Research paperAnalyses and comparisons of telomerase activity and telomere length in human T and B cells: Insights for epidemiology of telomere maintenance
Introduction
Telomeres, DNA–protein complexes at the end of eukaryotic chromosomes, shorten with each cell division. Cells with critically short telomeres can enter replicative senescence, a state of irreversible cell growth arrest. