Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2008 May 5.
Published in final edited form as: J Immunol. 2008 Mar 15;180(6):3775–3781. doi: 10.4049/jimmunol.180.6.3775

Telomerase Is Involved in IL-7-Mediated Differential Survival of Naive and Memory CD4+ T Cells1

Yinhua Yang 1, Jie An 1, Nan-ping Weng 1,2
PMCID: PMC2367009  NIHMSID: NIHMS44861  PMID: 18322183

Abstract

IL-7 plays an essential role in T cell maintenance and survival. The survival effect of IL-7 is thought to be mediated through regulation of Bcl2 family proteins. After a comparative analysis of IL-7-induced growth and cell death of human naive and memory CD4+ T cells, we observed that more memory CD4+ T cells underwent cell division and proceeded to apoptosis than naive cells in response to IL-7. However, IL-7-induced expressions of Bcl2 family members (Bcl2, Bcl-xL, Bax, and Bad) were similar between naive and memory cells. Instead, we found that IL-7 induced higher levels of telomerase activity in naive cells than in memory cells, and the levels of IL-7-induced telomerase activity had a significant inverse correlation with cell death in CD4+ T cells. Furthermore, we showed that reducing expression of telomerase reverse transcriptase and telomerase activity significantly increased cell death of IL-7-cultured CD4+ T cells. Together, these findings demonstrate that telomerase is involved in IL-7-mediated differential survival of naive and memory CD4+ T cells.

Balancing survival and death of lymphocytes is a vital task of the immune system necessary to maintain its function throughout life (1). IL-7, produced by stromal cells, plays a key role in promoting proliferation and survival of T cells in the periphery (2-4). However, the requirements for proliferation and survival for naive and memory T cells appear different. Naive T cells require IL-7 and a weak interaction between TCR and self-peptide/MHC to mediate survival and proliferation (5, 6) while survival and homeostatic proliferation of memory CD4+ T cells can be supported by IL-7 in the absence of TCR signals (7, 8). Furthermore, memory CD4+ T cells appear to be faster in entering the cell cycle than naive CD4+ T cells in response to IL-7 in vitro (9).

The engagement of IL-7 with its receptors: IL-7Rα and common γ-chain, on T cells leads to activation of the JAK1/JAK3- and STAT5-signaling pathways (10). One of the consequences of STAT5 signaling is activation of S-phase kinase-associated protein 2 and inhibition of protein kinase Cθ, which in turn leads to down-regulation of cyclin-dependent kinase inhibitor p27kip1. The down-regulation of p27kip1 then ensures that T cells enter the cell cycle (11). Another consequence of STAT5 signaling is the up-regulation of antiapoptotic Bcl2 family members (Bcl2 and Bcl-xL) and reduced expression of proapoptotic members (Bax and Bad) (12, 13). Thus, the survival effect of IL-7 is believed to be mainly mediated by Bcl2 family members.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric repeats and prevents excessive loss of telomeres during successive cell divisions (14). Telomerase consists of two core components: a catalytic subunit (telomerase reverse transcriptase (TERT)3) and a telomerase RNA component (TERC). Although the expression of TERC is ubiquitously expressed, the expression of TERT is tightly regulated and is considered as a limiting factor for telomerase function (15). In T cells, little or no telomerase activity is detected at the resting stage but high levels of telomerase activity are induced after mitotic stimulation such as cross-linking TCR and cytokines including IL-7 (16-18). Furthermore, telomerase appears to serve as a key regulator of the replicative lifespan of T cells (19, 20). In addition to its telomere-lengthening function, telomerase has also been reported to protect cells from apoptosis in neuronal cells (21), fibroblasts (22), and tumor cell lines (23). Luiten et al. (24) reported that TERT-transduced human CD4+ T cell clones were more resistant to oxidative stress-induced apoptosis. It is not known how telomerase protects cells against apoptosis and whether telomerase plays a role in IL-7-induced normal T cell survival.

To understand the role of telomerase in IL-7 function in T cells, we analyzed IL-7-induced proliferation, cell death, and telomerase expression in human naive and memory CD4+ T cells. We found that memory cells had faster cell division and more cell death than naive cells in response to IL-7, and that naive cells had higher induced telomerase activity than memory cells. The levels of induced telomerase activity in CD4+ T cells were inversely correlated with cell death. We further demonstrated that reducing TERT expression and telomerase activity increased cell death in IL-7-cultured CD4+ T cells. Thus, telomerase plays a previously unrecognized role in IL-7-mediated survival of CD4+ T cells.

