An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity

Telomerase is a ribonucleoprotein complex with a well-established function of adding telomeric DNA to the ends of linear chromosomes. In addition to associated factors, the two core human telomerase enzyme components are the reverse transcriptase protein hTERT, and the RNA-templating component hTR (also called hTER or hTERC). Many studies have associated telomerase activity levels in resting adult human peripheral blood mononuclear cells (PBMCs) with different health and disease states. For example, inherited telomerase mutations resulting in haploinsufficiency cause telomere syndromes characterized by lung fibroses, cancer predisposition, and bone marrow failure. In wild-type (WT) individuals, low telomerase activity in resting PBMCs is associated with risk factors for aging-related diseases and chronic stress,^1-3  suggesting that low telomerase might indicate or promote certain disease states. Although increases in resting PBMC telomerase activity have been associated with meditation, healthy lifestyle changes, decreased measures of psychological distress,^4-6  and decreased low-density lipoprotein,⁷  the combination of short white blood cell telomeres and high telomerase activity has also been associated with a variety of disease risk factors including chronic psychological stress.^8-12  Hence, regulating telomerase activity levels is important for maintaining health and proper immune function, but the complicated relationships observed between telomerase levels in PBMCs and health and disease states indicate the need for a fuller understanding of roles of telomerase components.

The PBMC studies described above compared average telomerase activity from heterogeneous populations containing several different cell types and are potentially confounded by changes in fractions of specific cell types. In-vitro studies, including the present study, have investigated associations between telomerase activity levels and immune cell function in individual cell types. For example, CD4 T lymphocytes greatly modulate telomerase activity,^13,14  from very low levels in the resting state to large increases of telomerase enzymatic activity, hTERT messenger (m)RNA, and hTR upon stimulation^14-16 ; as cell proliferation slows, the levels of all three decrease.^16-19  Furthermore, when T-cell proliferation in vitro is hindered by cortisol, actinomycin D, cycloheximide, or herbimycin A, telomerase activity is also reduced.^16,20  Although CD4 T-cell proliferation and telomerase activity correlate, it is unknown whether telomerase activity is necessary for, or even quantitatively coupled to, this proliferative response. Because proliferation upon stimulation is an essential function of CD4 T cells, understanding the role of telomerase in T-cell proliferation is important for understanding normal T-cell and immune function.

We report here that, unexpectedly, hTR specifically is important for short-term CD4 T-cell survival. Although the level of telomerase activity is important for long-term CD4 T-cell survival, this work identifies a new telomere-independent and telomerase activity–independent function of telomerase RNA in immune cells that we postulate acts in a cell-protective, antiapoptotic pathway that can be influenced by stress or other regulatory factors.

Human buffy coats from 9 healthy donors between 17 and 25 years old were purchased from Stanford Blood Center. PBMCs were isolated from buffy coats by centrifugation with Ficoll-Paque Plus (GE Healthcare), and CD4 T cells were isolated from PBMCs using the Untouched CD4⁺ T Cell Isolation Kit II, Human (Miltenyi). Cells were stimulated 24 hours after isolation with 50 μL of Dynabeads Human T-Activator CD3/CD28 (Life Technologies) per 1 million CD4⁺ T cells and cultured in RPMI 1640 with 10% fetal bovine serum, 1% penicillin and streptomycin, and 1% glutamine with 10 ng/mL interleukin-2. Cells were transduced with lentivirus 24 hours after stimulation. Two micrograms per milliliter of puromycin was added 24 hours after transduction and kept in culture during the course of the experiment. Live cells and percentage live cells determined by trypan blue exclusion were counted with the TC20 Automated Cell Counter (BioRad). Dexamethasone treatment was for 72 hours at 1μM.

The lentiviral vector system was provided by Didier Trono (University of Geneva, Geneva, Switzerland²¹ ). Lentivirus and short hairpin (sh)RNA expression vectors were prepared as described previously.²²  The following lentiviruses were generated from those previously described by Li et al,²²  but the cytomegalovirus (CMV) promoter was replaced with the elongation factor 1-α promoter driving either puromycin resistance or green fluorescent protein (GFP): empty vector, scrambled-sequence shRNA (shScramble), and shTR1. shTERT was generated in the same lentiviruses based on the target sequence from Listerman et al.²³  shTR2 was generated based on a previously published target sequence.²⁴  shBIM was generated based on the previously published target sequence.²⁵  CMV-GFP-IRES-puromycin and CMV-TERT-IRES-puromycin lentiviruses were described in Listerman et al.²³  The hTR overexpression constructs were generated by polymerase chain reaction (PCR) amplification of U3-hTR-500 from the following plasmids from Kathleen Collins (University of California, Berkeley, CA) and cloned into the pHR vector with elongation factor 1-α driving puromycin resistance: pBS’U3-hTR-500, pBS’U3-Δ96-7-500, pBS’U3-C204G-500, pBS’U3-G305A-500, and pBS’U3-hTR-U64-500.^26,27  Lentivirus was titered by quantitative reverse-transcription PCR. See the supplemental Methods, available on the Blood Web site, for protocol.

Cell cycle state was measured by staining with Vybrant DyeCycle Green (Life Technologies) according to the manufacturer’s instructions.

Apoptosis was measured with Caspase-Glo 3/7, Caspase-Glo 8, and Caspase-Glo 9 assay kits (Promega) according to the manufacturer’s instructions. Luminescence was read by the Veritas Microplate Luminometer (Turner BioSystems).

Relative telomerase activity was measured by the real-time quantitative telomere repeat amplification protocol (RQ-TRAP), and samples were determined to be in the linear range of this assay.^28,29 

hTR RNA levels and mRNA levels were measured as described by Listerman et al.²⁹  See supplemental Table 2 for primers used.

Cells were mounted on coated glass Cytoslides (Thermo Scientific) by centrifugation (Cytospin; Thermo Scientific). Cells were fixed and permeabilized. Immunostaining was performed with the primary antibody anti-phospho-Histone H2A.X (05-636; Millipore) or pAb anti-53BP1 (NB100-304; Novus Biologicals) and with the secondary antibody Alexa Fluor 594 (Molecular Probes). Primary antibodies were diluted 1:500; secondary antibodies were diluted 1:750. DNA was visualized with 4,6-diamidino-2-phenylindole (Life Technologies). IF was followed by telomere fluorescence in situ hybridization (FISH) as described by Diolaiti et al³⁰  without pepsin treatment. The telomeric peptide nucleic acid (PNA) probe used was FAM-OO-ccctaaccctaaccctaa (Panagene) at 0.5 μg/mL. All images were obtained using a DeltaVision Real-Time Deconvolution Microscope (Applied Precision) with the 100×/1.4 NA Plan Apo objective (Olympus). Images were acquired in 0.5-μM increments, deconvoluted, Z-projected in softWoRx (Applied Precision), and adjusted for brightness and contrast in the image processing program Fiji.³¹  Telomeric and DNA damage foci and telomeric and DNA damage integrated intensity were measured with CellProfiler image analysis software (www.cellprofiler.org; pipelines available on request).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting were conducted on whole cell lysates using the Hybond-P PVDF Membrane (GE Healthcare) with either the Western Lightning Plus ECL kit (Perkin Elmer) or the SuperSignal West Femto kit (Thermo Fisher) according to the manufacturers’ instructions.

The following primary antibodies were used: Rabbit anti-Bid (human) at 1:1000 (Cell Signaling Technology 2002); Rabbit anti-Bim at 1:1000 (Cell Signaling Technology 2819); Rabbit anti-Puma at 1:1000 (Cell Signaling Technology 4976); Rabbit anti-Bad at 1:1000 (Santa Cruz Biotechnology sc-943); mouse anti-p53 (Abcam PAb 240); and mouse anti-GAPDH at 1:20 000 (Millipore MAB374). The following secondary antibodies were used: goat anti-rabbit at 1:2000 (Jackson Immunoresearch 111-035-144) and sheep anti-mouse (Jackson Immunoresearch 515-035-003) at 1:2000 for p53 and 1:10 000 for GAPDH.

