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*
Division of Clinical Oncoimmunology, Ludwig Institute for Cancer Research, University Hospital, Lausanne, Switzerland;
Swiss Institute for Cancer Research (ISREC), Epalinges, Switzerland;
Department of Probe Applications, Dako A/S, Glostrup, Denmark; and
§
Department of Biochemistry, Public University of Navarra, Pamplona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAckowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAckowledgementsReferences |
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surface expression
in response to Ag stimulation (3). Expression of other
surface markers is generally unchanged, with a few exceptions, notably
the decline in levels of the costimulatory receptor CD28
(4, 5, 6). Senescent CD4+ T cells are
still able to secrete a number of cytokines when stimulated
(5), and senescent CD8+ T cells
retain high levels of Ag-specific cytotoxicity (3). The impossibility to propagate T lymphocytes indefinitely limits their use for clinical applications that require large cell numbers such as adoptive transfer therapy of infections and malignancies. Why cultured T cells stop proliferating at a certain point remains unclear. In fibroblasts, telomere shortening during cell division is the molecular clock that triggers the entry of cells into senescence (7, 8). Expression of the enzyme telomerase allows germline cells and tumor cells to maintain telomere length during proliferation. Ectopic expression of human telomerase catalytic subunit (hTERT)3 is sufficient to permit some cell types, such as fibroblasts, retinal pigment epithelial cells, and endothelial cells (7, 8), to avoid senescence and to proliferate indefinitely. These immortalized cells continue to display normal cellular functions and do not undergo changes characteristic of malignant transformation (9, 10). Other cell types require alterations in cell cycle regulatory elements, besides telomerase expression, for immortalization. In particular, blocking of the Rb/p16 pathway together with telomerase expression are required to immortalize human keratinocytes and breast epithelial cells (11).
Telomerase expression can be detected in hemopoietic stem cells, where it is transiently up-regulated during in vitro cytokine-driven expansion (12, 13). Telomerase activity is also detectable in different stages of T lymphocyte differentiation (14). In vitro, mature T cells transiently express telomerase activity in response to stimulation with specific Ag (15), mitogens, or anti-CD3/anti-CD28 Abs (14, 16, 17). However, if the same cultures are subjected to repeated cycles of stimulation, the peak of telomerase activity becomes progressively lower (16). The ability of lymphocytes to express telomerase activity is exceptional among normal differentiated human cells. It has been proposed that up-regulation of telomerase allows T lymphocytes to preserve their replicative potential during clonal expansion so that memory cells with a strong capacity for expansion can be generated (18). However, a negative correlation between telomere length and donor age has been observed in CD4+ T lymphocytes, and peripheral blood T lymphocytes with a memory phenotype have shorter telomeres than naive cells (19, 20). Telomeres also shorten in in vitro cultured lymphocytes (19, 20), and both telomere length and CD28 expression decline with the number of cell divisions (5, 21). To investigate whether ectopic hTERT expression leads to immortalization of normal human CTL we infected bulk cultures or clones of human CD8+ lymphocytes with a retrovirus transducing an hTERT cDNA clone. We report that although the transduced cells express high levels of telomerase and maintain telomere length, they do not have a significantly longer life span.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAckowledgementsReferences |
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PBMC were obtained from healthy donors by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), and CD8+ cells were purified by magnetic cell sorting using a miniMACS device (Miltenyi Biotec, Auburn, CA). The CD8+ CTL clones NH 55 and LAU 203 0.3/3 were obtained by limiting dilution culture in the presence of PHA, irradiated allogeneic feeder cells, and IL-2 as previously described (22). Clone NH 55 was obtained from peptide-stimulated PBMC of normal donor NM (23) and clone LAU 203 0.3/3 from tumor-infiltrating lymph node cells of melanoma patient LAU 203 (24).
