerase activity of CD4+ and
CD8+ T cells obtained from blood and lymph nodes from
HIV-infected individuals was comparable, and telomerase activity in
unseparated bone marrow samples from AIDS patients was not increased
compared with healthy individuals. However, we can at this time
formally not exclude the possibility of high telomerase activity at the single-cell level in precursor cells.
Furthermore, we showed that in patients treated with reverse
transcriptase inhibitors, telomerase activity might be inhibited. Such
a decrease in telomerase activity is not expected as a result of
disease progression as illustrated by the changes in telomerase activity in the patients in Table 1. In addition, telomerase activity
in PBMC from four untreated HIV-infected individuals did not change
within 3 years of infection (unpublished observation, 1997). Alterations of the normal banding pattern in the
TRAP assay is expected in the presence of nucleoside analogs, because
the elongation of the TS primer by the reverse transcriptase part of
telomerase will be stopped at random by competition of the nucleoside
analogs. Analog-specific alterations of the normal banding pattern have
been reported when telomerase activity of Tetrahymena
thermophilia was assayed in the presence of nucleoside analogs.20 Culturing T-cell lines in the presence of AZT or dideoxyguanine (ddG), however, did result in inhibition
of telomerase activity without a disturbed banding
pattern.21 Our results showed a disturbed banding pattern
in the automatic sequencer (Fig 3A) and a smear in the original TRAP
assay (data not shown) when telomerase was assayed in cell extracts
from two of the patients treated with nucleoside analogs, while the
signal disappeared in three other treated patients. Thus, this
indicates that nucleoside analogs given in therapeutic dosage are
present in sufficient amounts in cell extracts to inhibit telomerase
activity in the TRAP assay, which indicates that the intracellular
concentration of the nucleoside analogs is sufficient to inhibit
telomerase activity in vivo. Indirect effects of treatment are less
likely to play a role because treatment was not potent enough to
increase CD4+ T-cell counts and suppress viral load during
follow-up. The decreased telomerase activity did not result in
accelerated loss of telomere length after 2 years of treatment. Because
telomerase antagonists have been proposed as anticancer
drugs,22 it might be interesting to study the long-term
effects of telomerase inhibition by nucleoside analogs on telomere
length and development of AIDS-related malignancies.
In conclusion, telomerase activity and upregulation in HIV-1 infection,
which are in the normal range, are not related to telomere length loss
and thus cannot explain differential telomere length loss in
CD4+ and CD8+ T cells.
| |
ACKNOWLEDGMENT |
We thank Dr A. van't Wout, N. Kootstra, Dr H. Schuitemaker, and M. Westers for providing us with tissue samples; our colleagues at the
Municipal Health Service, N. Albrecht and J. Maas, for providing us
with fresh blood samples from cohort participants; Dr F. de Wolf and M. Bakker from the Academical Medical Hospital for data on viral load; and
Dr G. Pantaleo for stimulating discussions.
| |
FOOTNOTES |
Submitted April 30, 1998; accepted October 1, 1998.
Supported by Grant No. 1008 from the Dutch AIDS Foundation and by the
Dutch Cancer Foundation. This study was conducted as part of the
Amsterdam Cohort Studies on AIDS.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Frank Miedema, PhD, Central
Laboratory of the Netherlands Red Cross Blood Transfusion Service,
Department of Clinical Viro-Immunology, Plesmanlaan 125, 1066 CX
Amsterdam, The Netherlands; e-mail: miedema{at}clb.nl.
| |
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Abstract
Introduction