All statistical analyses were performed using Prism (GraphPad Software). Significant differences were assessed by either 1-way analysis of variance (ANOVA) or by unpaired Student t tests as indicated. A cutoff of P < .05 was used to determine significance.

Previous work has demonstrated that dexamethasone treatment reduces T-cell survival and telomerase activity in short-term in-vitro experiments.^4-6,20  We tested directly whether increasing telomerase components mitigated the effects of dexamethasone-induced apoptosis on T-cell survival.

We overexpressed WT hTR or different disease-causing enzymatically inactive mutants of hTR (Δ96-7, G305A, and hTR-U64) from a lentiviral vector in stimulated CD4 T cells incubated for 72 hours in the presence or absence of dexamethasone, a corticosteroid that induces Bim-mediated apoptosis in T cells.^32,33  The hTR Δ96-7 mutation, located in the pseudoknot in the 5′ half of hTR, abolishes telomerase enzymatic activity but allows normal hTERT binding.²⁷  The G305A substitution point mutation in the stem of the P6.1 stem loop in the 3′ half of hTR also completely abolishes catalytic activity and reduces binding to hTERT by 80%. As a control for overexpression of a noncoding RNA, we used the hTR-U64 fusion chimeric RNA, which contains the 5′ half of hTR (pseudoknot and template regions), but the 3′ half (CR 4/5 and box H/ACA regions) is replaced by the H/ACA domain of the similarly sized U64 small nucleolar (sno)RNA. This fusion RNA is stably expressed but cannot bind hTERT and thus does not confer telomerase activity.²⁷ 

Without dexamethasone, overexpressing WT hTR increased telomerase activity without increasing TERT mRNA (Figure 1A-B), suggesting that either hTERT exists in the cell without hTR or that hTR overexpression stabilizes hTERT protein (Figure 1A-B). The increased telomerase activity did not protect against dexamethasone-induced apoptosis (Figure 1C, comparing solid to checkered red bars). As expected, overexpression of control vectors or the catalytically inactive hTR mutants did not increase telomerase activity or affect hTERT mRNA levels (Figure 1A-B). Surprisingly however, overexpressing the G305A point mutant hTR, but not WT hTR or hTR-U64, protected against dexamethasone-induced apoptosis (Figure 1C, comparing solid to checkered bars). Thus, protection from dexamethasone-induced apoptosis by catalytically inactive overexpressed hTR occurs and requires one or more regions located in the 3′ half of hTR.

We determined whether fully WT-sequence hTR, in a catalytically inactive state, can protect against dexamethasone-induced apoptosis. To increase the fraction of catalytically inactive endogenous WT hTR, we knocked down hTERT using shRNA. hTERT knockdown alone was sufficient to prevent dexamethasone-induced apoptosis (Figure 1D, comparing solid to checkered pink bars). This dexamethasone effect also extended to a natural corticosteroid: shRNA targeting hTERT also protected against apoptosis induced by the clinically shorter-acting hydrocortisone, whereas the control shScramble or hTR knockdown did not (supplemental Figure 1). When hTERT knockdown was combined with hTR overexpression, either WT hTR or G305A hTR protected against dexamethasone-induced apoptosis. Thus, when hTERT levels are reduced, both endogenous and overexpressed WT hTR, as well as overexpressed G305A hTR, can protect against dexamethasone-induced apoptosis (Figure 1D). Again, hTR-U64 fusion RNA overexpression, even when combined with hTERT knockdown, failed to protect against dexamethasone-induced apoptosis. The finding that knocking down hTERT alone protected against apoptosis suggests that the overexpressed hTR-U64 fusion interfered with the ability of endogenous WT hTR to protect against apoptosis.

In summary, WT hTR, when catalytically inactive, protects CD4 T cells from dexamethasone-induced apoptosis, and hTR-U64 fusion RNA interferes with this protection.

Because increasing catalytically inactive hTR protected against dexamethasone-induced apoptosis, we predicted that, conversely, reducing endogenous hTR would increase apoptosis. We knocked down telomerase in human CD4 T cells, using lentiviral vectors targeting hTERT (shTERT), and two different anti-hTR shRNAs targeting the hTR templating sequence (shTR1) or the hTR pseudoknot structure (shTR2), previously reported to specifically knock down telomerase RNA in human cancer cell lines and primary fibroblasts.^22,24  After 14 days, each of these shRNAs (see supplemental Figure 2 for optimization procedures) equally reduced telomerase activity levels (approximately 80%) compared with two different controls: empty lentivector or shScramble (Figure 2A). Knockdown of either core component of telomerase did not affect the transcriptional steady-state level of the other (Figure 2B). Notably, only hTR knockdown, and not hTERT knockdown, resulted in fewer live cells compared with empty vector, shScramble, or shTERT, as measured by trypan blue staining (Figure 2C, day 15). This result was confirmed by using CD4 T cells isolated from 6 additional donors in 8 independent experiments (data not shown) and by using GFP vectors instead of puromycin-resistant vectors (supplemental Figure 3). Furthermore, hTR knockdown did not affect the percentages of naïve, central memory, effector memory, or Th1 CD4 T cells (supplemental Figure 4). Additionally, the reduction of live cells with hTR knockdown was observed when starting with only naïve CD4 T cells (supplemental Figure 5). The reduction in live cells was not due to disruption of progression through the cell cycle (Figure 2D). Instead, hTR knockdown, but not hTERT knockdown, induced apoptosis, measured by increases in caspase-3/7 and caspase-9 activities, with no change detected in caspase-8 activity (Figure 2E). Together, these results indicate that hTR knockdown activated the intrinsic apoptotic pathway in stimulated CD4 T cells, whereas hTERT knockdown did not.

Western blotting measures of protein levels of the apoptotic proteins Bim, Bad, Bid, Puma, and p53 showed upregulation of both Puma, which triggers Bim upregulation, and Bim, which triggers caspase-9 activation, with hTR knockdown compared with shScramble and shTERT (Figure 2F). No changes were observed in p53 levels, suggesting that PUMA is triggered through a p53-independent and DNA damage–independent pathway.^32,34  Combining Bim depletion with hTR knockdown increased cell viability (Figure 2G), confirming that Bim may be at least partially responsible for the apoptosis induced by hTR knockdown.

Additional experiments eliminated extrinsic factors as contributing to hTR knockdown–induced apoptosis. Media from hTR knockdown cells did not induce apoptosis in control cells (supplemental Figure 6). Similarly, when hTR was knocked down with lentiviral vectors expressing GFP, survival of surrounding GFP⁻ cells was not affected (supplemental Figure 3). Furthermore, genome-wide microarray analysis of hTR or hTERT knockdown compared with shScramble did not reveal any transcriptional changes of genes involved in apoptosis, suggesting that hTR knockdown triggers apoptosis at a posttranscriptional level. After validation by quantitative PCR, only three immune-function genes were statistically significantly upregulated by hTR knockdown compared with shScramble: CXCL10, CXCL11, and IL6. Because these genes are not directly involved in apoptosis, this upregulation may instead result from apoptosis. Interestingly, several snoRNAs significantly changed with hTR knockdown compared with hTERT knockdown, suggesting potential involvement of snoRNA pathways (supplemental Figure 7). The combined results indicate that in stimulated CD4⁺ T cells, hTR knockdown was sufficient to induce cell-intrinsic Bim-mediated apoptosis.

