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The Journal of Immunology, 2006, 177: 3657-3668.
Copyright © 2006 by The American Association of Immunologists, Inc.

Mechanisms Regulating the Proliferative Potential of Human CD8+ T Lymphocytes Overexpressing Telomerase1

Olivier Menzel*, Marco Migliaccio*, Darlene R. Goldstein{dagger}, Sophie Dahoun{ddagger}, Mauro Delorenzi*,§ and Nathalie Rufer2,*

* National Center of Competence in Research Molecular Oncology, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; {dagger} Institut de Mathématiques, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; {ddagger} Division of Medical Genetics, Geneva University Hospital, Geneva, Switzerland; and § Swiss Institute of Bioinformatics, Lausanne, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division, eventually leading to a state of cellular senescence. Ectopic expression of telomerase results in the extension of their replicative life spans without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Using CD8+ T lymphocyte clones overexpressing telomerase, we investigated the molecular mechanisms that regulate T cell proliferation. In this study, we show that early passage T cell clones transduced or not with human telomerase reverse transcriptase displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in human telomerase reverse transcriptase-transduced cells with extended life spans. These cells, despite maintaining high expression levels of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. Strikingly, late passage T cells overexpressing telomerase accumulated the cyclin-dependent inhibitors p16Ink4a and p21Cip1 that have largely been associated with in vitro growth arrest. We conclude that alternative growth arrest mechanisms such as those mediated by p16Ink4a and p21Cip1 still remained intact and regulated the growth potential of cells independently of their telomere status.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Like most human somatic cells, T lymphocytes also have a finite life span. They undergo replicative senescence after 30–70 population doublings (PDs)3 of in vitro culture and remain alive as long as IL-2 is periodically added (reviewed in Ref. 1). Upon entering the state of senescence, cells undergo a plethora of changes in morphology with increased cell volume and loss of original shape (reviewed in Ref. 2) accompanied by irreversible structural alterations in the nuclear heterochromatin (3). This state, well defined in epithelial cells, is thought to play a protective mechanism against the development of tumors (reviewed in Ref. 4).

One of the better understood causes of replicative senescence involves telomere attrition. Telomeres are specialized structures capped by cellular proteins at the end of eukaryotic chromosomes and are important in maintaining the integrity of chromosomes. In most somatic cells, telomere shortening during cell division represents a molecular clock that triggers the entry of cells into senescence (5). In fibroblasts, the presence of one or a few critically short telomeres induces a DNA damage-like response resulting in proliferation arrest through the ATM/p53/p21Cip1 (hereafter p21) pathway (6, 7). Despite transient telomerase activity upon mitogenic and Ag stimulation (8), T lymphocytes also show progressive telomere loss with age and with cellular replication in vitro (9, 10, 11). In particular, the telomere length of Ag-experienced effector T cells in normal healthy individuals is found to be significantly shorter than that of naive cells (12). Thus, telomere-dependent restrictions in replicative potential are more likely to occur in effector T cells than in other cells of the hemopoietic system, because the shortest telomeres were observed in those cells in individuals over 60 years old (11).

An alternative mechanism that has been largely associated with irreversible growth arrest depends on the expression of the cyclin-dependent kinase inhibitor p16Ink4a (hereafter p16) (13, 14, 15). The p16 protein inhibits phosphorylation of the retinoblastoma protein pRb by cyclin D-CDK4/6, thus preventing entry into the cell cycle (reviewed in Ref. 16). This protein is normally not expressed at detectable levels in most adult tissues (reviewed in Ref. 17). However, it can be induced in vitro in various situations of stress and is highly expressed in epithelial cells and in some fibroblast strains that senesce despite having long telomeres (18, 19, 20, 21). We recently reported that p16 expression could directly be induced as a consequence of T lymphocyte activation (22) and was not related to activation-induced cell death (23). Importantly, the accumulation of p16 was responsible for the exit of a significant fraction of T cells from the proliferative population, thus limiting their numerical expansion in vitro (22).

Replicative senescence in various human cell types, including fibroblasts and endothelial cells (24, 25, 26), can be prevented by the ectopic expression of human telomerase reverse transcriptase (hTERT). In particular, transduction of the hTERT gene resulted in the extension of the proliferative life span of cells without inducing changes associated with transformation (27, 28). In line with these results, ectopic hTERT expression was sufficient to significantly prolong the in vitro life span of CD8+ and CD4+ T lymphocyte clones (29, 30, 31, 32). Overexpression of hTERT in those T cells did not alter their Ag specificity, their phenotypes, or their functional properties (29, 31, 32, 33). Despite a lack of transformation characteristics, it is still unclear whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Two recent reports suggested that the ectopic expression of hTERT in fibroblasts, in addition to extending the replicative life span, also mediated the expression of growth-controlling genes (34, 35). In T lymphocytes, telomerase overexpression was not only associated with resistance to oxidative stress (31) but also to loss of genetic stability (36) and the development of major chromosomal aberrations (37), resulting in growth arrest of the transduced cells. For example, with regard to the use of "immortalized" T lymphocytes for adoptive immunotherapy these findings emphasize the urgent need to further investigate the molecular mechanisms controlling the proliferation of such cells.

In the present study, we report striking differences in the control of the expansion potential between the following: 1) early passage CD8+ T cell clones (30 PDs) overexpressing or not overexpressing hTERT; 2) late passage hTERT-transduced T cell clones with elongated telomeres and extended life span (>150 PDs); and 3) normal aged T cell clones (70–80 PDs). Our data further indicate that both p16 and p53/p21 cell cycle checkpoints are still functional in a significant proportion of long-term cultured clones overexpressing telomerase. Because these cells accumulate p16 and p21 similarly as aging T cells, expression of these proteins can be mediated through a mechanism that is independent of telomere attrition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of hTERT-transduced CD8+ T cell clones

Human CD8+ T cell clones expressing either hTERT/GFP or GFP alone were obtained and cultured following serial rounds of subcloning steps as previously described (30). Briefly, the progeny of one single naive CD8+CD45RA+CD27+ T cell clone obtained by limiting dilution was transduced with the murine stem cell virus-based retroviral vector containing the gene of the enhanced GFP (Clontech Laboratories) under the control of the phosphoglycerate kinase promoter with or without the full-length human telomerase catalytic subunit (hTERT) cDNA. T cells expressing high levels of GFP were sorted by a FACS-Star Plus flow cytometer (BD Biosciences) and further recloned. Cells were seeded onto Terasaki 60-well plates (1 cell/well) in 20 µl of RPMI 1640 medium supplemented with 8% human serum and 150 U/ml recombinant human IL-2 (a gift from GlaxoSmithKline) and stimulated with 1 µg/ml PHA (Sodiag) plus 0.5 x 106/ml irradiated allogenic PBMCs (3000 rad) as feeder cells. After 10 days of culture, growing clones were transferred to 96 U-bottom plates, and 4 days later (i.e., 2 wk after subcloning) cells were stimulated again to obtain routinely 1–4 x 106 cells per clone. Cells were counted to determine the number of PDs, and the subcloning procedure was repeated until no further subclones could be obtained (GFP control clones) or until the seventh recloning step (hTERT/GFP clones). Transduction and subcloning procedures were performed on CD8+ naive T cell clones isolated from two different healthy donors (HDs) aged between 30 to 35 years (HD1 and HD2).

Telomere fluorescence in situ hybridization (FISH) and flow cytometry FISH

The average length of telomere repeats at chromosome ends from in vitro cultivated T cell clones was measured by FISH and flow cytometry as previously described (10, 11). Telomere fluorescence was calculated by subtracting the mean fluorescence of the background control (no probe) from the mean fluorescence obtained from cells hybridized with the telomere probe after calibration with FITC-labeled fluorescent beads (Quantum TM-24 premixed; Bangs Laboratories) and conversion into molecules of equivalent soluble fluorochrome (MESF) units. The following equation was used to estimate the telomere length in base pairs: bp = MESF x 0.495 (10, 38).

G-banding and karyotyping

To produce metaphase spreads derived from the T cell clones used in this study, cells (1 x 106 ml) were stimulated using PHA and feeder cells as described above and expanded in 150 U/ml rIL-2. At day 5 of culture, 0.1 mg/ml colcemid (Invitrogen Life Technologies) was added. After 1 h, cells were harvested, incubated for 20 min in a hypotonic 75 mM KCl solution, and fixed using a 1:3 methanol/acetic acid mixture. Metaphase spreads were prepared by dropping the fixed-cell suspension on glass slides. G-banding of metaphase slides was done by 0.25% trypsin (Invitrogen Life Technologies) and analyzed using Alphelys software.

Cell culture and cell proliferation assay

Cultures of T cell clones were obtained by seeding the T cells onto 24-well culture plates (2 x 106 cells in 2 ml/well) in RPMI 1640 medium supplemented with 8% human serum and 150 U/ml rIL-2 (GlaxoSmithKline) and stimulated using PHA (Sodiag) and feeder cells as described above. Culture medium was checked daily and changed when required. The stimulation procedure was repeated every 15 days of culture. PDs were determined by the periodic counting of living cells using trypan blue to exclude dying cells. When indicated, samples of 1 x 106 cells were resuspended in 500 µl of PKH26 (Sigma-Aldrich) solution (4 x 10–6 M in Diluent C), incubated for 5 min at room temperature, washed three times, and seeded into 24-well plates. Half of PKH26-labeled cells were stimulated with PHA and feeder cells, whereas the other half was used as nonproliferative control cells.

Affymetrix gene expression analysis

Gene expression profiling was performed on young T cell clones expressing or not expressing hTERT, aged control T cell clones, and hTERT-transduced T cell clones with extended life span upon mitogen stimulation and cell culture expansion as described above. Cells from young control T cell clones (HD2; young-GFP) did not expand properly when compared with the other clones and were not included in the transcriptional profiling. This result is most likely due to unknown side events that occurred during the shipping and storage of this particular sample. Mycoplasma-free cells were harvested after 15–18 days of mitogen stimulation, and <10% of the expanded cells were annexin V positive. RNA from ~30 x 106 T cells was extracted, purified, and used for cDNA synthesis following standard procedures (<www.affymetrix.com>). Two separate Affymetrix gene expression experiments were performed. The first set of Affymetrix data comprised T cell clones generated from HD1 (hybridized to Affymetrix U133A GeneChip), whereas the second one was done on T cell clones isolated from HD2 (Affymetrix U133Plus 2.0). Triplicates (01–03) were performed for each clonal condition (young-control, young-hTERT, aged, and extended clones) and for both sets of Affymetrix chips (corresponding to HD1 and HD2). Each unit of these chips is represented by a set of oligonucleotides called a probe set. The U133Plus2.0 GeneChip contains the 22,215 human probe sets of the U133A chip that represent ~12,000 distinct annotated transcripts (distinct UniGene clusters). The microarray data set discussed in this publication have been deposited in National Center for Biotechnology Information’s Gene Expression Omnibus (<www.ncbi.nlm.nih.gov/geo/>) and are accessible through Gene Expression Omnibus Series accession number GSE5144.

Microarray data analysis

The R statistical environment was used for data analysis with the open source Bioconductor (<www.bioconductor.org>) packages affy, affyPLM, cluster, limma, and multtest. Array quality was assessed in affyPLM using robust multiarray analysis quality control measures (39). One chip was excluded (young-hTERT-expressing clone HD1-02) due to poor quality. Normalized RNA abundance estimates were obtained by robust multiarray analysis (40). The method uses quantile normalization (41) and yields estimates of abundance for each probe set on a base 2 log scale. For comparisons between the two sets of data, intensities for each data set and each probe set were centered to the mean of the medians of the conditions. Differentially expressed genes between pairs of conditions were identified based on log base 2 fold change and log-odds ratios (B-statistics) (42, 43).

Intracellular staining, BrdU incorporation assays, and flow cytometry analysis

For p16 analysis, cell pellets from 0.25 x 106 cells were fixed in PBS with 2% paraformaldehyde, washed, and permeabilized in 90% ethanol. Following 30 min of incubation at 4°C, cells were washed and 50 µl of total cell suspension was mixed in 5 µl of PE-conjugated mAbs against human p16 or the corresponding isotype controls (BD Biosciences). Cells were incubated for 60 min at room temperature, washed in 1xPBS with 3% FCS, and analyzed on a FACSCalibur flow cytometer (BD Biosciences). The percentage of p16-positive cells was determined as recently described (22). For BrdU incorporation studies, 106 cell samples were incubated in the presence of 10 µM BrdU for 1 h at 37°C. Cells were then fixed, permeabilized, treated with DNase to expose BrdU epitopes, and stained with the allophycocyanin-conjugated anti-BrdU Ab and 7-aminoactinomycin D (7-AAD) using the BrdU flow kit (BD Biosciences). Where indicated, anti-p16 Ab, or the corresponding isotype control, was also added for simultaneous intracellular staining.

Western blot analysis

Protein expression in total cell extracts from cultured T cell clone samples obtained between days 10 and 15 after stimulation was analyzed by Western blotting as described (22). Briefly, 3 x 106 cells were spun down and lysed, and cell extract corresponding to 8 x 105 cells was loaded in each well. Proteins were resolved on a 12% PAGEr Duramide precast gel (Cambrex), and membranes were blocked and probed with anti-p21 and anti-tubulin Abs (Santa Cruz Biotechnology). Quantification of p21 and tubulin expression was done using the FluorChem IS-8900 digital imaging system (Alpha Innotech) and the AIDA software (Raytest). Where indicated, quiescent 4 x 106 T cells were exposed to UV irradiation with 10, 15, or 20 J/cm2. Following irradiation, growth medium was immediately added and cells were incubated for an additional 20 h. After incubation, cells were harvested for protein extraction, and the ratio of p21 to tubulin was quantified as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
hTERT-transduced CD8+ T lymphocytes with an extended life span display long telomeres and normal karyotypes

We previously found that T cells overexpressing hTERT displayed significant elongation of telomeres (30). These experiments were performed on transduced CD8+ naive T cell clones obtained from a healthy individual (HD1). To further investigate telomere length dynamics in hTERT-expressing T cells over long-term culture, CD8+ naive T cell clones derived from a second healthy donor (HD2) expressing either hTERT/GFP or GFP alone were generated following serial rounds of subcloning steps (Fig. 1A). All tested GFP and hTERT/GFP CD8+ T cell subclones (subcloning steps II–VII) had lost the expression of the costimulatory CD28 receptor (Ref. 30 and data not shown) while retaining the ability to kill the Fc receptor-bearing tumor cell line P815 in a CD3 mAb-mediated redirected killing assay (30). Ectopic expression of hTERT in T cell clones derived from both HD1 and HD2 resulted in constitutive telomerase expression (data not shown) and significant extension of their replicative life spans (>150 PD; n = 36; (30). Conversely, GFP control T subclones showed low to undetectable levels of telomerase activity, and further subclones could only be generated until the fourth recloning step. These clones were considered as aged or presenescent as opposed to the T cell clones with prolonged life spans upon hTERT transduction, which were defined as extended (Fig. 1A).


Figure 1
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  		FIGURE 1. Telomere length and karyotype analysis of transduced CD8+ T cell clones. A, The progeny of one naive CD8+ T cell clone (subcloning step I) was transduced with murine stem cell virus-based retroviral vectors containing the gene for GFP with or without the full-length hTERT cDNA. All clones expressing either hTERT/GFP (•) or GFP alone ({circ}) were obtained following serial rounds of subcloning steps (II–VII). All hTERT-derived T cell subclones generated previously (HD1; Ref. 30 ) and, in this study, (HD2; n = 36) showed extended replicative life span (longer than or equal to that of subcloning step VI). B, Telomere fluorescence analysis in early (young with or without hTERT) and late passage (aged-GFP and extended-hTERT) CD8+ T cell clones derived from two healthy donors (HD1 and HD2). The dotted line was arbitrarily set at the mean telomere fluorescence obtained from the extended clones and allows direct comparison between samples. The telomere fluorescence was converted to kilobase values as described in Materials and Methods. C, G-banding karyogram of a representative metaphase isolated from early and late passage T cell clones expressing or not expressing hTERT. Both young T cell clones depicted a normal 46,XY karyotype, whereas karyotype analysis of a metaphase derived from extended and aged clones showed a trisomy for chromosome 22 and several minute chromosomes, respectively (see arrows).

 
The average length of telomere repeats at chromosome ends was quantified in early and late passage T cell clones expressing or not expressing hTERT by flow FISH (Fig. 1B). In young T cell clones at equivalent ages of culture, ectopic expression of telomerase resulted in a significant increase in telomere lengths. This augmentation was particularly pronounced for the hTERT-transduced clones derived from the second donor. Although aged T cell clones displayed the lowest levels of telomere fluorescence, their corresponding telomere lengths were still heterogeneous and varied between 2.1 kb (HD1) and 5.4 kb (HD2). In contrast, most hTERT-expressing T cell clones cultured for extended periods displayed telomere length elongations of up to 12 kb (n = 20), similar to those found in cord blood T lymphocytes (10). Together, these data confirm our previous finding (30) that increased hTERT expression allows elongation of telomere ends and extension of life span.

We next performed cytogenetic analysis on young, aged, and extended T cell clones generated from both healthy donors using G-band preparations (Fig. 1C). Early passage of GFP alone and hTERT/GFP-transduced T cell clones all contained 46,XY normal chromosomes (12 analyzed metaphases for each subclone). A detailed analysis of >140 metaphases derived from late passage T cell clones revealed a normal karyotype in ~90% of hTERT-expressing cells and >80% of senescing cells (Table I). Occurrences of distinct chromosomal translocations or trisomies were among the most frequent abnormalities observed in those clones. Overall, our data are in line with the notion that long-term cultures accumulate some but yet limited chromosomal aberrations over time (1).


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  		Table I. Cytogenetic analysis by G-banding and karyotypinga

 
hTERT-transduced CD8+ T cell clones with an extended life span are characterized by reduced growth kinetics in response to stimulation

Ectopic hTERT expression has been shown to enhance the expansion potential of T cells (29, 30, 31, 32, 33, 36). To investigate more precisely the dynamics of cell proliferation in these cells, we conducted a quantitative flow cytometry analysis of the proliferative response to the mitogenic lectin PHA using PKH26 as an indicator of cell division (Fig. 2A). Both of the early passage T cell clones, expressing or not expressing hTERT, underwent similar increased expansion when compared with late passage T cells. Expansion was particularly evident on day 4 after stimulation. In contrast, the proportion of cells that did not start to proliferate in response to mitogens was higher in the aged and extended T cell clones as observed on days 4 and 5 upon activation. We could not detect significant differences in the proportion of cells undergoing apoptosis (Annexin-V positive) among the latter clones when compared with the young one (data not shown).


Figure 2
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  		FIGURE 2. Expansion potential of early and late passage CD8+ T cell clones transduced with hTERT. A, PKH26 fluorescence histograms at days 3, 4, and 5 after mitogen stimulation (shaded histograms). Open histograms, PKH26 intensity obtained from each respective unstimulated T cell clone. Two representative examples of late passage T cell clones are depicted. The dotted line indicates the proportion of PKH26bright undivided cells according to the second aged GFP T cell clone at day 3. The graphs are representative of five independent experiments. B, Growth kinetics of young (with or without hTERT), extended (with hTERT), and aged (GFP) T cell clones were assessed by periodic cell counting of living cells (PDs) following three rounds of mitogen stimulation (see arrows) and by using the following formula: PD (day x; day y) = (log(average cell count at day y) – (log(average cell seeded at day x))/log2. One representative experiment of four is shown.

 
We next determined the growth kinetics of early and late passage T cells by counting living cells at regular intervals after mitogenic stimulation (Fig. 2B). Young T cell clones, whether they expressed hTERT or not, showed equal growth kinetics and underwent at least 10 PDs following three rounds of stimulation. Thus, our data indicate that hTERT expression does not confer increased growth advantage in those age-matched cells, which is in line with a previous report by Luiten et al. (31). Intriguingly, a significant decline in the number of PDs was repetitively observed in hTERT-transduced T cells with extended life spans (Fig. 2B). Finally, senescing T cell clones frequently displayed the lowest growth potential.

Altogether, these results are consistent with a higher proportion of young T cells being recruited into cell cycle as opposed to late passage (extended and aged) cells (Fig. 2A). They also support the notion that alternative growth arrest mechanisms may regulate the proliferation potential of cells that are cultured over prolonged periods of time. Of note, several aged control T cell clones stopped proliferation while these experiments were being performed. Despite reduced expansion potential, however, such replicative growth arrest was not observed in hTERT-transduced cells cultured over prolonged periods of time.

Increased hTERT expression in early passage CD8+ T cells does not result in marked global changes in gene expression

To assess any biological differences at the molecular level, we next compared the gene expression profile of 22,215 transcripts in early and late passage CD8+ T lymphocytes overexpressing hTERT with their corresponding control cells. Clones isolated from two different healthy donors were expanded before being subjected to two independent sets of Affymetrix gene-profiling experiments as described in Materials and Methods.

We first performed a hierarchical clustering to study the overall similarity of gene expression patterns between young, aged, and extended T cell clones (Fig. 3A). High reproducibility was found for the triplicates (replicates 01, 02, and 03) derived from each distinct clonal condition. Strikingly, similar gene expression patterns were observed between the clones belonging to the same category, young, aged or extended, and could accordingly be defined into three distinct clusters or groups. The primary source of the generated cells, HD1 or HD2, had no influence on the clustering, because T cell clones from the same category but different donors displayed closely related gene profiles and still clustered together. In particular, within the young cluster the T cell clones expressing or not expressing hTERT formed a relatively homogeneous group (HD1; Fig. 3A). Changes between early passage T cell clones (young-control HD1, young-hTERT HD1, and young-hTERT HD2) were further explored using two-dimensional scatter plots of the differences between the gene-wise mean normalized intensities (Fig. 3B). As shown in Fig. 3A, the overall expression patterns were highly similar with no statistically significant differences among the young clones whether or not they expressed increased levels of telomerase, indicating that in young T cell clones telomerase expression does not induce major biological changes at the molecular level.


Figure 3
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  		FIGURE 3. Overall similarity of gene expression patterns by hierarchical clustering (A) and two-dimensional scatter-plots (B). A, Hierarchical clustering was based on Euclidean distances between samples and the Ward clustering method. Triplicates (01–03) from young (with or without hTERT), extended (with hTERT), and aged (GFP) T cell clones derived from HD1 and HD2 were included in the data analysis. One chip was excluded (young-hTERT-expressing clone HD1-replicate 02) due to poor quality. Samples from the young control (GFP) T cell clone of HD2 are missing (see Materials and Methods). Of note, hierarchical clustering identifies three major groups of clones: young vs extended vs aged. B, Overall pairwise similarity between conditions was explored with two-dimensional scatter plots of the differences between gene-wise mean normalized intensities ({Delta}1, {Delta}2, and {Delta}3), using the 22,215 common U133A probe sets.

 
Differential gene expression patterns in hTERT-transduced CD8+ T cell clones with an extended life span

To investigate in detail the gene expression profiles of extended T cell clones, we next generated heat maps to illustrate the patterns of expression for each clonal condition and included genes showing at least a 2-fold differential in expression. Again, the global expression patterns were best described as three main clusters of expression, including young, extended, and aged cells, independently of HD1 and HD2 (Fig. 4A). Overexpression of telomerase in T cell clones with prolonged life span resulted in considerable alteration of their transcriptional profile. Strikingly, the cluster formed by these cells displayed a gene expression pattern that placed them between young and aged T cells. An important proportion of key genes involved in the regulation and progression of the cell cycle were found up-regulated in both young and extended clones in respect to aged cells (Fig. 4AI and Table II). Among these genes, several are crucial for the G1-S phase (Cdc2, FoxM1, KI67), S phase (Top2A, KI67, TIMS, and cyclin-F) and G2-M transitions (Cdc2, cyclin-B, and cyclin-F).


Figure 4
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  		FIGURE 4. Gene expression profiles in early and late passage CD8+ transduced T cell clones. A, Heat maps illustrating expression patterns of genes showing at least a two-fold differential in expression (1403 probe sets). For visualization of the relative changes in each gene, the values of each gene were color-coded such that green represents the strongest down-regulation (–1) relative to the average for that gene and red represents the strongest up-regulation (+1). I represents genes that were mostly found up-regulated in young and extended clones in respect to aged cells, whereas II represents genes up-regulated in late passage (extended and aged) clones in respect to young cells. Of note, for most of the genes shared between young and extended T clones (Table II) the trend of expression was in favor of the younger clones expressing or not expressing hTERT, whereas for the genes shared between extended and aged clones (Table III) the trend of expression was toward normal old cells. B, Analysis of the expression of TCF4 transcription factor at mRNA (array) and protein (Western blot) level. Two representative probe sets (full and dashed line) are depicted for HD1 (gray) and HD2 (black). Extracts from equal numbers of cells (8 x 105 cells) were analyzed on Western blots. A representative example is depicted. Data collected from five independent experiments included clones of both HD1 and HD2 (n = 22) and are expressed as densitometric ratios of TCF4 and tubulin normalized to young GFP T cell clones. Experiments based on young control clones derived from HD2 were performed on subclone II instead of subclone I (see Material and Methods).

 

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  		Table II. Genes significantly up-regulated in young and extended T cell clones in respect to aged T cell clonesa

 
Remarkably, extended T cell clones with increased telomerase also shared genes that were found up-regulated in normal aged cells (Fig. 4AII and Table III). Thus, in contrast to early passage young clones, both aged and extended cells expressed higher levels of the CDK inhibitors p21 and p16, DNA-damage-inducible GADD45A, {alpha}-actinin (ACTN1; which promotes cell adhesion), and WIG1, a p53 target gene encoding for a growth inhibitory zinc finger protein. Of note, the gene profile of the aged control clones derived from HD1 revealed the highest differential gene down-regulation (Fig. 4AI) as well as up-regulation (Fig. 4AIIb). This observation correlated well with the data showing that this particular clone exhibited the shortest telomeres (Fig. 1) and had reduced growth potential (data not shown) when compared with the corresponding clone from HD2, most likely indicating a more advanced in vitro senescent state.


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  		Table III. Genes significantly up-regulated in extended and aged T cell clones in respect to young T cell clonesa

 
No major changes in the expression of genes involved in DNA damage repair (e.g., ATR, ATM, Rad17, Rad9, and p53), telomere-associated factors (e.g., tankyrases, NBS, Pot1, rif1), or apoptosis (e.g., Bcl2 and caspases) were observed between young, extended, and aged clones (data not shown). Moreover, only very few genes were found differentially expressed in hTERT-transduced T cell clones with an extended life span with respect to young and aged clones (Fig. 4AIII arrow and Table IV). Among these genes, the TCF4 gene (also known as ITF2, E2-2, and SEF2-1) appears of interest because it was mapped to chromosome 18p21.1 and encodes for a basic helix-loop-helix DNA binding protein of 667 amino acids involved in transcriptional regulation (44). Increased TCF4 expression was further observed at the protein level by Western blotting (Fig. 4B), confirming our mRNA expression arrays.


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  		Table IV. Genes significantly up-regulated in extended T cell clonesa

 
Late passage CD8+ T cell clones transduced or not transduced with hTERT express high levels of p16 protein associated with cell cycle arrest

Our finding that increased hTERT-transduced T cells cultured over extended periods expressed higher levels of p16 and p21 mRNAs suggests that alternative mechanisms besides telomere attrition may also mediate the replicative growth arrest of cells. Consistent with this notion, we recently reported that p16 limits the proliferative potential of bulk CD8+ T lymphocytes responding to mitogen stimulation by preventing a fraction of cells to enter into the cell cycle (22). To address whether de novo p16 expression was associated with the intermediate proliferative expansion observed in extended T cells overexpressing hTERT, we determined p16 expression levels by intracellular staining (Fig. 5A). High levels of p16 were found in a substantial fraction (>50%) of late passage T cell clones that expressed (extended) or did not express (aged) hTERT. In contrast, early passage T cells displayed reduced levels of the p16 protein. When p16 expression was assessed on days 4, 7, and 11 after mitogen stimulation, the percentage of p16-containing cells declined during the first four days after activation (Fig. 5A), most likely due to an increase in p16-negative proliferating cells (22). This decline in p16 expression was particularly severe in the young cells from HD1. However, the proportion of p16-expressing cells began to increase from day 5 onward, indicating de novo synthesis of this protein (22). This increase in p16 expression was observed in most tested clones, with a particular emphasis on late passage T cell expressing or not expressing the telomerase that contained the highest amounts of p16 by day 11 after stimulation (Fig. 5A).


Figure 5
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  		FIGURE 5. Expression of the p16 protein in early and late passage CD8+ T cell clones transduced or not transduced with telomerase. A, Levels of p16 expression were measured 0, 4, 7, and 11 days after mitogenic stimulation, and the percentage of p16-positive cells was calculated as described in Materials and Methods. Shaded and open histograms, The fluorescence obtained with p16 mAbs and its corresponding isotype control, respectively. Allogeneic PBMCs (GFP-negative) were discarded by gating the cells in the corresponding plots. The FL1 (GFP) and FL2 (p16) channels were carefully compensated using the samples stained with the isotype control Abs. Representative examples of HD1- and HD2-derived T cell clones are depicted. B, BrdU incorporation (1-h pulse) and DNA content (7-AAD; linear scale) analysis on gated p16-negative and p16-positive young (with or without hTERT), extended (with hTERT), and aged (GFP) T cell clones at day 7 after stimulation. The proportions (percentages) of cells in G0/G1 (BrdU-negative 7AADlow), G2/M (BrdU-negative 7-AADhigh), and S (BrdU-positive) phases of cell cycle, respectively, are depicted. One representative experiment of four is shown. Experiments based on young control clones derived from HD2 were performed on subclone II instead of subclone I (see Materials and Methods).

 
We next investigated the consequence of p16 expression during cell cycle replication of young, extended, and aged T cell clones. Using BrdU incorporation assays combined with p16 intracellular staining, we measured the fraction of cells that was in or entered into the S phase of the cell cycle during a 1-h pulse at day 7 after stimulation (Fig. 5B). The p16-positive gated T cells were predominantly composed of noncycling cells (BrdU-negative). In contrast, a significant fraction of p16-negative gated T cells had incorporated BrdU (up to 30% for the extended cells) and progressed through the S phase of the cell cycle (BrdU-positive; 7-AAD-positive). Similar results were found at day 4 after stimulation (data not shown). Altogether, the accumulation of p16 occurs preferentially in late passage T cell clones independently of telomerase expression. These findings also indicate that high levels of p16 expression in late passage T cells may account for their diminished proliferative potential upon stimulation by impeding a significant proportion of cells from entering into the cell cycle.

The p21 protein accumulates in late passage CD8+ T cell clones and is expressed in response to UV-induced premature senescence

To explore the role of p21 during in vitro cultures of early and late passage T cell clones expressing or not expressing telomerase, levels of p21 protein were next quantified by Western blotting. Two distinct p21 expression patterns could be defined based on the characterization of >40 T cell clones. As depicted in the first example (Fig. 6A), drastic p21 accumulation was found in late passage CD8+ T cell clones as opposed to p21 levels from early passage cells. However, in contrast to these data, several additional T cell clones irrespective of their extent of cell division did not reveal major changes in their p21 expression levels (Fig. 6A). Despite high heterogeneity in p21 expression within clonal T cells, our results indicate that constitutive expression of telomerase in T lymphocytes did not prevent the induction of p21 expression in a significant fraction of the generated clones.


Figure 6
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  		FIGURE 6. Expression of p21 protein in early and late passage CD8+ T cell clones transduced or not with telomerase. A, To compare protein levels per cell between different samples, extracts from equal numbers of cells (8 x 105 cells) were analyzed on Western blots. Two representative examples of p21 expression in young, extended, and aged clones are depicted. Data collected from eight independent experiments included clones of both HD1 and HD2 (corresponding to a total of 44 clones) and are expressed as densitometric ratios of p21 and tubulin normalized to the young GFP T cell clones. Of note, accumulation of the p21 protein was observed in 9 of 14 extended T cell clones and in 8 of 12 aged T cell clones, relative to the young GFP control clones. B and C, Effect of UV irradiation on the expression of the active forms of the p53 protein (p53P-Ser15) (B) and the p21 protein (C) in young, extended, and aged T cell clones. Where indicated, extracts from equal numbers of cells were analyzed by Western blotting 20 h after UV irradiation with 10, 15, or 20 J/cm2. A representative example is shown. C, Data collected from six distinct experiments and including HD1- and HD2-derived clones (n = 31) are expressed as densitometric ratios of p21 and tubulin normalized to each respective control cells (no UV). Of note, results from the four clones that have highly up-regulated p21 upon UV treatment were obtained within the same experiment.

 
Human somatic cells exposed to various forms of cell stress, including hyperoxia, {gamma}-irradiation, or UV light, undergo premature cell cycle arrest via activation of the p53/p21 pathway (reviewed in Ref. 45). In this study we investigated whether this pathway was still active in hTERT-transduced T cell clones with extended life spans by using UV irradiation. Early and late passage T cell clones constitutively expressing or not expressing hTERT responded to UV irradiation by activating p53 (Fig. 6B). Apart from a few exceptions, most T cell clones were only able to induce low-to-mild levels of p21 expression in response to UV treatment (Fig. 6C). Importantly, the induction of similar levels of p21 expression was observed in young, extended, and aged T cell clones, indicating no significant differences as a consequence of long-term culture and/or hTERT ectopic expression. These data also demonstrate that p53/p21 pathway is functional in all four distinct clonal conditions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Overexpression of telomerase in human CD8+ and CD4+ T cells has repetitively been found to confer a net growth advantage by extending the replicative capacity of the transduced cells (29, 30, 31, 32, 33). However, recent observations revealed that hTERT expression does not always prevent the overall loss of telomeric DNA (33, 36), eventually leading to genomic instability and replicative senescence (36, 37). In contrast to these studies, we generated CD8+ hTERT-transduced T cell subclones with significant telomere length elongation (up to 12 kb) and extended life spans (>150 PD). Despite such extreme life spans, the majority of these clones retained a normal 46,XY karyotype (Fig. 1 and Table I), indicating that cells with elongated telomeres upon induced telomerase expression may be reasonably protected against eventual loss of genetic stability. Altogether, our T cell clones with stable telomere ends provide a powerful model for studying whether telomerase possesses additional functions by modulating the growth potential of cells independently of their telomere status.

A major finding in this study is that overexpression of telomerase in transduced CD8+ T cell clones does not promote nor enhance their proliferation in response to mitogen-mediated stimulation. Similar growth kinetics were observed in early passage T cell clones whether they expressed hTERT or not, which is in agreement with the similar overall transcription profiles (Fig. 3) and proteome profiles (L. Thadikkaran, D. Menzel, J.-D. Tissot, and N. Rufer, manuscript in preparation) found in both young cells. Thus, these results indicate that ectopic expression of telomerase had, apart from telomere elongation, only minimal effects on early passage cells. Strikingly, late passage CD8+ T cell clones overexpressing telomerase displayed reduced proliferative expansion between that of young and aged cells following mitogenic activation (Fig. 2). Detailed expression-profiling analysis revealed that T cell clones with extended life spans not only shared many genes associated with the cell cycle progression of young cells but also up-regulated several cell cycle-regulating genes such as p16, GADD45A, and p21 in common with aged cells (Tables II and III). Because these cells strongly expressed both positive and negative cell cycle-related modulators, our data provide a rational explanation for their intermediate proliferative potential.

Although there was no net increase in the growth capacity of hTERT-transduced CD8+ T cell clones upon mitogen stimulation, they still displayed prolonged life spans, with some of these clones being maintained over 200 PDs in culture. An important question that remained was whether telomerase-dependent induction of genes contributed to this extension. Comparative transcription profiles on T cell clones derived from two different healthy donors showed that only a limited number of genes with significant differences were identified in long-term cultured hTERT-expressing cells, in line with a recent study by Roth et al. (36). Among these genes, the transcription factor TCF4 (44), which is also found up-regulated at the protein level, is of potential interest because of its involvement in T lymphocyte development (46). Remarkably, T cells with prolonged life spans and stable telomeres were able to maintain specific gene expression associated with cell cycle progression (Table II). However, because they were expressed at comparable levels in early passage T cells transduced or not transduced with hTERT, these observations further indicate that telomerase by itself is unlikely to promote such expression. Alternatively, telomere attrition may directly modulate the expression of genes linked to cell cycle progression. In line with this view, we found that these genes were down-regulated in aged control cells from HD1 when compared with those in the corresponding HD2 clones (Table III), correlating with shorter average telomere lengths (2 vs 5 kb) and diminished growth potential (data not shown). Thus, aged clones from HD1 displayed critically short telomeres and were found close to replicative senescence, in contrast with hTERT-transduced clones having extended life spans and stabilized telomeres (12 kb). Collectively, these results provide strong evidence that telomere attrition rather than hTERT may modulate the expression of critical genes associated with cell cycle progression.

Intriguingly, our results and those of others (29, 31, 32, 36) do not exactly recapitulate those found for hTERT-transduced epithelial cells in which telomerase could induce expression of growth-controlling genes such as epithelial growth factor receptor (35) or epiregulin (34) and enhance cell proliferation (35, 47). Expression of hTERT also promoted regenerative properties of bone marrow cells (48) and delayed cell cycle exit in cardiac muscle (49). A possible explanation for the discordant results found with the fibroblast study (35) is that enhanced proliferation potential was only observed in transduced cells grown in minimally defined medium in the absence of exogenous growth factors and not in cells grown under standard culture conditions (which was the case for the T cells used in this study). Of note, we did not observe increased growth of hTERT-expressing T cell clones when cells were maintained in low amounts of rIL-2 (data not shown). Thus, these observations may indicate that ectopic expression of telomerase has different consequences in lymphocytes and epithelial cells, requiring further investigations involving direct comparison.

Several studies reported that the extension of telomeres by ectopic expression of hTERT does not prevent transduced cells from becoming senescent. This finding has been clearly shown both in epithelial cells (18, 19, 50) and in bulk CD8+ T lymphocytes (22, 51) that stop to proliferate even though the lengths of their telomeres are well beyond the critical limit. Thus, it is becoming apparent that immortalization is not necessarily the final outcome of telomerase overexpression. In this study we show that hTERT-transduced T cell clones with prolonged life spans acquired several biological properties that are typically found in senescing clones following long-term culture. In particular, they accumulated functional p16 and p21 CDK inhibitors. These observations demonstrate that mitogen-induced p16 expression and UV-induced p21 expression can be mediated through mechanisms that are independent of telomere attrition. This finding is consistent with the growing body of data showing that distinct senescence programs can progress in parallel (7), eventually leading to the growth arrest of in vitro cultured cells.

Another point to be mentioned is the powerful readout of p16 intracellular staining that allowed us to perform several comprehensive studies of p16 expression dynamics either in subsets of CD8+ T lymphocytes (22, 23) or in hTERT-expressing CD8+ T cells (the present study). We demonstrated that, in cultured T lymphocytes, p16 expression is not a process that uniformly occurs in all cells but that de novo p16 appears only in a fraction of cells that have been actively cycling (22). Activation-induced p16 expression is found almost exclusively within the G0/G1 and G2/M phases of the cell cycle (Ref. 22 and Fig. 5). Intriguingly, accumulation of the p16 protein preferentially occurred in naive-derived as opposed to prime-derived T lymphocytes (23). We also observed low but readily detectable levels of p16 in freshly isolated ex vivo CD8+ T lymphocytes (23). Collectively, we proposed that activation-induced p16 expression might represent an alternative process to apoptosis by limiting the proliferation potential of recently activated naive-derived T lymphocytes upon antigenic priming. In this study we report that long-term cultured T cell clones expressing or not expressing telomerase preferentially accumulated p16 protein when compared with early passage clones. Thus, in contrast to the p16-induced expression in naive T cells that occurred as early as 8–10 days upon ex vivo stimulation, expression of p16 in cells kept in culture for extended periods of time (>100 PDs) is most likely triggered by a different process that has been commonly associated with in vitro aging and senescence in other somatic cells (18, 19, 20, 21). Further work is needed to elucidate the underlying mechanisms that trigger p16 expression either in ex vivo naive T lymphocytes or in aged in vitro cultured T cells.

Finally, our data indicate that telomere-independent arrest mechanisms not only remain intact but may also control and limit to some extent the proliferative expansion of hTERT-transduced lymphocytes. These results, however, contrast with those from recent reports (52, 53) in which hTERT-immortalized fibroblasts during continuous growth in vitro acquired several mutations (e.g., in the p53 gene) as well as a deletion in the p16 gene that may favor the appearance of clones carrying potentially malignant alterations. Moreover, one cannot formally exclude the possibility that the growth arrest observed in a significant fraction of hTERT-expressing T lymphocytes may be due to excessive telomerase activity acting as a hyperproliferative signal in transduced cells and inducing a senescent-like phenotype in a manner similar to that seen following overexpression of oncogenic Ras or Raf (54).

One of the major barriers to the widespread use of Ag-specific cytolytic T lymphocytes in adoptive transfer therapy remains the finite life span of these cells. Whether tumor-reactive CD8+ T cells that ectopically express telomerase could now be used as cell source for adoptive immunotherapy against infection and cancer remains unclear at this point. Nevertheless, our results regarding the safe use of hTERT-transduced T lymphocytes are encouraging, because they indicate that alternative growth arrest mechanisms such as those mediated through p16 and p21 are still functional in these cells and regulate their growth potential.


   Acknowledgments
 
We are thankful to Drs. Joachim Lingner, Daniel Speiser, and Jean-Daniel Tissot for critically reading the manuscript, and to Patricia Corthésy-Henrioud, Patrick Reichenbach, and Séverine Reynard for excellent technical assistance. We thank Dr. Patrick Descombes for advice on chip hybridization and the Genomics Platform of the National Center of Comprehensive Research Frontiers in Genetics (Medical University Center, Geneva, Switzerland) for use of their Affymetrix workstation. We thank Dr. Robert Weinberg (Massachusetts Institute for Technology, Boston, MA) for providing the full-length human hTERT cDNA.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This study was sponsored and supported by the Swiss National Center of Competence in Research in Molecular Oncology, a research instrument of the Swiss National Science Foundation, a grant from the Arthur Andersen Foundation, and Swiss National Science Foundation Grant 3100A0-105929. Back

2 Address correspondence and reprint requests to Dr. Nathalie Rufer, Swiss Institute for Experimental Cancer Research, 155 Chemin des Boveresses, CH-1066 Epalinges, Switzerland. E-mail address: Nathalie.Rufer{at}isrec.ch Back

3 Abbreviations used in this paper: PD, population doubling; FISH, fluorescence in situ hybridization; HD, healthy donor; hTERT, human telomerase reverse transcriptase; 7-AAD, 7-aminoactinomycin D. Back

Received for publication April 26, 2006. Accepted for publication July 7, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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