IL-15 Activates Telomerase and Minimizes Telomere Loss and May Preserve the Replicative Life Span of Memory CD8+ T Cells In Vitro
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免疫学杂志 2005年第7期
Abstract
The preservation of the replicative life span of memory CD8+ T cells is vital for long-term immune protection. Although IL-15 plays a key role in the homeostasis of memory CD8+ T cells, it is unknown whether IL-15 regulates the replicative life span of memory CD8+ T cells. In this study, we report an analysis of telomerase expression and telomere length in human memory phenotype CD8+ T cells maintained by IL-15 in vitro. We demonstrate that IL-15 is capable of activating telomerase in memory CD8+ T cells via Jak3 and PI3K signaling pathways. Furthermore, IL-15 induces a sustained level of telomerase activity over long periods of time, and in turn minimizes telomere loss in memory CD8+ T cells after substantial cell divisions. These findings suggest that IL-15 activates stable telomerase expression and compensates telomere loss in memory phenotype CD8+ T cells, and that telomerase may play an important role in memory CD8+ T cell homeostasis.
Introduction
Memory lymphocytes are generated from naive lymphocytes after the interaction with Ag. The long-lived memory lymphocytes appear to be dependent on cytokines such as IL-2, IL-4, IL-7, and IL-15 (1, 2, 3, 4). IL-15 is capable of promoting proliferation and long-term survival of memory CD8+ T cells in an Ag-independent fashion (1, 3, 5). The interaction of IL-15 with its receptor complex leads to activation of Jaks and subsequently Stats (6) as well as activation of PI3K (7) and other signaling pathways (8). Strikingly, despite apparent differences in the initial ligand/receptor interaction, both IL-15 and TCR engagement are capable of inducing similar synthesis of effector molecules and induction of cytotoxicity in memory phenotype CD8+ T cells (9). These findings indicate that the function of IL-15 in CD8+ memory T cells is not only a growth factor but also an Ag-independent activator. However, the long-term impact of IL-15 on the replicative life span of memory CD8+ T cells is unclear.
Telomeres are the end structure of eukaryotic chromosomes that consist of a tandem hexanucleotide repeats (TTAGGG)n and a number of associated proteins (10). During cell division, incomplete replication of the terminal DNA results in a loss of 50–200 bases of telomere repeats with each division (11, 12). The progressive shortening of telomeres occurring in cells undergoes substantial cell division without proper compensation. When telomere length in a cell is critically shortened, chromosomal instability leads to cell cycle arrest or senescence and/or apoptosis (13). Telomerase is a unique RNA-dependent DNA polymerase, which is capable of synthesizing terminal telomeric repeats and thus compensating the loss of telomeres due to cell divisions (14). Initially, studies suggested that telomerase is expressed in germline cells, where telomere maintenance is critical to fertility and survival of species, and in transformed cells with unlimited growth capacity, but not in normal somatic cells (15). However, subsequent studies indicate that telomerase is expressed in several types of normal somatic cells, such as T lymphocytes (16, 17, 18), although the levels and regulation of telomerase activity differ among these cells.
The function of telomerase in regulation of telomere length and replicative life span of lymphocytes has been examined recently (19). In long-term cultured T cells, telomerase activity is associated with stabilizing telomere length (20, 21, 22). Telomere length was stable while telomerase activity was high; telomere loss became apparent while telomerase activity was diminished at later time points of the culture and T cells became senescent or underwent apoptosis. However, exogenous introduction of telomerase catalytic subunit (hTERT) into T cells can stabilize telomere length and preserve proliferative capability (23, 24, 25). Furthermore, mutation of the telomerase RNA template gene results in an inherited disorder, dyskeratosis congenita, with manifestations that include immune defects, bone marrow failure, and premature graying (26). Together, these findings indicate the importance of telomere structure in cellular replicative life span, and telomerase in preserving telomere length and thus prolonging the cellular life span.
One of the central issues of immunological memory is how memory cells are maintained over long periods of time in vivo without loss of their replicative capacity. Because IL-15 plays key roles in the homeostasis of memory CD8+ T cells, we examined the role of IL-15 in regulation of the replicative capacity of memory CD8+ T cells. In this study, we report an analysis of growth, telomerase expression, and telomere length in long-term cultured memory phenotype CD8+ T cells. Our findings suggest that IL-15 is capable of inducing telomerase activity that sustains over a longer period of time than that induced by TCR cross-linking. The induction of telomerase results in minimizing the loss of telomere length of memory CD8+ T cells. Thus, IL-15 may preserve the replicative life span of proliferating memory CD8+ T cells.
Materials and Methods
Isolation of memory phenotype CD8+ T cells
Blood was collected from normal donors at the apheresis unit of the National Institute on Aging Clinical Core Facility under an approved protocol (MRI2003-054; Cytophoresis of Volunteer Donors). Isolation of memory phenotype CD8+ T cells from blood was previously described (9). In brief, PBMC were isolated through centrifugation under a Lympho-Ficoll gradient, and memory phenotype CD8+ T cells were isolated from PBMCs via an immunomagnetic separation. The purity of isolated memory phenotype CD8+ T cells (CD8+CD27+CD45RA–) was >90% based on FACS analysis and 5% of the residual cells were CD8+CD27+CD45RAlow cells. In some cases, memory phenotype CD8+ T cells were isolated by a cell sort with the purity >99%.
Culture of naive and memory phenotype CD8+ T cells
Freshly isolated memory phenotype CD8+ T cells were cultured with either anti-CD3 plus anti-CD28 beads (anti-CD3/CD28) (gift from Dr. C. June, University of Pennsylvania, Philadelphia, PA) or IL-15 at 50 ng/ml (PeproTech) in vitro in RPMI 1640 with 10% FBS and penicillin (10 U/ml)/streptomycin (10 μg/ml). Anti-CD3/CD28 is applied once at the beginning of the culture in most cases; multiple anti-CD3/CD28 stimulation prolonged culture time by 7–10 days. IL-15 was added at the beginning of culture. The concentration of cells in the culture was checked every 5 days and maintained at 1–3 million/ml. At approximately every 10 days, half of the medium was removed and replenished with fresh medium containing IL-15 (50 ng/ml). Cell growth was measured in two ways. First, cells were enumerated using a hemocytometer to assess cell numbers under each of the stimulation conditions. The second method used a dye, CFSE (Molecular Probes), to track the number of cell divisions. For analyzing the specificity of IL-15-induced telomerase, naive and memory phenotype CD8+ T cells were isolated and cultured in the presence of 50 ng/ml IL-15 or IL-7 (PeproTech) for 5 days, and cells were harvested for determining telomerase activity.