Short or deprotected telomeres can trigger a p53-mediated DNA damage response to induce apoptosis or senescence in normal cells.^35,36  Although the short duration of our experiments and the lack of p53 upregulation (Figure 2F) argued against short or damaged telomeres as the trigger for the observed hTR knockdown–induced apoptosis, we directly analyzed telomere shortening and telomere damage. Telomere length distributions, determined by measuring the integrated intensity of individual telomeric PNA foci, showed no significant shortening or change in length distribution in shTERT, shTR1, or shTR2 cells compared with each other and compared to no virus, empty vector, and shScramble (Figure 3A and supplemental Figure 8A). We also found no significant difference in the numbers or length frequency distributions of telomeres detected per area in control, hTERT knockdown, or hTR knockdown cells (Figure 3B and supplemental Figure 8B). This finding ruled out the possibility that the shortest (and therefore most likely uncapped) telomeres were not detected by this method. As a positive control, shRNA-mediated knockdown of TIN2, a known telomere-protective shelterin protein, resulted in significant telomere PNA-FISH signal reductions and concomitantly decreased telomeres detected per area (supplemental Figure 9 A-B). Thus, neither hTERT nor hTR knockdown induced significant telomere shortening in the time frame of our experiments.

To assess whether DNA damage at the telomeres occurred independent of telomere length, we measured colocalization between telomeres and the DNA damage pathway proteins γH2AX and 53BP1; such colocalizations are termed TIFs (telomere DNA damage-induced foci). hTR knockdown did not induce an increase in γH2ax or 53BP1 TIFs per telomere or TIFs per DNA damage foci (Figure 3C-D; supplemental Figure 8C-D) or increase the total amount γH2ax of 53BP1 foci in nuclei regardless of localization (Figure 3E and supplemental Figure 8E), in contrast to the shTIN2-positive control (supplemental Figure 9C-E). These results together provide additional evidence that hTR knockdown does not induce apoptosis via telomere shortening or a telomeric DNA damage response in the time frame of our experiments.

We also manipulated hTERT, without manipulating the endogenous hTR, to further test the hypothesis that hTR in a state incapable of supporting telomerase enzymatic activity is the form of hTR that protects from apoptosis. Overexpression of full-length, enzymatically competent hTERT increased telomerase activity, indicating that some hTR normally exists in CD4 cells unbound to hTERT. We predicted that the overexpressed hTERT would increase the fraction of hTR assembled into catalytically active telomerase, thus reducing the level of catalytically inactive hTR. Consistent with this prediction, full-length hTERT overexpression decreased the live cell number and increased caspase-3/7 activity (Figure 4). We overexpressed the β⁻splice variant of hTERT protein. This natural major isoform of hTERT lacks a portion of the telomerase enzyme active site but binds hTR efficiently, assembling it into a catalytically inactive protein-hTR complex.²⁹  Overexpression of either full-length or β⁻hTERT did not affect total hTR levels. Overexpression of the β isoform did not increase telomerase activity, as expected,²⁹  or apoptosis (Figure 4). The data showing that hTR complexed with β⁻hTERT did not impede hTR antiapoptotic function further support the hypotheses that hTR in catalytically inactive form protects from apoptosis and that binding of hTR per se does not affect its antiapoptotic role.

Because hTERT overexpression alone was sufficient to induce apoptosis under endogenous hTR conditions, we predicted that apoptosis would be rescued by cooverexpression of catalytically inactive hTR mutants but not WT hTR. Cooverexpressing WT hTR and hTERT while increasing telomerase activity (Figure 5A-B, comparing solid to checkered red bars) decreased live cell counts (Figure 5C) and increased apoptosis compared with cooverexpression of hTR and GFP (Figure 5D). These results suggest that hTERT overexpression was high enough to bind up the overexpressed hTR molecules to increase telomerase activity, preventing accumulation of catalytically inactive hTR. In marked contrast, cooverexpressing Δ96-7 hTR or G305A hTR and hTERT protected against hTERT-induced apoptosis (Figure 5D). Hence, a catalytically inactive hTR, bound either efficiently (Δ96-7) or poorly (G305A) to full-length hTERT, can protect against hTERT overexpression–induced apoptosis. Again, overexpression of the hTR-U64 fusion failed to protect against hTERT-induced apoptosis (Figure 5D), further supporting the results showing that the hTR 3′ portion is necessary for the antiapoptotic role of hTR.

Various studies have suggested nontelomeric roles for hTERT that implicate it in proliferation, apoptosis, and mitochondrial function (for references, see Listerman et al²³ ), in addition to maintaining telomeres. Only two studies, however, have proposed telomerase-independent roles for hTR: regulating p53-dependent cell growth in cancer cell lines and fibroblasts,²⁴  and regulating differentiation of myeloid cells in zebrafish.³⁷  Here, we have presented evidence indicating that hTR, specifically, has a new role in protecting stimulated human CD4 T cells from apoptosis only when hTR is in an enzymatically inactive state. In this context, enzymatic activity is defined in terms of the canonical telomeric DNA polymerization reaction performed by the core active hTERT-hTR telomerase complex. We further report that the 3′ portion of hTR is necessary for protecting against hTERT overexpression–induced or corticosteroid-induced apoptosis.

Our unexpected results from multiple shRNA and overexpression experiments, summarized in Table 1, provide data consistent with hTR existing in more than one functional form: the well-known enzymatically active form that complexes with catalytically competent hTERT and other factors to synthesize telomeric DNA and elongate telomeres, and one or more enzymatically inactive forms that protect from apoptosis. In this model (Figure 6), full-length hTERT overexpression, which reduces endogenous catalytically inactive hTR by converting it into catalytically active hTR, induces apoptosis. In contrast, overexpressing hTR mutants, or catalytically dead β⁻hTERT, does not induce apoptosis. This model (Figure 6) is plausible given structural studies indicative of multiple conformations for hTR: a predicted active pseudoknot conformation with a triple helix that is stabilized by hTERT and required for telomerase activity, and an inactive “open” pseudoknot conformation.^38-40  We propose that hTR, possibly in an open inactive conformation, can interact with other factors to protect against apoptosis.

The disease-linked hTR mutants used here disrupt distinct hTR structures: Δ96-7 disrupts the pseudoknot triple helix and the G305A mutation disrupts the P6.1 stem loop. Both create catalytically inactive hTR that can bind hTERT either well (Δ96-7) or poorly (G305A). Our finding that overexpression of catalytically inactive G305A hTR, but not WT hTR or full- length hTERT, protects from dexamethasone-induced apoptosis indicates that in the time frame of our experiments, increasing telomerase enzymatic activity does not protect against apoptosis. The portion of hTR present in G305A (but missing from hTR-U64) is necessary for this protection. Furthermore, hTR protection from apoptosis is not specific to the P6.1 disruption of G305A, because in the setting of hTERT knockdown, endogenous or overexpressed WT hTR protected against dexamethasone-induced apoptosis. In addition, binding to full-length hTERT per se does not prevent hTR from protecting against apoptosis, because overexpressed catalytically inactive Δ96-7 mutant, which binds hTERT, protects from hTERT-induced apoptosis. In summary, our data support a model in which catalytically inactive hTR protects from apoptosis and the 3′ half of hTR is necessary for this protection.

Our data that hTERT overexpression induces apoptosis might superficially appear to contradict previous studies reporting that hTERT overexpression extends replicative lifespan in lymphocytes.^41-43  However, differences in experimental time lines (and potentially culture conditions) are likely to account for these discrepancies. Previous studies used cells cultured for up to 300 days, whereas our studies lasted 1 to 2 weeks. It is possible that hTERT overexpression might initially induce apoptosis by reducing the amount of catalytically inactive hTR but, in long-term culture, extend lifespan through better telomere maintenance. Hence, the present study suggests that although increasing telomerase activity might increase the replicative lifespan of immune cells, increasing the available catalytically inactive hTR can additionally increase T-cell survival by protecting against apoptosis. It is also possible that hTR and hTERT exert the effects on apoptosis described here via different mechanisms.