Materials and Methods

Isolation and culture of CD4+ T cell subsets

Peripheral blood was collected from healthy adults of the National Institute on Aging Clinical Research Branch under Intramural Research Program-approved protocols (MRI2003-054). The procedure for isolation of human naive and memory CD4+ T cells by cell sort was previously described (25). In brief, naive and memory CD4+ T cells were enriched by immunomagnetic beads through negative removal of other types of cells in PBMC, and followed by cell sort (Molflow; DakoCytomation). Naive and memory CD4+ T cells were based on their surface expression of CD45RA, and central and effector memory cells were based on CD45RA and CCR7 or CD62L. The purity of naive and memory cells was over 95%. Naive and memory cells were cultured with IL-7 (50 ng/ml; PeproTech) with RPMI 1640 medium, 10% FBS, and penicillin (10 U/ml)/streptomycin (10 μg/ml) (Invitrogen Life Technologies) for specified time before harvest for analysis.

Analysis of IL-7Rα expression, proliferation, and apoptosis

IL-7Rα expression was determined by an anti-IL-7Rα Ab (PE conjugated; Immunotech) and analyzed by FACScan (BD Biosciences). The effect of IL-7 exposure time on IL-7Rα expression was determined by replacing IL-7-containing medium with regular medium without IL-7 at specified time points, and IL-7Rα expression was analyzed at the seventh day of culture. Cell proliferation in response to IL-7 was determined by a CFSE (Invitrogen Life Technologies) tracking method. Proliferation index was analyzed by ModFit LT 3.0 Mac (Verity House Software). Cell apoptosis and death were determined by annexin V (ANXA5) and 7-aminoactinomycin D (7-AAD) staining and analyzed by CellQuest (BD Biosciences). The dead cells were defined by the positive staining of 7-AAD or specified.

Telomerase activity measurement assay

A modified telomerase assay was applied as previously described (18). In brief, 0.5 × 106 freshly isolated or in vitro-treated live cells were lysed. Telomerase activity was normalized to the equivalent number of live cells and was then determined by the ratio of the intensity of amplified telomere repeats and the intensity of internal control.

Quantitative RT-PCR

The procedure of quantitative RT-PCR was previously described (26). Total RNA was isolated from freshly isolated and IL-7-treated naive and memory CD4+ T cells by a RNA STAT-60 kit (Tel-Test) according to the manufacturer’s instructions. A total of 500 ng of total RNA from each sample was used for reverse transcription at 42°C for 1 h followed by inactivation of reverse transcriptase at 70°C for 15 min. RNase H was used to destroy RNA by incubating at 37°C for 30 min. A total of 100 ng of each cDNA sample was used for real-time PCR according to the manufacturer’s instruction (Applied Biosystems). The gene-specific primers were designed by the Primer Express 3.0 software (Applied Biosystems) and synthesized by Integrated DNA Technologies. The primer sequences are listed as follows (forward and reverse): acyl-coenzyme A oxidase 1 (Acox1): 5′-TTAACGAAGGCATTGGTCAAGG-3′ and 5′-ATGCCAGTGTTTGCAGTCCAG-3′; Bad: 5′-GAGTGACGAGTTTGTGGACTCCTT-3′ and 5′-TGT GCCCGCGCTCTTC-3′; Bax: 5′-GAAGCTGAGCGAGTGTCTCAAG-3′ and 5′-GTCCACGGCGGCAATC-3′; Bcl2: 5′-TCATGTGTGTGGAGAGCGTCA-3′ and5′-ACAGTTCCACAAAGGCATCCC-3;Bcl-xL:5′-CGGCGGCTGGGATACTTTTG-3′ and 5′-AGCGGTTGAAGCGTTCCTGG-3′; human TERT (hTERT): 5′-CGTACAGGTTTCACGCATGTG-3′ and 5′-ATGACGCGCAGGAAAAATGT-3′; ribonuclear protein L-32: 5′-AACCCAGAGGCATTGACAACAG-3′ and 5′-TCACTTGTTGCACATCAGCAGC-3′. The relative expression of genes was conducted by normalizing the threshold cycle of each gene to the threshold cycle value of ribonuclear protein L-32 first, and then the ratio to the levels of freshly isolated naive cells or unstimulated cells as specified. The mean ± SEM was used for presentation.

Western blot

Cells were lysed in Kyriakis lysis buffer and 40 μg of proteins from each sample was used for Western blot. The dilutions were as follows: anti-p27kip1, 1/1000; anti-Bcl-2, 1/500; anti-Bcl-xL, 1/1000; anti-Bad, 1/500; and anti-Bax, 1/250 (BD Biosciences); and anti-β-actin, 1/10000 (Sigma-Aldrich). HRP-linked secondary Abs (mouse and rabbit) were 1/5000 (GE Health Sciences). Signals were detected using the ECL detection system (GE Health Sciences). The quantitation was conducted by collecting images (FluorChem; Alpha Innotech), normalized loading based on the intensity of β-actin, and calculated using UN-SCAN-IT software (Silk Scientific).