Tracking the cell cycle number via CFSE
The procedures of CFSE labeling and analysis were previously described (27). In brief, freshly isolated memory phenotype CD8+ T cells at 2 x 106 cells/ml were incubated with 10 μM CFSE at 37°C for 10 min, washed twice with 10 ml of RPMI 1640 medium, and stimulated under the same conditions described above for unlabeled memory phenotype CD8+ T cells. The CFSE profiles were analyzed using flow cytometry at days 5, 15, and beyond. Cells that had undergone three, six, and eight divisions were isolated by cell sorting for assessment of telomere length and telomerase activity.
Inhibition of telomerase activation
Inhibitors for Jak3 (Jak3 inhibitor I, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline), PI3K (LY294002), AKT (SH-6), and JNK (SB600125) were purchased from EMD. The concentrations of each inhibitor used in this study were based on the previous report (28) as well as our own experiment. Memory phenotype CD8+ T cells were cultured in the presence of IL-15 (50 ng/ml) for 5–7 days. Then, inhibitors were added (1 μg/ml Jak3 inhibitor I, 5 μM LY294002, 5 μM SH-6, and 1 μM SB600125) and incubated for an additional 24 h before harvest. The viability of treated cells was analyzed by trypan blue exclusion assay (Invitrogen Life Technologies), and telomerase activity of these cells was determined by telomeric repeat amplification protocol (TRAP)3 assay.
Telomerase activity
Telomerase activity is measured using TRAP as previously described with some modification (15, 29). We have conjugated the Ts primer with a fluorescent dye, TAMRA. The amplified telomere DNA products were visualized, and the fluorescent intensities were captured using a fluorescent imaging system (Typhoon 9410; Amersham Biosciences). Quantification of the image files to calculate telomerase activity was done using ImageQuant software.
Telomere length
Telomere lengths were measured by two different methods. We used the Flow-fluorescent in situ hybridization (FISH) (30, 31) for analyzing long-term cultured cells and the Southern blot technique (32) for sorted cells. In brief, freshly harvested cells were permeabilized and hybridized with FITC-PNA probe (PNA-(C3TA2)3 probe; PerkinElmer Biosystems) at 83°C for 10 min. Cells were then allowed to hybridize in the dark at room temperature for 2 h. After two washes, the cells were stained with propidium iodide (Sigma-Aldrich) to check for positive nuclear staining. The statistical mean was measured for probed and unprobed measurements, and the difference was taken to be the intensity measurement of telomere staining by the FITC probe. PBMCs from a normal donor were used for normalization of analysis done at different times.
Southern blot analysis of mean telomeric restriction fragment (TRF) length was done with sorted cells after a certain number of cell divisions as previously described (32). Briefly, genomic DNA was isolated from sorted CD8+ memory T cells by using a DNA isolation kit (Gentra Systems), and digested with HinfI and RsaI (Roche Molecular Biochemicals). One microgram of digested DNA was loaded per well of a 0.5% agarose gel and separated by electrophoresis. The gel was dried at 65°C for 1.5 h, denatured, and neutralized. The hybridization was conducted using a 32P-end-labeled oligonucleotide (CCCTAA)4 probe, at 46°C overnight. After three rounds of washing (5x SSC/0.1% SDS, 2x SSC/0.1% SDS, and 3.2 M tetramethylammonium chloride/0.1% SDS) at 45°C, the gel was analyzed by phosphor imager (Typhoon 9410; Amersham Biosciences). Mean TRF length was calculated as described previously (11).
Results
IL-15 induces long-term growth of memory phenotype CD8+ T cells in vitro
Both anti-CD3/CD28 Abs and IL-15 are capable of inducing cell divisions of memory phenotype CD8+ T cells. To compare the growth kinetics of memory phenotype CD8+ T cells in response to anti-CD3/CD28 Abs and IL-15 stimulation, we analyzed memory phenotype CD8+ T cells of a half-dozen of normal donors, and found that anti-CD3/CD28-stimulated cells underwent cell division and expansion faster than did IL-15-treated memory phenotype CD8+ T cells in the first 5 days (Fig. 1A). In general, >90% of the cells have divided at least once after 5-day culture of anti-CD3/CD28 stimulation, whereas 10% of cells divided in the same period of time under IL-15 (Fig. 1A). This difference was mostly diminished after 15 days of culture where 90% of cells underwent at least 1 division under IL-15 and few cells have not divided at least once under anti-CD3/CD28 stimulation (Fig. 1A). Interestingly, whereas memory phenotype CD8+ T cells grew 25–40 days in culture after repeated anti-CD3/CD28 stimulation (a single stimulation lasted 25–35 days, and subsequent anti-CD3/CD28 stimulation led to another 5–7 days of growth), memory phenotype CD8+ T cells grew 45–80 days in culture under IL-15 even though the cell division rate decreased at the late time points. Although the expansion of memory phenotype CD8+ T cells estimated by a total population change under anti-CD3/CD28 or IL-15 is moderate (three to five mean population doublings) (Fig. 1B), the majority of cells underwent 6–8 divisions over 15–25 days of culture based on CFSE analysis (Fig. 1A). When cells underwent 6 divisions, they were isolated and relabeled with CFSE and cultured in the presence of IL-15; we found that these cells underwent an additional 4–6 divisions in 10–15 days (data not shown). Thus, the number of cell divisions estimated by mean population doublings is likely underestimated, and the actual number of cell divisions of those survival memory phenotype CD8+ T cells in long-term culture under IL-15 is probably 15–20 cell divisions.
FIGURE 1. IL-15 induced long-term growth of memory CD8+ T cells. A, Comparison of proliferation of memory phenotype CD8+ T cells in response to anti-CD3/CD28 and IL-15 (50 ng/ml) using CFSE. One representative result of five independent experiments is shown at days 5 and 15 after stimulation. Cell division numbers are indicated in the bottom of the figure. B, Long-term culture of memory phenotype CD8+ T cells in the presence of anti-CD3/CD28 or IL-15 (50 ng/ml). One representative growth curve of seven independent donors is shown.