Purified fresh CD8+ T cells and
CD8+ CTL clones were plated at
105 cells/well in Iscove’s medium (Life
Technologies, Basel, Switzerland) supplemented with 10% human serum,
asparagine, arginine, and glutamine in the presence of 150 U/ml
recombinant human IL-2 (a gift from Glaxo, Geneva, Switzerland) and
stimulated with 1 µg/ml PHA (Life Technologies) plus irradiated
allogeneic PBMC (3000 rad) as feeder cells, as described elsewhere
(22). In the case of freshly isolated
CD8+ T cells, additional cycles of stimulation
were performed at intervals of 2 or 3 wk. Population doublings were
determined by weekly counts of viable cells. To estimate the mean
number of cell divisions vs time, we stained cell samples with the
diacetate form of the carboxyfluorescein succinimidyl ester (CFSE;
Molecular Probes, Eugene, OR) once or twice per week, and analyzed them
by flow cytometry 6 h, as well as 4 and 7 days after staining
(25, 26). A senescent population of clone NH 55 was
stained at the same times and used as a standard. Living cells were
gated and peaks corresponding to cells that had undergone a definite
number of divisions were identified in the fluorescence histograms.
From a histogram with N peaks, the mean number of cell
divisions (MNCD) was calculated according to the formula
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To induce endogenous telomerase activity, complete PBMC (106 cells/ml) were stimulated with immobilized anti-CD3 (OKT3, 10 µg/ml) plus soluble anti-CD28 (CK248, 1 µg/ml) in the presence of 150 U/ml of IL-2. After 5 days of culture, CD8+ cells were isolated by magnetic sorting, and telomerase activity was determined.
Retrovirus construction and infection
The hTERT retroviral construct was made by PCR amplification of
full-length hTERT cDNA that was inserted into the mouse stem cell virus
(MSCV) pac vector (27) provided by Dr. D. R. Littman
(New York University Medical Center, New York, NY). Amphotropic viruses
were generated by the transfection of this plasmid into 
NX cells
(provided by Dr. G. Nolan, Stanford University, Stanford, CA) as
described (28). CD8+ bulk cultures
and clones were infected 3 days after stimulation according to a spin
infection protocol (29) repeated three times at daily
intervals. The efficiency of transduction, estimated by staining with
specific anti-hTERT Ab (see below), varied from 1 to 5%.
Transduced cells were selected in 1.0 µg/ml puromycin for 1
wk.
Telomerase repeat amplification protocol (TRAP) assays and immunofluorescence
Nuclear extracts were prepared as described (30)
and TRAP was performed according to (31) using the
ACX-anchored return primer. Relative signal intensity of the repeat
bands was measured by phosphor imaging analysis. To compare telomerase
activity of the different cell populations, the intensity of the
signals from individual reactions was normalized to the cell
equivalents of extract added. Signals that fell into the range in which
there was a linear correlation between signal intensity and cell
equivalents were used for quantification (see Fig. 1
C). For indirect
immunofluorescence, cells were diluted in complete culture medium,
dropped onto glass slides coated with polylysine, and incubated for
2 h at 37°C. After washing and fixation with 4%
paraformaldehyde in PBS for 15 min at room temperature, cells were
permeabilized in 0.1% Triton X-100 in PBS for 10 min before blocking
with 2% BSA/0.1% Tween 20 in PBS for 30 min at room temperature.
After washing, cells were incubated with primary Abs for 1 h at
room temperature. Mouse anti-nuclear pore Ab (32) was
a gift from Dr. Susan Gasser (Swiss Institute for Experimental Cancer
Research, Epalinges, Switzerland). Rabbit anti-hTERT Ab (0.4
µg/ml) was affinity-purified and will be described elsewhere (M.
Amacker and J. Lingner, manuscript in preparation). Incubation with
secondary Abs, Alexa 488-conjugated goat anti-rabbit Ab (Molecular
Probes), and Cy3-conjugated goat anti-mouse Ab (The Jackson
Laboratory, Bar Harbor, ME) were conducted for 45 min at room
temperature. Images were acquired on a Zeiss LSM 410 confocal
microscope (Fig. 2A) and on a
Coolview Photonics CCD Zeiss Axiophot microscope (Fig. 2B).
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To measure cellular telomere length, cells were hybridized in situ with a fluorescent telomere-specific peptide nucleic acid probe, according to previously described, slightly modified protocols (20, 33, 34). To follow the evolution of a single population of lymphocytes during long-term culture, cell samples were frozen at different time points and analyzed in a single flowFISH experiment. Frozen cells (105-5 x 105) were thawed, washed in PBS, and resuspended to 105 cells/100 µl of a hybridization mixture (Dako, Glostrup, Denmark) containing 70% dimethylformamide and a telomere-specific FITC-conjugated (C3TA2)3 peptide nucleic acid probe. After 10 min at 82°C samples were incubated overnight at room temperature in the dark. Control samples were resuspended in hybridization solution without probe to obtain background fluorescence values. After hybridization, cells were spun down and washed twice with 4 ml PBS at 40°C for 10 min and finally resuspended in PBS containing 0.1% BSA, 10 µg/ml RNase A (Boheringer Mannheim, Indianapolis, IN), and 0.1 µg/ml propidium iodide (Calbiochem-Novabiochem, La Jolla, CA). After 4 h at room temperature in the dark, cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Sunnyvale, CA) or stored at 4°C before analysis. Signals from FITC-labeled beads (Quantum premixed; Flow Cytometry Standards, San Juan, PR) were measured at the beginning and at the end of every experiment. Signals were acquired in linear scale mode, and events were gated according to propidium iodide fluorescence to restrict analysis to cells with diploid DNA content, as described before (33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAckowledgementsReferences |
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We isolated CD8+ T cells from peripheral blood of healthy donors by magnetic sorting and stimulated them with irradiated allogeneic PBMC and PHA to trigger cell proliferation. Three days later, the cell population was infected with an amphotropic MSCV virus (27) containing the puromycin N-acetyl transferase gene (conferring resistance to puromycin) under the control of the phosphoglycerol kinase promoter and a complete hTERT cDNA under the control of the viral long terminal repeat. As expected from the work of others (7, 8), primary human lung fibroblasts infected with the same virus expressed telomerase activity and did not undergo senescence (data not shown).
Infected lymphocytes were selected in puromycin, and telomerase
activity was measured in nuclear extracts of resistant cells by TRAP.
As shown in Fig. 1
, A and C, transduced
lymphocytes expressed high levels of telomerase activity, similar to
those found in fibroblasts transduced with the same construct. Enzyme
activity was 
80 times higher than that in cells derived from the
same lymphocyte population but transduced with a control vector. These
results indicate that the introduced hTERT cDNA is expressed in T
lymphocytes as efficiently as in fibroblasts, and that in both cell
types hTERT is the component that limits telomerase activity. Nuclear
extracts from cells transduced with the empty vector or the hTERT
construct were prepared 3 wk after stimulation with PHA, when the
transiently induced endogenous telomerase activity had declined to
barely detectable levels. For any given extract there was a linear
correlation between the signals due to telomerase products (as measured
by phosphor imager) and the amount of extract added (Fig. 1B), indicating that the efficiency of the PCR step did not
vary between reactions. Addition of increasing amounts of extract from
vector-transduced, telomerase-negative cells to a TRAP reaction with
3000 cell equivalents of hTERT-transduced lymphocytes did not
significantly affect the level of telomerase activity measured (Fig. 1D), indicating that at the concentrations used, lymphocyte
extracts do not inhibit telomerase activity.
To compare maximal endogenous telomerase activity with the one due to
the transgene, we also performed TRAP assays with extracts of
CD8+ T cells activated with immobilized
anti-CD3 Ab plus soluble anti-CD28 Ab, a method that has been
reported to efficiently induce telomerase activity in cultured human T
cells (14, 17). Extracts were prepared 5 days after
stimulation, at the peak of induced telomerase activity. As shown in
Fig. 1, the activity detected in activated normal lymphocytes was
approximately four times lower than that in hTERT-transduced cells.