Inhibition of telomerase in primary CD4+ T cells by small hairpin RNA (shRNA)

Human telomerase shRNA and control plasmids were obtained from the Addgene Plasmid Repository. A total of 10 μg of shRNA plasmid, together with 10 μg of retroviral packaging plasmids, were cotransfected into the BOSC23 packaging cell line by FuGENE6 Transfection Reagent (Roche Applied Science) according to the manufacturer’s instruction. Retroviruses were harvested from culture supernatant after 2-3 days of transfection via centrifugation at 2000 rpm for 10 min and then filtration by Millex-HV filter (Millipore). Primary CD4+ T cells were cultured in RPMI 1640 medium containing 50 ng/ml IL-7, 10% FBS, penicillin (10 U/ml)/streptomycin (10 μg/ml) (Invitrogen Life Technologies) for 12 days before viral infection. The procedure for viral infection of IL-7-cultured CD4+ T cells was as follows: 1) 24-well plates were coated with RetroNectin (TaKaRa Bio) at 4°C overnight, then 300 μl of virus-containing medium was added to the plates followed by centrifugation at 2,000 rpm, 30°C for 3 h. This viral coating procedure was repeated twice to concentrate the virus on the plates. 2) A total of 1 × 106 of IL-7-cultured CD4+ T cells was added to each well after removal of virus-containing medium and incubated at 37°C with 5% CO2 for 2.5 days. Cells were then harvested for staining with ANXA5/7-AAD and analyzed by FACS. Simultaneously, proteins were isolated for telomeric repeat amplification protocol (TRAP) assay and Western blot, and RNAs were isolated for real-time RT-PCR analysis.

Statistical analysis

The differences of biological parameters between naive and memory CD4+ T cells were analyzed by the Student t test. A value of p < 0.05 was considered as significant.

Results

Memory CD4+ T cells exhibit faster proliferative response than do naive cells to IL-7

To investigate the responses of naive and memory T cells to IL-7, we compared cell proliferation between naive and memory CD4+ T cells using a cell division tracking dye, CFSE. A fraction of memory cells underwent one division but naive cells did not divide at day 7, and substantial cell divisions were observed in both naive and memory cells at day 14 of IL-7 culture (Fig. 1A). To quantitate the cell divisions, we compared the proliferation index (PI), a measure for cell division as defined by the total number of cells after culture divided by the computed number of original parent cells, and found that the PI was significantly higher in memory cells than in naive cells at day 7 (p = 0.001) and day 14 (p = 0.006). We further analyzed the subsets of memory cells (central memory and effector memory T cells) and found that the proliferative response to IL-7 was a little slower in effector memory T than in central memory T cells, but the difference was not significant (data not shown). Interestingly, the cell numbers did not increase significantly in memory cells over naive cells after 14 days of culture with IL-7 (Fig. 1B). In contrast, similar proliferation profiles were observed between naive and memory cells after anti-CD3 plus anti-CD28 stimulation (Fig. 1C). It was recently reported that p27Kip1 serves as a key regulator in IL-7-induced proliferation (11). We therefore examined the level of p27Kip1 in naive and memory cells after IL-7 culture and found that the p27Kip1 level was similar in naive and memory cells before IL-7 culture (Fig. 1, D and E). At day 7 of culture, a significant down-regulation of the p27Kip1 level was found in memory cells (25% reduction, p < 0.05) but not in naive cells (Fig. 1, D and E). Only at day 14 of culture was the p27Kip1 level significantly down-regulated in naive cells (35% reduction, p < 0.05) while p27Kip1 was back to the baseline level in memory cells indicating the exit of memory cells from the cell cycle as reported recently (9) (Fig. 1, D and E). The earlier down-regulation of the p27Kip1 level in memory cells than in naive cells agrees with the faster IL-7-induced proliferation of memory cells than naive cells.

FIGURE 1.

FIGURE 1

Open in a new tab

Memory CD4+ T cells divide faster than naive CD4+ T cells in response to IL-7. A, A representative CFSE profile of days 7 and 14 of IL-7-cultured naive and memory CD4+ T cells were presented from 10 independent experiments. PI was calculated based on CFSE profiles using ModFit LT 3.0 software. B, Cell recovery after 14-day IL-7 culture of naive and memory CD4+ T cells. Cells were stained with trypan blue and live cells were counted under the microscope using a hematometer. Mean and SEM were presented (n = 14). C, A representative CFSE profile of day 5 of anti-CD3/CD28-stimulated naive and memory CD4+ T cells. D, A representative Western blot image of p27kip1 and β-actin levels was shown from three independent experiments. E, The mean of relative quantities of p27kip1 protein and SEM were presented (n = 3).

Memory CD4+ T cells express higher levels of IL-7Rα than do naive cells

To understand the mechanism of different proliferative responses between naive and memory CD4+ T cells to IL-7, we measured the level of IL-7Rα expression before and after IL-7 culture. We found that freshly isolated memory cells had significantly higher levels of IL-7Rα expression than did resting naive cells (mean fluorescent intensity (MFI) = 362 and 274, respectively; p < 0.01) (Fig. 2, A and B). After the exposure of IL-7, the levels of IL-7Rα expression were down-regulated in both naive and memory cells. Although removal of IL-7 from culture led to partial restoration of IL-7Rα expression, memory cells had a higher level of restored IL-7Rα expression than naive cells (Fig. 2C). The high level of IL-7Rα expression in memory cells may provide an explanation for the earlier entry into the cell cycle of memory cells than naive cells in response to IL-7.

FIGURE 2.

FIGURE 2

Open in a new tab

IL-7Rα expression is higher on memory than on naive CD4+ T cells. A, A representative histogram of IL-7Rα expression on freshly isolated naive and memory CD4+ T cells from 16 independent experiments was shown. Filled: unstained cells, dark line: naive cells, and gray line: memory cells. B, Statistic analysis of IL-7Rα expression on naive and memory CD4+ T cells (n = 16). C, IL-7Rα expression in naive and memory CD4+ T cells before and after culture with IL-7 at different length of time (1-7 days). IL-7 was presence in the culture for an indicated day(s), then IL-7-containing medium was removed and replaced with fresh medium without IL-7 for the remaining time. Cells were harvested at day 7 for analysis of IL-7Rα expression by FACS.

Naive CD4+ T cells have a better survival rate than memory cells in response to IL-7

To determine whether there is any difference in IL-7-mediated survival between naive and memory CD4+ T cells, we compared cell death and the expression of genes involved in survival (Bcl2 family members). We observed significantly more death in memory cell populations than in naive cells after day 14 of IL-7 culture based on ANXA5 and 7-AAD staining (Fig. 3, A and B). We then analyzed expression of Bcl2 family members by quantitative realtime RT-PCR and by Western blot. We found that freshly isolated naive and memory cells expressed similar levels of Bcl2, Bclxl, Bax, and Bad at the mRNA levels (Fig. 3C) and at the protein levels (Fig. 3, D and E). Although the expression of these Bcl2 family members was changed at various degrees after IL-7 culture, their overall expression levels were similar between naive and memory cells at mRNA (Fig. 3C) and at protein levels (Fig. 3, D and E). Thus, IL-7-induced expression of Bcl2 family members does not appear to be the reason for the differential cell death of naive and memory CD4+ T cells in response to IL-7.

FIGURE 3.

FIGURE 3

Open in a new tab

More cell death is found in memory than in naive CD4+ T cells after IL-7 culture. A, Cell death of IL-7-cultured naive and memory CD4+ T cells. Cell death was analyzed by ANXA5 and 7-AAD staining and a representative dot plot was presented. B, Mean dead cell percentages of IL-7-cultured naive and memory CD4+ T cells at day 14 (n = 13, p < 0.05). C, Gene expression of four Bcl2 family members in naive and memory CD4+ T cells before and after IL-7 culture. ■, Naive cells; □, memory cells. The mean and SEM were shown (n = 6). D, A representative Western blot image of four Bcl2 family members was shown. E, The mean of relative quantities of Bcl2 family proteins and SEM were presented (n = 4).

IL-7 induces higher levels of telomerase activity in naive than in memory CD4+ T cells

The role of telomerase in regulation of cell survival has been reported (21-24) and it was also reported that IL-7 is capable of inducing telomerase activity in human T cells (17, 27). To determine whether IL-7 differentially regulates telomerase in naive and memory CD4+ T cells, we compared expression of telomerase catalytic subunit (TERT) and telomerase activities. IL-7 induced up-regulation of TERT in naive and memory cells but the levels of induced TERT were not significantly higher in naive cells than in memory cells (Fig. 4A). However, IL-7 induced significantly higher telomerase activity in naive cells than in memory cells (Fig. 4, B and C). As telomerase can be activated without increase of TERT expression in T cells (28), this difference suggests that naive cells activate telomerase enzyme more efficiently than do memory cells. Because telomerase activity was measured by live cell equivalents, to rule out the potential detrimental effect of dead cells in the cell lysate, we used sorted live naive and memory cells (14 days of IL-7 culture) for telomerase assay and confirmed that the sorted IL-7-cultured naive cells had significantly higher levels of telomerase activity than did the memory cells (Fig. 4, D and E).