Recently, long noncoding RNAs were shown to regulate gene expression and proliferation in lymphocytes.⁴⁴  Possible mechanisms for the antiapoptotic effect of catalytically inactive hTR could also involve binding to factors or being processed into microRNAs. The hTR 3′ portion could be important for these mechanisms, for localization, or for both, because the hTR-U64 fusion RNA would be localized to the nucleolus, whereas the other hTR RNA constructs we investigated are not strictly nucleolar. Both hTR and the fusion hTR-U64 RNA have a 3′ H/ACA dyskerin-binding domain. The observed interference by hTR-U64 with the antiapoptotic role of endogenous hTR in the setting of hTERT knockdown suggests that hTR-U64 can compete with endogenous hTR for dyskerin, which might be important for hTR to protect against apoptosis, potentially via hTR targeting for pseudouridinylation RNA, as do other snoRNAs.⁴⁵ 

Our findings predict that human disease states of telomere syndromes,⁴⁶  which include immunodeficiency, might be more severe with mutations reducing hTR versus catalytically inactive hTR mutants or hTERT insufficiency mutants. Mutations in dyskerin, which reduce hTR levels, have more severe disease phenotypes than other telomere syndrome mutations.⁴⁶  More specifically, a familial mutation in which the 74 nucleotides of the 3′ end of hTR were deleted (and which presumably decreased hTR levels) caused lymphocytes from the patients to show increased baseline levels of apoptosis.⁴⁷  Hence, we propose that mutations that reduce hTR levels can cause more severe telomere syndromes through two mechanisms: telomere shortening due to low telomerase (previous studies) and increased lymphocyte apoptosis due to reduced hTR levels (present study). Furthermore, we propose that bone marrow failure, the most common cause of death in individuals who have telomere syndromes, may not be explained solely by stem cell depletion through shortening telomeres but also by continued drains on the hematopoietic stem cell reserves to replace decreasing levels of immune cells caused by low hTR level–induced apoptosis as well as shortening telomeres.

Many studies have shown associations between telomerase levels in vivo and human health and disease. Studies have associated healthy lifestyle and behaviors with increased telomerase activity in PBMCs and, conversely, shown that corticosteroid treatment reduces T-cell survival and telomerase activity.^4-6,20  Our findings suggest a new way, other than via telomere maintenance, in which altering hTR levels regulates immune function. As new techniques become available for primary human immune cells, future studies focusing on hTR genome editing, hTR localization, and investigating potential hTR targets and interacting partners will be integral to identifying mechanisms by which hTR influences apoptosis.

We thank Beth Cimini for designing the Cell Profiler programs used to analyze telomere length and TIF data; Eric Verdin and Emmanuelle Passegue for insightful comments on experimental design; Kathleen Collins and Michael McManus for plasmids; Richard Novak for microarray analysis; and Jue Lin, Bradley Stohr, Morgan Diolaiti, and Richard Novak for comments on the manuscript.

This work was supported in part by the National Institutes of Health National Cancer Institute (grants CA096840 and AG030424). F.S.G. was supported by the Graduate Education in Medical Sciences training program at University of California, San Francisco (http://physio.ucsf.edu/GEMS) and the David & Annette Jorgensen Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Contribution: F.S.G. and E.H.B. conceived the study, designed the experiments, and wrote the manuscript; and F.S.G. performed the experiments.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for F.S.G. is Department of Molecular Biology and Immunobiology, Harvard Medical School, Boston, MA.

Correspondence: Elizabeth H. Blackburn, Department of Biophysics and Biochemistry, University of California, San Francisco, Room S-312F Genentech Hall, 600 16th St, San Francisco, CA 94158; e-mail: elizabeth.blackburn@ucsf.edu.