This difference could reflect low endogenous enzyme activity in all
activated normal cells, or high telomerase levels restricted to a small
fraction of cells. To address this question we stained cells, by
indirect immunofluorescence, with an Ab specific for hTERT (M.Amacker
and J. Lingner, manuscript in preparation). Observation by confocal
microscopy showed a pattern of distinct spots in the nucleus, as
previously described for tumor cells (35), in >90% of
the lymphocytes transduced with hTERT, whereas only background
fluo-rescence was detected in nontransduced control cells (Fig. 2A). No strongly stained cells were detectable among
lymphocytes activated with anti-CD3/anti-CD28 Abs (data not
shown), indicating that the difference between the average telomerase
activity of the transduced and activated control lymphocytes is mainly
due to a lower level of endogenous hTERT in all normal activated cells.
Transduced cells could be expanded for several weeks with no loss of
hTERT expression.
In addition to bulk cultures of freshly isolated
CD8+ T lymphocytes we infected two Ag-specific
CTL clones with the same retroviral hTERT construct. Clone NH 55
recognizes influenza matrix (FluMA) peptide 58–66 (23),
whereas clone LAU 203 0.3/3 is specific for the tumor-associated Ag
Melan-A26–35 (24) (see
Materials and Methods). Light microscopy of cells stained
with anti-hTERT Abs revealed that >90% of hTERT-transduced cells
expressed high levels of hTERT protein, whereas control cells did not
show any staining above background (Fig. 2B). At the
resolution obtained with the light microscope the punctuate pattern
seen in Fig. 2A is not resolved.
Ectopic hTERT expression does not prevent growth arrest of CD8+ T lymphocytes
To determine whether ectopic hTERT expression extended the
lifespan of T lymphocytes, we expanded cells infected with MSCV virus
containing hTERT cDNA or with the empty vector (carrying only the
puromycin resistance gene), as well as nontransduced cells, by periodic
stimulation with PHA plus irradiated PBMC in the presence of IL-2.
Living cells were counted at least once a week to determine the number
of population doublings. Bulk cultures of nontransduced
CD8+ T lymphocytes underwent 16–19 population
doublings before proliferation ceased, in agreement with previous
reports (36, 37). Cell populations expressing the hTERT
transgene stopped growing after a similar number of population
doublings as matched control populations (Fig. 3, A and B). After
growth arrest, cells not only failed to proliferate when restimulated
with PHA and irradiated feeder cells, but massive cell death by
apoptosis was often observed instead (data not shown). The apparent lag
in the growth kinetics of the transduced population shown in Fig. 3A probably reflects the effect of inhibitory products
generated by dying cells eliminated by the puromycin selection. Note
that no lag is observed when growth curves of cells transduced with the
hTERT construct and with the empty virus are confronted (Fig. 3B).
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, A and B).
CFSE staining of cells after growth arrest revealed that the cells had
ceased to divide. This indicates that the constant number of living
cells in the arrested cultures is not the consequence of equilibrium
between cell division and cell death.
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We used flowFISH to monitor telomere length in cultured
CD8+ T lymphocytes. The technique quantifies, by
flow cytometry, the fluorescent signals from individual cells after in
situ hybridization with a fluorescent peptide nucleic acid probe
specific for telomere repeats (20, 33, 34). Frozen
aliquots of cells collected at different times from the same population
were all processed in the same experiment. Cells were stained with
propidium iodide, and telomere fluorescence was measured only in G1
phase cells. Representative histograms are shown in Fig. 5. The fluorescence of every
individual cell is proportional to the total telomere length of its
chromosomes. An estimate of the mean telomere length is obtained by
comparing cellular signals with those from fluorescent bead standards,
applying the equation derived by Rufer et al. (20).
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, C and D). In contrast,
transduction with hTERT cDNA resulted in a progressive increase in
telomere length. When the cells stopped proliferating, telomeres had
reached an average length slightly higher than the peak observed 2 wk
after the first stimulation. The coefficient of variation of all the
fluorescence histograms is similar, indicating that the decrease in
telomere length during culture as well as the increase after hTERT
transduction affects the entire population of cells and not only a
subpopulation. This homogeneity correlates well with the uniform
staining of the transduced cell populations with anti-hTERT Abs.
Telomere fluorescence measurements in transduced and nontransduced CTL
clones after 30 days of culture show that ectopic hTERT expression
results in telomere lengthening also in these cells (Fig. 4, C and D). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAckowledgementsReferences |
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As in fibroblasts (7), ectopic expression of hTERT induces telomere lengthening in T cells. But unlike fibroblasts or endothelial cells (39), hTERT-transduced T lymphocytes cease to proliferate after a similar number of population doublings or cell divisions as control cells. This indicates that telomere shortening is not the only process that limits the life span of T cells. However, it cannot be excluded that one or a few telomeres continue to shorten in hTERT-transduced cells and trigger growth arrest when they reach a critical size. To rule out this unlikely hypothesis it will be necessary to quantify individual telomere length by Q-FISH (40).
Our results are reminiscent of the finding that immortalization of human keratinocytes and breast epithelial cells depends on additional changes besides hTERT expression, namely, on inactivation of the Rb protein/p16 pathway (11). One explanation for the different requirements for the immortalization of fibroblasts, on the one hand, and certain epithelial cells, on the other, is that the culture conditions for the latter are suboptimal and lead to activation of the p16/Rb stress pathway. A similar explanation may apply to T lymphocytes. Progressive p16 accumulation during T cell in vitro proliferation has indeed been reported (41), but the culture system used did not allow restimulation of cells with PHA, suggesting that it was inadequate for maximal expansion of T cells by this method.
An alternative explanation for the growth arrest of
CD8+ T lymphocytes under the conditions used in
our experiments may be that the stimulation requirements of T
lymphocytes change during clonal expansion as part of a differentiation
process of naive into memory cells. Thus, the apparent senescence of
these cells may not be due to the intrinsic limitation of the
proliferative capacity of the cells, but rather to the loss of the
ability to proliferate in response to the stimuli provided (PHA +
feeder cells plus IL-2). One change that might reduce responsiveness is
the decrease in the expression of the costimulatory receptor CD28 in T
cells that have undergone a high number of divisions. It has been
reported that CD28-mediated costimulation is necessary to prevent cell
death during T cell activation (42) and that CD28
expression declines during in vitro culture and is virtually absent in
senescent cells (43). We have compared CD28 expression on
lymphocytes transduced with hTERT or with the control vector, at
different times of culture and in senescent cells (data not shown). In
both populations we observed a similar gradual loss of CD28 expression
during culture. Loss of CD28 may be one of the causes why
growth-arrested T cells respond to restimulation with PHA and feeder
cells by apoptosis rather than proliferation. Down-regulation of other
costimulatory molecules such as CD134 (OX-40) and CD154 (CD40L), or IL
receptors such as IL-4R, IL-7R, and IL-15R and the IL-2R common 
chain (4) may also affect responsiveness of these cells to
TCR-mediated signaling. According to this hypothesis, transduction of
CD8+ T cells with components that restore their
capacity to respond to the same signals as naive cells should allow the
cells to proliferate further until their telomeres become critically
short. We predict that, as in fibroblasts, the latter limit can indeed
be overcome by ectopic expression of hTERT. Thus, hTERT may be required
but is not sufficient to immortalize human T lymphocytes.
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Ackowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAckowledgementsReferences |
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NX cells, and to Don Littman for the MSCV plasmid.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Markus Nabholz, Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland.
3 Abbreviations used in this paper: hTERT, human telomerase catalytic subunit; hTER, human telomerase RNA template subunit; CFSE, 5-carboxyfluorescein diacetate-succinimidyl ester; MSCV, mouse stem cell virus; TRAP, telomerase repeat amplification protocol.
Received for publication March 14, 2000. Accepted for publication August 8, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAckowledgementsReferences |
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