FIGURE 4.

FIGURE 4

Open in a new tab

IL-7 induces higher telomerase activity in naive than in memory CD4+ T cells. A, Relative levels of TERT gene expression were presented as mean and SEM (n = 6). B, IL-7 induced higher levels of telomerase activity in naive than in memory CD4+ T cells. A representative gel image of telomerase assay in naive and memory CD4+ T cells was shown. C, Quantitative analysis of induced telomerase activity in naive and memory CD4+ T cells after IL-7 culture. Mean and SEM of C were presented (n = 13). D, A representative gel image of telomerase assay from sorted live naive and memory CD4+ T cells after 14 days of IL-7 culture was presented. E, Mean and SEM of D were presented (n = 5).

Telomerase is associated with IL-7-induced cell survival

To further determine whether telomerase regulates IL-7-induced survival of naive and memory cells, we compared the levels of telomerase activity and cell death of 22 IL-7-cultured naive and memory CD4+ T cell samples. We found that samples with a high percentage of cell death (mean percentage of cell death = 20%, n = 12) had three times lower levels of telomerase activity than samples with the low percentage of cell death (mean percentage of cell death = 9%, n = 10) (Fig. 5). This suggested that there is a correlation between IL-7-induced levels of telomerase activity and the degree of CD4+ T cell survival. To directly determine the role of telomerase in IL-7-mediated CD4+ T cell survival, we reduced TERT expression via introducing shRNA specific for TERT (shTERT) in IL-7-cultured CD4+ T cells. We found that shTERT significantly and specifically down-regulated the levels of TERT mRNA but not a control gene (ACOX1) (Fig. 6A). We also found a significant reduction of telomerase activity in shTERT-transduced cells as compared with the mock shRNA (shVECT) transduced cells (Fig. 6, B and C). These CD4+ T cells with reduced expression of hTERT and telomerase activity had a reduced cell recovery (Fig. 6D) and significantly higher level of cell death as compared with shVECT-transduced cells (p < 0.05) (Fig. 6E). Thus, it is evident that telomerase (TERT) is required for IL-7-mediated survival of CD4+ T cells.

FIGURE 5.

FIGURE 5

Open in a new tab

Correlation of IL-7-induced telomerase activity and cell death in CD4+ T cells. The high death group had an average of 20% dead cells (n = 12) while the low death group had an average of 9% dead cells (n = 10). Telomerase activity was determined in each samples based on live cell equivalent, and cell death was determined by ANXA5 and 7-AAD staining. The Student t test was used and p < 0.01.

FIGURE 6.

FIGURE 6

Open in a new tab

Levels of IL-7-induced telomerase determine the degree of CD4+ T cell survival. A, Reduction of TERT expression via shTERT transduction. Mean and SEM of TERT expression were presented (n = 6). ACOX1, a housekeeping gene, was used as control for shTERT- and shVECT-transduced cells. B, Reduction of telomerase activity in shTERT-transduced cells as compared with the control. A representative image of telomerase activity of shTERT and control vector-transduced CD4+ T cells. C, Quantitative analysis of reduced telomerase activity in shTERT-transduced cells as compared with the control (n = 6). D, Cell recovery after retroviral transductions (shRNA of TERT and control vector), n = 6. E, Higher percentages of apoptotic and dead cells in shTERT-transduced CD4+ T cells than in shVECT-transduced cells. Mean and SEM were presented (n = 6).

Discussion

The proliferation and survival effects of IL-7 were comparatively analyzed in human naive and memory CD4+ T cells here. Our findings suggest that naive and memory CD4+ T cells have differential responses to IL-7. Memory cells had a faster entry in cell cycle than did naive cells, supported by the higher level of expression of IL-7Rα and faster down-regulation of p27kip1 in memory cells than in naive cells. Intriguingly, memory cells also had greater amounts of cell death than naive cells. Although the survival effect of IL-7 is believed to be mainly through regulation of the members of Bcl2 family in T cells, we showed here that telomerase also plays a previously unrecognized role in regulating IL-7-mediated survival of CD4+ T cells as down-regulation of telomerase results in increased death of IL-7-cultured CD4+ T cells. Together, these findings suggest that telomerase is involved in regulation of IL-7-mediated survival of CD4+ T cells through a distinct pathway from Bcl2 or acting downstream of the Bcl2 pathway.

IL-7 induced consistently lower levels of telomerase activity in memory than in naive CD4+ T cells through the course of a 14-day culture. This difference appears neither at the mRNA levels of hTERT nor due to a general feature of telomerase expression of memory CD4+ T cells as telomerase activity was induced at a comparable level between naive and memory CD4+ T cells after cross-linking TCR and costimulatory receptor (29). Thus, the lower levels of IL-7-induced telomerase activity in memory cells could be due to a wide range of events that affect telomerase activity including posttranscriptional or posttranslational modifications of hTERT. A better understanding of the regulation of telomerase activity in T cells will allow further determination of the difference of IL-7-induced telomerase activity in naive and memory CD4+ T cells.