IL-15 induces telomerase activity in memory phenotype CD8+ T cells
To investigate whether IL-15 is capable of inducing telomerase, we isolated memory phenotype CD8+ T cells from normal donors by cell sort and cultured these sorted cells with different concentrations of IL-15 (from 100 to 1.5 ng/ml) for 5 days. We found that IL-15 is capable of inducing telomerase activity in memory phenotype CD8+ T cells, and that the levels of induced telomerase activity are dependent on the concentration of IL-15 (Fig. 2A). We then examined the kinetic changes of telomerase activity in a time course, and found that telomerase activity started to appear after 48 h of IL-15 culture (50 ng/ml) (Fig. 2B). To analyze the specificity of IL-15-induced telomerase in memory phenotype CD8+ T cells and compared with another cytokine IL-7, we found that IL-15 induced significantly higher levels of telomerase activity in memory than in naive phenotype CD8+ T cells (Fig. 2C). In contrast, IL-7 induced limited levels of telomerase activity in naive and memory phenotype CD8+ T cells (Fig. 2C).
FIGURE 2. Activation of telomerase in memory phenotype CD8+ T cells by IL-15. A, IL-15 dose-dependent activation of telomerase in memory phenotype CD8+ T cells. A series of 2-fold dilutions of IL-15 (from 100 to 3.1 ng/ml) was used in culture of memory phenotype CD8+ T cells. After 5-day culture, cells were lysed and assayed for telomerase activity by TRAP assay, with each reaction using cell lysate of 5000 cell equivalents. One representative result from three different individuals is shown. IC indicates the internal control of the assay. B, Time course of telomerase activation in memory phenotype CD8+ T cells under IL-15. Freshly isolated memory phenotype CD8+ T cells were cultured in the presence of 50 ng/ml IL-15 and harvested at indicated time and measured for telomerase activity. One representative result from three different individuals is shown. C, Activation of telomerase in naive and memory phenotype CD8+ T cells by IL-15 and IL-7. One representative result from three different individuals is shown.
Next, we compared telomerase activity of memory phenotype CD8+ T cells that were stimulated with anti-CD3/CD28 Abs or IL-15 in long-term culture. Both stimulation conditions induced telomerase in memory phenotype CD8+ T cells, but they induced different patterns of telomerase expression (Fig. 3A). As a whole population, the levels of telomerase activity were generally lower in IL-15-cultured cells than in anti-CD3/CD28-stimulated cells. Anti-CD3/CD28 induced a rapid activation of telomerase, which peaked around 3 days, followed by a short period of stable high level activity (5–10 days), and a gradual loss of the activity. In contrast, IL-15-induced telomerase activity features a modest initial induction (5–10 days), followed by a sustained moderate activity over a longer period of time (30–40 days), and an eventual decrease of the activity at the end of the long-term culture. Interestingly, cells from some donors even showed higher levels of telomerase activity in the late time of culture (Fig. 3A). The physiological significance of this is not clear.
FIGURE 3. Telomerase expression pattern in long-term cultured memory phenotype CD8+ T cells. A, Comparison of telomerase activation in divided memory phenotype CD8+ T cells stimulated between anti-CD3/CD28 and IL-15. Freshly isolated and stimulated memory CD8+ T cells were labeled with CFSE and cells that underwent three, six, and eight divisions were isolated by cell sort. Cells were lysed and assayed for telomerase activity. The TRAP assay was conducted using cell lysates at 5000 and 2500 cell equivalents. IC indicates the internal control of the assay. B, Quantitation of telomerase activity in stimulated memory cells relative to unstimulated memory cells in A. C, Comparison of telomerase activity in long-term cultured memory CD8+ T cells stimulated with ant-CD3/CD28 or IL-15. One representative result of three independent donors is shown.
It has been reported that memory CD8+ T cells expressing IL-2R respond to IL-15 more efficiently than those memory phenotype CD8+ T cells that do not express IL-2R (33). Because the number of IL-2R-expressing cells are varied among different donors (data not shown), the proliferative response of memory phenotype CD8+ T cells cultured under IL-15 reflect only those IL-2R-expressing cells. Thus, the lower levels of telomerase activity observed in IL-15-stimulated memory CD8+ T cells may also reflect the mixed responding populations. To further investigate the difference of induced levels of telomerase activity by anti-CD3/CD28 Abs and IL-15, we isolated responding cells that underwent three, six, and eight cell divisions using CFSE labeling and cell sort, and measured telomerase activity in these sorted cells. Interestingly, the levels of telomerase activity are substantially higher in actively dividing cells than in a whole population under IL-15 stimulation, and are similar between IL-15 and anti-CD3/CD28 Abs (Fig. 3, B and C). Furthermore, although different levels of telomerase activity were found between the sorted actively dividing cells and a whole population, there was no significant difference in telomerase activity among actively dividing memory phenotype CD8+ T cells that had undergone different numbers of cell divisions (Fig. 3, B and C).
Signaling requirements of IL-15-induced telomerase activation in memory phenotype CD8+ T cells
The action of IL-15 is dependent on the signaling pathways through its receptors. The Jaks/Stat5 and PI3K/AKT are known signaling pathways involved in IL-15 action (6, 7). To determine whether these signaling pathways are required for IL-15-induced telomerase activation in memory phenotype CD8+ T cells, we used specific inhibitors for Jak3 and PI3K. We found that treatment of inhibitors to Jak3 (Jak3 inhibitor I) or PI3K (LY294002) or AKT (SH-6) for 1 day after 5-day pretreatment of IL-15 significantly reduced telomerase activity in memory CD8+ T cells. In general, 30% of telomerase activity remained after treatment with each inhibitor (Fig. 4, A and B), whereas there was no obvious toxicity to treated cells (C). In contrast, treatment with inhibitor to JNK (SB600125) did not show any effects in telomerase inhibition (Fig. 4, A and B). Combination of inhibitors to PI3K and Jak3 resulted in increased inhibition (data not shown), suggesting that Jak3 and PI3K/AKT pathways play a major role in IL-15-induced activation of telomerase, and other pathways may contribute to the minor role in telomerase activation in memory CD8+ T cells.
FIGURE 4. Signaling requirement of IL-15-induced telomerase activation in memory phenotype CD8+ T cells. A, Repression of telomerase activity after treatment of inhibitors to PI3K, AKT, and Jak3. One representative from three independent experiments is shown. B, Quantitation of relative inhibition of telomerase activity in memory phenotype CD8+ T cells after 1-day treatment of inhibitors to PI3K, AKT, and Jak3. Mean of three donors and SEM are shown. C, Viability of inhibitor-treated memory phenotype CD8+ T cells. Mean of three donors and SEM are shown.
IL-15 maintains telomere length of long-term cultured memory phenotype CD8+ T cells
To determine whether telomerase activity induced by IL-15 is capable of compensating the loss of telomere length, we analyzed telomere length of memory phenotype CD8+ T cells after a certain number of cell divisions and at multiple time points in long-term culture. We used CFSE for isolation of memory phenotype CD8+ T cells that underwent six and eight cell divisions after IL-15 or anti-CD3/CD28 stimulation via cell sort, and measured the telomere length (terminal telomere restriction fragments) by Southern analysis. If there is a loss of 50–200 bp per cell division, one would expect a loss of 400-1600 bp of telomeres after eight cell divisions. However, the average loss of telomeres of five donors is 110 bp (SE is 170 bp) in IL-15-cultured memory phenotype CD8+ T cells (Fig. 5A). Thus, telomere lengths are relatively stable in long-term cultured memory phenotype CD8+ T cells.
FIGURE 5. Telomere length dynamics in long-term cultured memory phenotype CD8+ T cells. A, Telomere lengths were analyzed in memory phenotype CD8+ T cells before and after six and eight cell divisions with stimulation of anti-CD3/CD28 or IL-15. Freshly isolated memory phenotype CD8+ T cells were collected along stimulated memory phenotype CD8+ T cells that underwent six and eight divisions isolated by cell sort. Telomere length was measured as mean TRF by Southern blot analysis. D1–D5 indicates five different donors. B, Telomere length changes in long-term cultured memory phenotype CD8+ T cells. Flow-FISH was used for telomere length measurement. One representative result of four independent donors is shown.
We also examined telomere length in long-term cultured memory phenotype CD8+ T cells at multiple time points by Flow-FISH. Again, we found telomere lengths were relatively stable after some initial loss over long-term culture (Fig. 5B). In contrast, telomere length was significantly decreased in memory phenotype CD8+ T cells at late time points of culture despite initial gain of telomeres under anti-CD3/CD28 stimulation (Fig. 5B). This demonstrates that the difference in regulation of telomerase activity is associated with the difference in telomere length regulation in long-term cultured memory phenotype CD8+ T cells under IL-15 or TCR cross-linking.
Discussion
We have demonstrated in this study that IL-15 is capable of inducing telomerase activity through activation of Jak3 and PI3K/AKT signaling pathways in memory phenotype CD8+ T cells, and that IL-15-induced telomerase activity is associated with the relative stable telomere length in long-term cultured memory phenotype CD8+ T cells. Previous reports showed that exogenous expression of telomerase catalytic unit (hTERT) prevents telomere loss and extends the replicative life span of CD8+ and CD4+ T cells (23, 24, 25), suggesting that preservation of telomere structure can extend cellular replicative life span of cells. By activating and maintaining telomerase activity in memory CD8+ T cells, IL-15 may preserve the replicative life span of dividing memory CD8+ T cells.
Although both cross-linking of TCR/costimulatory receptors and IL-15 can also activate telomerase in memory CD8+ T cells, the difference in telomerase expression kinetics between these two stimulation conditions reflects their different and overlapping signaling pathways as well as physiological roles in memory CD8+ T cells. TCR and Ag interaction leads to activation of Syk (e.g., ZAP70), Src (e.g., Lck) protein tyrosine kinase families, increase of cytoplasmic free calcium [Ca2+]i, and PI3K/AKT pathways, whereas IL-15 induces Jak/Stats pathway and PI3K/AKT pathways. Physiologically, activation of T cells via TCR engagement leads to substantial expansion of effector cells, and the majority of these effector cells undergo apoptosis after the clearance of Ag. The role of telomerase in TCR-mediated activation is to preserve the telomere length of rapidly dividing effector cells at the phase of clonal expansion. In contrast, the role of IL-15 in memory CD8+ T cells is promoting survival and slow proliferation over the long term. The sustained levels of telomerase activity induced by IL-15 is designed to protect the loss of telomere repeats of continued slow proliferation of memory CD8+ T cells over life time. Thus, IL-15 not only promotes survival from death but also preserves telomere length and the capacity for replication of memory CD8+ T cells for future robust responses upon exposure to Ag.
Considering the importance of IL-15 in memory CD8+ T cell homeostasis (34, 35), it is not a surprise that IL-15 is capable of activating telomerase and compensating the loss of telomeres in actively dividing memory CD8+ T cells. Interestingly, IL-7, a key cytokine in T cell development and homeostasis, is also capable of inducing telomerase activity in T cells (36). Similarly to IL-15, IL-7 did not compensate completely the loss of telomere length in cultured naive CD4+ T cells, because some loss of telomere repeats was observed in 12-day cultures of CD4+ T cells maintained by IL-7 (36). We have analyzed whether IL-7 induces telomerase activity in CD8+ T cells in this study. Compared with IL-15 induced telomerase in memory phenotype CD8+ T cells, IL-7 only induced a limited level of telomerase in naive or in memory phenotype CD8+ T cells after 5 days treatment. Together, it is clear that cytokines involved in homeostasis of T cells (naive and memory cells) are capable of activating telomerase and compensating the erosion of telomeres during cell divisions, and thus preserving the replicative life span while inducing proliferation. It is worth noting that some cytokines can repress telomerase activity including IFN- (37) and TGF- (38). Thus, the status of telomerase expression in lymphocytes in vivo will depend on the net outcome of those positive and negative regulators.
In summary, we investigated the effect of IL-15 on the replicative life span of long-term cultured memory CD8+ T cells and presented two novel findings. First, IL-15 is capable of inducing telomerase expression in memory CD8+ T cells through activation of Jak3 and PI3K pathways. Second, telomere length was maintained at a relatively stable length in memory CD8+ T cells that underwent substantial divisions. In the absence of sufficient levels of telomerase, telomere shortening was observed in many types of normal cells undergoing active divisions including lymphocytes (11, 20, 21). We demonstrate that telomerase activity induced by IL-15 can protect telomere length in actively dividing memory CD8+ T cells and that IL-15 may preserve the replicative capacity of memory CD8+ T cells.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Drs. Dan Longo and Mark Mattson for reviewing the manuscript, Robert Wersto and Joe Chrest for the cell sort, and Karen Madara at National Institute on Aging leukophoresis unit for blood samples.
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 Y.L. and W.Z. contributed equally to this work.
2 Address correspondence and reprint requests to Dr. Nan-ping Weng, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Box 21, Baltimore, MD 21224. E-mail address: wengn@grc.nia.nih.gov
3 Abbreviations used in this paper: TRAP, telomeric repeat amplification protocol; FISH, fluorescent in situ hybridization; TRF, telomeric restriction fragment.
Received for publication May 18, 2004. Accepted for publication January 12, 2005.
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The preservation of the replicative life span of memory CD8+ T cells is vital for long-term immune protection. Although IL-15 plays a key role in the homeostasis of memory CD8+ T cells, it is unknown whether IL-15 regulates the replicative life span of memory CD8+ T cells. In this study, we report an analysis of telomerase expression and telomere length in human memory phenotype CD8+ T cells maintained by IL-15 in vitro. We demonstrate that IL-15 is capable of activating telomerase in memory CD8+ T cells via Jak3 and PI3K signaling pathways. Furthermore, IL-15 induces a sustained level of telomerase activity over long periods of time, and in turn minimizes telomere loss in memory CD8+ T cells after substantial cell divisions. These findings suggest that IL-15 activates stable telomerase expression and compensates telomere loss in memory phenotype CD8+ T cells, and that telomerase may play an important role in memory CD8+ T cell homeostasis.
Introduction
Memory lymphocytes are generated from naive lymphocytes after the interaction with Ag. The long-lived memory lymphocytes appear to be dependent on cytokines such as IL-2, IL-4, IL-7, and IL-15 (1, 2, 3, 4). IL-15 is capable of promoting proliferation and long-term survival of memory CD8+ T cells in an Ag-independent fashion (1, 3, 5). The interaction of IL-15 with its receptor complex leads to activation of Jaks and subsequently Stats (6) as well as activation of PI3K (7) and other signaling pathways (8). Strikingly, despite apparent differences in the initial ligand/receptor interaction, both IL-15 and TCR engagement are capable of inducing similar synthesis of effector molecules and induction of cytotoxicity in memory phenotype CD8+ T cells (9). These findings indicate that the function of IL-15 in CD8+ memory T cells is not only a growth factor but also an Ag-independent activator. However, the long-term impact of IL-15 on the replicative life span of memory CD8+ T cells is unclear.
Telomeres are the end structure of eukaryotic chromosomes that consist of a tandem hexanucleotide repeats (TTAGGG)n and a number of associated proteins (10). During cell division, incomplete replication of the terminal DNA results in a loss of 50–200 bases of telomere repeats with each division (11, 12). The progressive shortening of telomeres occurring in cells undergoes substantial cell division without proper compensation. When telomere length in a cell is critically shortened, chromosomal instability leads to cell cycle arrest or senescence and/or apoptosis (13). Telomerase is a unique RNA-dependent DNA polymerase, which is capable of synthesizing terminal telomeric repeats and thus compensating the loss of telomeres due to cell divisions (14). Initially, studies suggested that telomerase is expressed in germline cells, where telomere maintenance is critical to fertility and survival of species, and in transformed cells with unlimited growth capacity, but not in normal somatic cells (15). However, subsequent studies indicate that telomerase is expressed in several types of normal somatic cells, such as T lymphocytes (16, 17, 18), although the levels and regulation of telomerase activity differ among these cells.
The function of telomerase in regulation of telomere length and replicative life span of lymphocytes has been examined recently (19). In long-term cultured T cells, telomerase activity is associated with stabilizing telomere length (20, 21, 22). Telomere length was stable while telomerase activity was high; telomere loss became apparent while telomerase activity was diminished at later time points of the culture and T cells became senescent or underwent apoptosis. However, exogenous introduction of telomerase catalytic subunit (hTERT) into T cells can stabilize telomere length and preserve proliferative capability (23, 24, 25). Furthermore, mutation of the telomerase RNA template gene results in an inherited disorder, dyskeratosis congenita, with manifestations that include immune defects, bone marrow failure, and premature graying (26). Together, these findings indicate the importance of telomere structure in cellular replicative life span, and telomerase in preserving telomere length and thus prolonging the cellular life span.
One of the central issues of immunological memory is how memory cells are maintained over long periods of time in vivo without loss of their replicative capacity. Because IL-15 plays key roles in the homeostasis of memory CD8+ T cells, we examined the role of IL-15 in regulation of the replicative capacity of memory CD8+ T cells. In this study, we report an analysis of growth, telomerase expression, and telomere length in long-term cultured memory phenotype CD8+ T cells. Our findings suggest that IL-15 is capable of inducing telomerase activity that sustains over a longer period of time than that induced by TCR cross-linking. The induction of telomerase results in minimizing the loss of telomere length of memory CD8+ T cells. Thus, IL-15 may preserve the replicative life span of proliferating memory CD8+ T cells.
Materials and Methods
Isolation of memory phenotype CD8+ T cells
Blood was collected from normal donors at the apheresis unit of the National Institute on Aging Clinical Core Facility under an approved protocol (MRI2003-054; Cytophoresis of Volunteer Donors). Isolation of memory phenotype CD8+ T cells from blood was previously described (9). In brief, PBMC were isolated through centrifugation under a Lympho-Ficoll gradient, and memory phenotype CD8+ T cells were isolated from PBMCs via an immunomagnetic separation. The purity of isolated memory phenotype CD8+ T cells (CD8+CD27+CD45RA–) was >90% based on FACS analysis and 5% of the residual cells were CD8+CD27+CD45RAlow cells. In some cases, memory phenotype CD8+ T cells were isolated by a cell sort with the purity >99%.
Culture of naive and memory phenotype CD8+ T cells
Freshly isolated memory phenotype CD8+ T cells were cultured with either anti-CD3 plus anti-CD28 beads (anti-CD3/CD28) (gift from Dr. C. June, University of Pennsylvania, Philadelphia, PA) or IL-15 at 50 ng/ml (PeproTech) in vitro in RPMI 1640 with 10% FBS and penicillin (10 U/ml)/streptomycin (10 μg/ml). Anti-CD3/CD28 is applied once at the beginning of the culture in most cases; multiple anti-CD3/CD28 stimulation prolonged culture time by 7–10 days. IL-15 was added at the beginning of culture. The concentration of cells in the culture was checked every 5 days and maintained at 1–3 million/ml. At approximately every 10 days, half of the medium was removed and replenished with fresh medium containing IL-15 (50 ng/ml). Cell growth was measured in two ways. First, cells were enumerated using a hemocytometer to assess cell numbers under each of the stimulation conditions. The second method used a dye, CFSE (Molecular Probes), to track the number of cell divisions. For analyzing the specificity of IL-15-induced telomerase, naive and memory phenotype CD8+ T cells were isolated and cultured in the presence of 50 ng/ml IL-15 or IL-7 (PeproTech) for 5 days, and cells were harvested for determining telomerase activity.
Tracking the cell cycle number via CFSE
The procedures of CFSE labeling and analysis were previously described (27). In brief, freshly isolated memory phenotype CD8+ T cells at 2 x 106 cells/ml were incubated with 10 μM CFSE at 37°C for 10 min, washed twice with 10 ml of RPMI 1640 medium, and stimulated under the same conditions described above for unlabeled memory phenotype CD8+ T cells. The CFSE profiles were analyzed using flow cytometry at days 5, 15, and beyond. Cells that had undergone three, six, and eight divisions were isolated by cell sorting for assessment of telomere length and telomerase activity.
Inhibition of telomerase activation
Inhibitors for Jak3 (Jak3 inhibitor I, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline), PI3K (LY294002), AKT (SH-6), and JNK (SB600125) were purchased from EMD. The concentrations of each inhibitor used in this study were based on the previous report (28) as well as our own experiment. Memory phenotype CD8+ T cells were cultured in the presence of IL-15 (50 ng/ml) for 5–7 days. Then, inhibitors were added (1 μg/ml Jak3 inhibitor I, 5 μM LY294002, 5 μM SH-6, and 1 μM SB600125) and incubated for an additional 24 h before harvest. The viability of treated cells was analyzed by trypan blue exclusion assay (Invitrogen Life Technologies), and telomerase activity of these cells was determined by telomeric repeat amplification protocol (TRAP)3 assay.
Telomerase activity
Telomerase activity is measured using TRAP as previously described with some modification (15, 29). We have conjugated the Ts primer with a fluorescent dye, TAMRA. The amplified telomere DNA products were visualized, and the fluorescent intensities were captured using a fluorescent imaging system (Typhoon 9410; Amersham Biosciences). Quantification of the image files to calculate telomerase activity was done using ImageQuant software.
Telomere length
Telomere lengths were measured by two different methods. We used the Flow-fluorescent in situ hybridization (FISH) (30, 31) for analyzing long-term cultured cells and the Southern blot technique (32) for sorted cells. In brief, freshly harvested cells were permeabilized and hybridized with FITC-PNA probe (PNA-(C3TA2)3 probe; PerkinElmer Biosystems) at 83°C for 10 min. Cells were then allowed to hybridize in the dark at room temperature for 2 h. After two washes, the cells were stained with propidium iodide (Sigma-Aldrich) to check for positive nuclear staining. The statistical mean was measured for probed and unprobed measurements, and the difference was taken to be the intensity measurement of telomere staining by the FITC probe. PBMCs from a normal donor were used for normalization of analysis done at different times.
Southern blot analysis of mean telomeric restriction fragment (TRF) length was done with sorted cells after a certain number of cell divisions as previously described (32). Briefly, genomic DNA was isolated from sorted CD8+ memory T cells by using a DNA isolation kit (Gentra Systems), and digested with HinfI and RsaI (Roche Molecular Biochemicals). One microgram of digested DNA was loaded per well of a 0.5% agarose gel and separated by electrophoresis. The gel was dried at 65°C for 1.5 h, denatured, and neutralized. The hybridization was conducted using a 32P-end-labeled oligonucleotide (CCCTAA)4 probe, at 46°C overnight. After three rounds of washing (5x SSC/0.1% SDS, 2x SSC/0.1% SDS, and 3.2 M tetramethylammonium chloride/0.1% SDS) at 45°C, the gel was analyzed by phosphor imager (Typhoon 9410; Amersham Biosciences). Mean TRF length was calculated as described previously (11).
Results
IL-15 induces long-term growth of memory phenotype CD8+ T cells in vitro
Both anti-CD3/CD28 Abs and IL-15 are capable of inducing cell divisions of memory phenotype CD8+ T cells. To compare the growth kinetics of memory phenotype CD8+ T cells in response to anti-CD3/CD28 Abs and IL-15 stimulation, we analyzed memory phenotype CD8+ T cells of a half-dozen of normal donors, and found that anti-CD3/CD28-stimulated cells underwent cell division and expansion faster than did IL-15-treated memory phenotype CD8+ T cells in the first 5 days (Fig. 1A). In general, >90% of the cells have divided at least once after 5-day culture of anti-CD3/CD28 stimulation, whereas 10% of cells divided in the same period of time under IL-15 (Fig. 1A). This difference was mostly diminished after 15 days of culture where 90% of cells underwent at least 1 division under IL-15 and few cells have not divided at least once under anti-CD3/CD28 stimulation (Fig. 1A). Interestingly, whereas memory phenotype CD8+ T cells grew 25–40 days in culture after repeated anti-CD3/CD28 stimulation (a single stimulation lasted 25–35 days, and subsequent anti-CD3/CD28 stimulation led to another 5–7 days of growth), memory phenotype CD8+ T cells grew 45–80 days in culture under IL-15 even though the cell division rate decreased at the late time points. Although the expansion of memory phenotype CD8+ T cells estimated by a total population change under anti-CD3/CD28 or IL-15 is moderate (three to five mean population doublings) (Fig. 1B), the majority of cells underwent 6–8 divisions over 15–25 days of culture based on CFSE analysis (Fig. 1A). When cells underwent 6 divisions, they were isolated and relabeled with CFSE and cultured in the presence of IL-15; we found that these cells underwent an additional 4–6 divisions in 10–15 days (data not shown). Thus, the number of cell divisions estimated by mean population doublings is likely underestimated, and the actual number of cell divisions of those survival memory phenotype CD8+ T cells in long-term culture under IL-15 is probably 15–20 cell divisions.
FIGURE 1. IL-15 induced long-term growth of memory CD8+ T cells. A, Comparison of proliferation of memory phenotype CD8+ T cells in response to anti-CD3/CD28 and IL-15 (50 ng/ml) using CFSE. One representative result of five independent experiments is shown at days 5 and 15 after stimulation. Cell division numbers are indicated in the bottom of the figure. B, Long-term culture of memory phenotype CD8+ T cells in the presence of anti-CD3/CD28 or IL-15 (50 ng/ml). One representative growth curve of seven independent donors is shown.
IL-15 induces telomerase activity in memory phenotype CD8+ T cells
To investigate whether IL-15 is capable of inducing telomerase, we isolated memory phenotype CD8+ T cells from normal donors by cell sort and cultured these sorted cells with different concentrations of IL-15 (from 100 to 1.5 ng/ml) for 5 days. We found that IL-15 is capable of inducing telomerase activity in memory phenotype CD8+ T cells, and that the levels of induced telomerase activity are dependent on the concentration of IL-15 (Fig. 2A). We then examined the kinetic changes of telomerase activity in a time course, and found that telomerase activity started to appear after 48 h of IL-15 culture (50 ng/ml) (Fig. 2B). To analyze the specificity of IL-15-induced telomerase in memory phenotype CD8+ T cells and compared with another cytokine IL-7, we found that IL-15 induced significantly higher levels of telomerase activity in memory than in naive phenotype CD8+ T cells (Fig. 2C). In contrast, IL-7 induced limited levels of telomerase activity in naive and memory phenotype CD8+ T cells (Fig. 2C).
FIGURE 2. Activation of telomerase in memory phenotype CD8+ T cells by IL-15. A, IL-15 dose-dependent activation of telomerase in memory phenotype CD8+ T cells. A series of 2-fold dilutions of IL-15 (from 100 to 3.1 ng/ml) was used in culture of memory phenotype CD8+ T cells. After 5-day culture, cells were lysed and assayed for telomerase activity by TRAP assay, with each reaction using cell lysate of 5000 cell equivalents. One representative result from three different individuals is shown. IC indicates the internal control of the assay. B, Time course of telomerase activation in memory phenotype CD8+ T cells under IL-15. Freshly isolated memory phenotype CD8+ T cells were cultured in the presence of 50 ng/ml IL-15 and harvested at indicated time and measured for telomerase activity. One representative result from three different individuals is shown. C, Activation of telomerase in naive and memory phenotype CD8+ T cells by IL-15 and IL-7. One representative result from three different individuals is shown.
Next, we compared telomerase activity of memory phenotype CD8+ T cells that were stimulated with anti-CD3/CD28 Abs or IL-15 in long-term culture. Both stimulation conditions induced telomerase in memory phenotype CD8+ T cells, but they induced different patterns of telomerase expression (Fig. 3A). As a whole population, the levels of telomerase activity were generally lower in IL-15-cultured cells than in anti-CD3/CD28-stimulated cells. Anti-CD3/CD28 induced a rapid activation of telomerase, which peaked around 3 days, followed by a short period of stable high level activity (5–10 days), and a gradual loss of the activity. In contrast, IL-15-induced telomerase activity features a modest initial induction (5–10 days), followed by a sustained moderate activity over a longer period of time (30–40 days), and an eventual decrease of the activity at the end of the long-term culture. Interestingly, cells from some donors even showed higher levels of telomerase activity in the late time of culture (Fig. 3A). The physiological significance of this is not clear.
FIGURE 3. Telomerase expression pattern in long-term cultured memory phenotype CD8+ T cells. A, Comparison of telomerase activation in divided memory phenotype CD8+ T cells stimulated between anti-CD3/CD28 and IL-15. Freshly isolated and stimulated memory CD8+ T cells were labeled with CFSE and cells that underwent three, six, and eight divisions were isolated by cell sort. Cells were lysed and assayed for telomerase activity. The TRAP assay was conducted using cell lysates at 5000 and 2500 cell equivalents. IC indicates the internal control of the assay. B, Quantitation of telomerase activity in stimulated memory cells relative to unstimulated memory cells in A. C, Comparison of telomerase activity in long-term cultured memory CD8+ T cells stimulated with ant-CD3/CD28 or IL-15. One representative result of three independent donors is shown.
It has been reported that memory CD8+ T cells expressing IL-2R respond to IL-15 more efficiently than those memory phenotype CD8+ T cells that do not express IL-2R (33). Because the number of IL-2R-expressing cells are varied among different donors (data not shown), the proliferative response of memory phenotype CD8+ T cells cultured under IL-15 reflect only those IL-2R-expressing cells. Thus, the lower levels of telomerase activity observed in IL-15-stimulated memory CD8+ T cells may also reflect the mixed responding populations. To further investigate the difference of induced levels of telomerase activity by anti-CD3/CD28 Abs and IL-15, we isolated responding cells that underwent three, six, and eight cell divisions using CFSE labeling and cell sort, and measured telomerase activity in these sorted cells. Interestingly, the levels of telomerase activity are substantially higher in actively dividing cells than in a whole population under IL-15 stimulation, and are similar between IL-15 and anti-CD3/CD28 Abs (Fig. 3, B and C). Furthermore, although different levels of telomerase activity were found between the sorted actively dividing cells and a whole population, there was no significant difference in telomerase activity among actively dividing memory phenotype CD8+ T cells that had undergone different numbers of cell divisions (Fig. 3, B and C).
Signaling requirements of IL-15-induced telomerase activation in memory phenotype CD8+ T cells
The action of IL-15 is dependent on the signaling pathways through its receptors. The Jaks/Stat5 and PI3K/AKT are known signaling pathways involved in IL-15 action (6, 7). To determine whether these signaling pathways are required for IL-15-induced telomerase activation in memory phenotype CD8+ T cells, we used specific inhibitors for Jak3 and PI3K. We found that treatment of inhibitors to Jak3 (Jak3 inhibitor I) or PI3K (LY294002) or AKT (SH-6) for 1 day after 5-day pretreatment of IL-15 significantly reduced telomerase activity in memory CD8+ T cells. In general, 30% of telomerase activity remained after treatment with each inhibitor (Fig. 4, A and B), whereas there was no obvious toxicity to treated cells (C). In contrast, treatment with inhibitor to JNK (SB600125) did not show any effects in telomerase inhibition (Fig. 4, A and B). Combination of inhibitors to PI3K and Jak3 resulted in increased inhibition (data not shown), suggesting that Jak3 and PI3K/AKT pathways play a major role in IL-15-induced activation of telomerase, and other pathways may contribute to the minor role in telomerase activation in memory CD8+ T cells.
FIGURE 4. Signaling requirement of IL-15-induced telomerase activation in memory phenotype CD8+ T cells. A, Repression of telomerase activity after treatment of inhibitors to PI3K, AKT, and Jak3. One representative from three independent experiments is shown. B, Quantitation of relative inhibition of telomerase activity in memory phenotype CD8+ T cells after 1-day treatment of inhibitors to PI3K, AKT, and Jak3. Mean of three donors and SEM are shown. C, Viability of inhibitor-treated memory phenotype CD8+ T cells. Mean of three donors and SEM are shown.
IL-15 maintains telomere length of long-term cultured memory phenotype CD8+ T cells
To determine whether telomerase activity induced by IL-15 is capable of compensating the loss of telomere length, we analyzed telomere length of memory phenotype CD8+ T cells after a certain number of cell divisions and at multiple time points in long-term culture. We used CFSE for isolation of memory phenotype CD8+ T cells that underwent six and eight cell divisions after IL-15 or anti-CD3/CD28 stimulation via cell sort, and measured the telomere length (terminal telomere restriction fragments) by Southern analysis. If there is a loss of 50–200 bp per cell division, one would expect a loss of 400-1600 bp of telomeres after eight cell divisions. However, the average loss of telomeres of five donors is 110 bp (SE is 170 bp) in IL-15-cultured memory phenotype CD8+ T cells (Fig. 5A). Thus, telomere lengths are relatively stable in long-term cultured memory phenotype CD8+ T cells.
FIGURE 5. Telomere length dynamics in long-term cultured memory phenotype CD8+ T cells. A, Telomere lengths were analyzed in memory phenotype CD8+ T cells before and after six and eight cell divisions with stimulation of anti-CD3/CD28 or IL-15. Freshly isolated memory phenotype CD8+ T cells were collected along stimulated memory phenotype CD8+ T cells that underwent six and eight divisions isolated by cell sort. Telomere length was measured as mean TRF by Southern blot analysis. D1–D5 indicates five different donors. B, Telomere length changes in long-term cultured memory phenotype CD8+ T cells. Flow-FISH was used for telomere length measurement. One representative result of four independent donors is shown.
We also examined telomere length in long-term cultured memory phenotype CD8+ T cells at multiple time points by Flow-FISH. Again, we found telomere lengths were relatively stable after some initial loss over long-term culture (Fig. 5B). In contrast, telomere length was significantly decreased in memory phenotype CD8+ T cells at late time points of culture despite initial gain of telomeres under anti-CD3/CD28 stimulation (Fig. 5B). This demonstrates that the difference in regulation of telomerase activity is associated with the difference in telomere length regulation in long-term cultured memory phenotype CD8+ T cells under IL-15 or TCR cross-linking.
Discussion
We have demonstrated in this study that IL-15 is capable of inducing telomerase activity through activation of Jak3 and PI3K/AKT signaling pathways in memory phenotype CD8+ T cells, and that IL-15-induced telomerase activity is associated with the relative stable telomere length in long-term cultured memory phenotype CD8+ T cells. Previous reports showed that exogenous expression of telomerase catalytic unit (hTERT) prevents telomere loss and extends the replicative life span of CD8+ and CD4+ T cells (23, 24, 25), suggesting that preservation of telomere structure can extend cellular replicative life span of cells. By activating and maintaining telomerase activity in memory CD8+ T cells, IL-15 may preserve the replicative life span of dividing memory CD8+ T cells.
Although both cross-linking of TCR/costimulatory receptors and IL-15 can also activate telomerase in memory CD8+ T cells, the difference in telomerase expression kinetics between these two stimulation conditions reflects their different and overlapping signaling pathways as well as physiological roles in memory CD8+ T cells. TCR and Ag interaction leads to activation of Syk (e.g., ZAP70), Src (e.g., Lck) protein tyrosine kinase families, increase of cytoplasmic free calcium [Ca2+]i, and PI3K/AKT pathways, whereas IL-15 induces Jak/Stats pathway and PI3K/AKT pathways. Physiologically, activation of T cells via TCR engagement leads to substantial expansion of effector cells, and the majority of these effector cells undergo apoptosis after the clearance of Ag. The role of telomerase in TCR-mediated activation is to preserve the telomere length of rapidly dividing effector cells at the phase of clonal expansion. In contrast, the role of IL-15 in memory CD8+ T cells is promoting survival and slow proliferation over the long term. The sustained levels of telomerase activity induced by IL-15 is designed to protect the loss of telomere repeats of continued slow proliferation of memory CD8+ T cells over life time. Thus, IL-15 not only promotes survival from death but also preserves telomere length and the capacity for replication of memory CD8+ T cells for future robust responses upon exposure to Ag.
Considering the importance of IL-15 in memory CD8+ T cell homeostasis (34, 35), it is not a surprise that IL-15 is capable of activating telomerase and compensating the loss of telomeres in actively dividing memory CD8+ T cells. Interestingly, IL-7, a key cytokine in T cell development and homeostasis, is also capable of inducing telomerase activity in T cells (36). Similarly to IL-15, IL-7 did not compensate completely the loss of telomere length in cultured naive CD4+ T cells, because some loss of telomere repeats was observed in 12-day cultures of CD4+ T cells maintained by IL-7 (36). We have analyzed whether IL-7 induces telomerase activity in CD8+ T cells in this study. Compared with IL-15 induced telomerase in memory phenotype CD8+ T cells, IL-7 only induced a limited level of telomerase in naive or in memory phenotype CD8+ T cells after 5 days treatment. Together, it is clear that cytokines involved in homeostasis of T cells (naive and memory cells) are capable of activating telomerase and compensating the erosion of telomeres during cell divisions, and thus preserving the replicative life span while inducing proliferation. It is worth noting that some cytokines can repress telomerase activity including IFN- (37) and TGF- (38). Thus, the status of telomerase expression in lymphocytes in vivo will depend on the net outcome of those positive and negative regulators.
In summary, we investigated the effect of IL-15 on the replicative life span of long-term cultured memory CD8+ T cells and presented two novel findings. First, IL-15 is capable of inducing telomerase expression in memory CD8+ T cells through activation of Jak3 and PI3K pathways. Second, telomere length was maintained at a relatively stable length in memory CD8+ T cells that underwent substantial divisions. In the absence of sufficient levels of telomerase, telomere shortening was observed in many types of normal cells undergoing active divisions including lymphocytes (11, 20, 21). We demonstrate that telomerase activity induced by IL-15 can protect telomere length in actively dividing memory CD8+ T cells and that IL-15 may preserve the replicative capacity of memory CD8+ T cells.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Drs. Dan Longo and Mark Mattson for reviewing the manuscript, Robert Wersto and Joe Chrest for the cell sort, and Karen Madara at National Institute on Aging leukophoresis unit for blood samples.
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 Y.L. and W.Z. contributed equally to this work.
2 Address correspondence and reprint requests to Dr. Nan-ping Weng, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Box 21, Baltimore, MD 21224. E-mail address: wengn@grc.nia.nih.gov
3 Abbreviations used in this paper: TRAP, telomeric repeat amplification protocol; FISH, fluorescent in situ hybridization; TRF, telomeric restriction fragment.
Received for publication May 18, 2004. Accepted for publication January 12, 2005.
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