Engagement of TCR and costimulatory receptors leads to resting naive and memory CD4+ T cells entry and completion of a cell cycle within 24-48 h. In contrast, IL-7-induced proliferation of naive and memory CD4+ T cells takes >7 days. Although the initial signaling after IL-7 and its receptor interaction such as phosphorylation of STAT5 occurs within minutes, the subsequent events that result in proliferation are not fully understood. A continuous exposure to IL-7 appears necessary for proliferation of naive and memory CD4+ T cells (9), suggesting that this length of time may be required for accumulating signal strength to reach a threshold of cell cycle commitment, similar to that observed in TCR-mediated activation (30), and/or for production of other factors to promote cell division. These two possibilities are not mutually exclusive, and future experiments will be needed to test them and to better understand the mechanisms of cytokine-mediated homeostatic proliferation of T cells.

The extra telomere-lengthening function of telomerase or TERT involving antiapoptotic activity has been reported in different types of cells (21-23). In CD4+ T cells, Luiten et al. (24) reported that an ectopic TERT expression does not change the susceptibility to activation-induced cell death but confers better resistance to apoptosis induced by oxidative stress. Despite these findings, the mechanism of a telomerase-mediated survival effect is not completely understood. It has been observed that TERT-transduced T cells contained higher frequencies of Bcl-2-expressing cells and fewer caspase-3-expressing cells as compared with wild-type cells (24). Furthermore, Mandal and Kumar (31) reported that IL-2 treatment induced both Bcl-2 expression and telomerase activity in an IL-2-dependent cytotoxic T cell line, CTLL-2. These findings suggest a correlation of Bcl2 expression and telomerase activity. In addition to telomerase activity, TERT expression has also been linked to antiapoptotic activity in cancer cells, involving inhibition of p53 (23, 32). Here, we found that IL-7-induced telomerase activity accounts for the differential survival of IL-7-cultured naive and memory CD4+ T cells. Although there is increased expression of Bcl2 and Bcl-xL in IL-7-cultured naive and memory CD4+ T cells, increased cell death in memory cells does not correlate with the levels of induced Bcl2 family proteins but rather with the lower levels of telomerase activity. Finally, down-regulation of telomerase in CD4+ T cells induced more cell death. However, it remains to be determined whether telomerase acts through a distinct pathway from Bcl2 or acts downstream of the Bcl2 pathway in its antiapoptotic function.

Significantly shortened telomeres can also trigger apoptosis (33). As telomere length differs among chromosomal ends (34) and a shortened telomere on a single chromosome can cause apoptosis (35), the central question that remains to be addressed is whether the telomerase-mediated antiapoptosis effect involves telomeres. In addition, it is also not clear whether TERT alone or the telomerase complex is required for the antiapoptosis activity. Evidence that TERT alone could promote cell growth and antiapoptosis argues for a telomere-lengthening independent mechanism (23, 36). Elucidating the mechanisms of telomerase in regulation of cell survival will offer the potential for clinical intervention such as vaccination.

Acknowledgments

We thank Drs. Richard Hodes and Mark Mattson for critical reading of the manuscript. We thank Francis J. Chrest, Cuong Nguyen, Christa Morris, Bob Wersto of National Institute on Aging (NIA) Flow Cytometry Unit for sorting cells, and Karen Madara of NIA Apheresis Unit for collecting bloods.

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.

Footnotes

1

Y.Y., J.A., and N.-p.W. were supported by the Intramural Research Program of the National Institute on Aging, National Institutes of Health.

3
Abbreviations used in this paper:
TERT
telomerase reverse transcriptase
ANXA5
annexin V
7-AAD
7-aminoactinomycin D
hTERT
human TERT
TRAP
telomeric repeat amplification protocol
PI
proliferation index
MFI
mean fluorescent intensity
Acox1
acyl-coenzyme A oxidase 1
sh
small hairpin

Disclosures

The authors have no financial conflict of interest.

References

  • 1.Marrack P, Kappler J. Control of T cell viability. Annu. Rev. Immunol. 2004;22:765–787. doi: 10.1146/annurev.immunol.22.012703.104554. [DOI] [PubMed] [Google Scholar]
  • 2.Bradley LM, Haynes L, Swain SL. IL-7: maintaining T-cell memory and achieving homeostasis. Trends Immunol. 2005;26:172–176. doi: 10.1016/j.it.2005.01.004. [DOI] [PubMed] [Google Scholar]
  • 3.Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance. J. Immunol. 2005;174:6571–6576. doi: 10.4049/jimmunol.174.11.6571. [DOI] [PubMed] [Google Scholar]
  • 4.Jiang Q, Li WQ, Aiello FB, Mazzucchelli R, Asefa B, Khaled AR, Durum SK. Cell biology of IL-7, a key lymphotrophin. Cytokine Growth Factor Rev. 2005;16:513–533. doi: 10.1016/j.cytogfr.2005.05.004. [DOI] [PubMed] [Google Scholar]
  • 5.Tan JT, Dudl E, LeRoy E, Murray R, Sprent J, Weinberg KI, Surh CD. IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc. Natl. Acad. Sci. USA. 2001;98:8732–8737. doi: 10.1073/pnas.161126098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kieper WC, Jameson SC. Homeostatic expansion and phenotypic conversion of naive T cells in response to self peptide/MHC ligands. Proc. Natl. Acad. Sci. USA. 1999;96:13306–13311. doi: 10.1073/pnas.96.23.13306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Seddon B, Tomlinson P, Zamoyska R. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells. Nat. Immunol. 2003;4:680–686. doi: 10.1038/ni946. [DOI] [PubMed] [Google Scholar]
  • 8.Li J, Huston G, Swain SL. IL-7 promotes the transition of CD4 effectors to persistent memory cells. J. Exp. Med. 2003;198:1807–1815. doi: 10.1084/jem.20030725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Swainson L, Verhoeyen E, Cosset FL, Taylor N. IL-7R α gene expression is inversely correlated with cell cycle progression in IL-7-stimulated T lymphocytes. J. Immunol. 2006;176:6702–6708. doi: 10.4049/jimmunol.176.11.6702. [DOI] [PubMed] [Google Scholar]
  • 10.Lin JX, Migone TS, Tsang M, Friedmann M, Weatherbee JA, Zhou L, Yamauchi A, Bloom ET, Mietz J, John S, et al. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity. 1995;2:331–339. doi: 10.1016/1074-7613(95)90141-8. [DOI] [PubMed] [Google Scholar]
  • 11.Li WQ, Jiang Q, Aleem E, Kaldis P, Khaled AR, Durum SK. IL-7 promotes T cell proliferation through destabilization of p27Kip1. J. Exp. Med. 2006;203:573–582. doi: 10.1084/jem.20051520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kim K, Lee CK, Sayers TJ, Muegge K, Durum SK. The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways. J. Immunol. 1998;160:5735–5741. [PubMed] [Google Scholar]
  • 13.Khaled AR, Li WQ, Huang J, Fry TJ, Khaled AS, Mackall CL, Muegge K, Young HA, Durum SK. Bax deficiency partially corrects interleukin-7 receptor α deficiency. Immunity. 2002;17:561–573. doi: 10.1016/s1074-7613(02)00450-8. [DOI] [PubMed] [Google Scholar]
  • 14.Blackburn EH. Switching and signaling at the telomere. Cell. 2001;106:661–673. doi: 10.1016/s0092-8674(01)00492-5. [DOI] [PubMed] [Google Scholar]
  • 15.Cong YS, Wright WE, Shay JW. Human telomerase and its regulation. Microbiol. Mol. Biol. Rev. 2002;66:407–425. doi: 10.1128/MMBR.66.3.407-425.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Weng N, Levine BL, June CH, Hodes RJ. Regulated expression of telomerase activity in human T lymphocyte development and activation. J. Exp. Med. 1996;183:2471–2479. doi: 10.1084/jem.183.6.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Soares MV, Borthwick NJ, Maini MK, Janossy G, Salmon M, Akbar AN. IL-7-dependent extrathymic expansion of CD45RA + T cells enables preservation of a naive repertoire. J. Immunol. 1998;161:5909–5917. [PubMed] [Google Scholar]
  • 18.Li Y, Zhi W, Wareski P, Weng NP. IL-15 activates telomerase and minimizes telomere loss and may preserve the replicative life span of memory CD8+ T cells in vitro. J. Immunol. 2005;174:4019–4024. doi: 10.4049/jimmunol.174.7.4019. [DOI] [PubMed] [Google Scholar]
  • 19.Rufer N, Migliaccio M, Antonchuk J, Humphries RK, Roosnek E, Lansdorp PM. Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential. Blood. 2001;98:597–603. doi: 10.1182/blood.v98.3.597. [DOI] [PubMed] [Google Scholar]
  • 20.Roth A, Yssel H, Pene J, Chavez EA, Schertzer M, Lansdorp PM, Spits H, Luiten RM. Telomerase levels control the lifespan of human T lymphocytes. Blood. 2003;102:849–857. doi: 10.1182/blood-2002-07-2015. [DOI] [PubMed] [Google Scholar]
  • 21.Fu W, Begley JG, Killen MW, Mattson MP. Anti-apoptotic role of telomerase in pheochromocytoma cells. J. Biol. Chem. 1999;274:7264–7271. doi: 10.1074/jbc.274.11.7264. [DOI] [PubMed] [Google Scholar]
  • 22.Gorbunova V, Seluanov A, Pereira-Smith OM. Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis. J. Biol. Chem. 2002;277:38540–38549. doi: 10.1074/jbc.M202671200. [DOI] [PubMed] [Google Scholar]
  • 23.Cao Y, Li H, Deb S, Liu JP. TERT regulates cell survival independent of telomerase enzymatic activity. Oncogene. 2002;21:3130–3138. doi: 10.1038/sj.onc.1205419. [DOI] [PubMed] [Google Scholar]
  • 24.Luiten RM, Pene J, Yssel H, Spits H. Ectopic hTERT expression extends the life span of human CD4+ helper and regulatory T-cell clones and confers resistance to oxidative stress-induced apoptosis. Blood. 2003;101:4512–4519. doi: 10.1182/blood-2002-07-2018. [DOI] [PubMed] [Google Scholar]
  • 25.Liu K, Li Y, Prabhu V, Young L, Becker KG, Munson PJ, Weng N. Augmentation in expression of activation-induced genes differentiates memory from naive CD4+ T cells and is a molecular mechanism for enhanced cellular response of memory CD4+ T cells. J. Immunol. 2001;166:7335–7344. doi: 10.4049/jimmunol.166.12.7335. [DOI] [PubMed] [Google Scholar]
  • 26.Fann M, Godlove JM, Catalfamo M, Wood Iii WH, Chrest FJ, Chun N, Granger L, Wersto R, Madara K, Becker K, et al. Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8+ T cell response. Blood. 2006;108:3363–3370. doi: 10.1182/blood-2006-02-005520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Wallace DL, Berard M, Soares MV, Oldham J, Cook JE, Akbar AN, Tough DF, Beverley PC. Prolonged exposure of naive CD8+ T cells to interleukin-7 or interleukin-15 stimulates proliferation without differentiation or loss of telomere length. Immunology. 2006;119:243–253. doi: 10.1111/j.1365-2567.2006.02429.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Liu K, Hodes RJ, Weng N. Telomerase activation in human T lymphocytes does not require increase in telomerase reverse transcriptase (hTERT) protein but Is associated with hTERT phosphorylation and nuclear translocation. J. Immunol. 2001;166:4826–4830. doi: 10.4049/jimmunol.166.8.4826. [DOI] [PubMed] [Google Scholar]
  • 29.Weng N, Levine BL, June CH, Hodes RJ. Regulation of telomerase RNA template expression in human T lymphocyte development and activation. J. Immunol. 1997;158:3215–3220. [PubMed] [Google Scholar]
  • 30.Viola A, Lanzavecchia A. T cell activation determined by T cell receptor number and tunable thresholds. Science. 1996;273:104–106. doi: 10.1126/science.273.5271.104. [DOI] [PubMed] [Google Scholar]
  • 31.Mandal M, Kumar R. Bcl-2 modulates telomerase activity. J. Biol. Chem. 1997;272:14183–14187. doi: 10.1074/jbc.272.22.14183. [DOI] [PubMed] [Google Scholar]
  • 32.Rahman R, Latonen L, Wiman KG. hTERT antagonizes p53-induced apoptosis independently of telomerase activity. Oncogene. 2005;24:1320–1327. doi: 10.1038/sj.onc.1208232. [DOI] [PubMed] [Google Scholar]
  • 33.Karlseder J, Broccoli D, Dai Y, Hardy S, de Lange T. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science. 1999;283:1321–1325. doi: 10.1126/science.283.5406.1321. [DOI] [PubMed] [Google Scholar]
  • 34.Lansdorp PM, Verwoerd NP, Van de Rijke FM, Dragowska V, Little MT, Dirks RW, Raap AL, Tanke HJ. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 1996;5:685–691. doi: 10.1093/hmg/5.5.685. [DOI] [PubMed] [Google Scholar]
  • 35.Hemann MT, Strong MA, Hao LY, Greider CW. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell. 2001;107:67–77. doi: 10.1016/s0092-8674(01)00504-9. [DOI] [PubMed] [Google Scholar]
  • 36.Sarin KY, Cheung P, Gilison D, Lee E, Tennen RI, Wang E, Artandi MK, Oro AE, Artandi SE. Conditional telomerase induction causes proliferation of hair follicle stem cells. Nature. 2005;436:1048–1052. doi: 10.1038/nature03836. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES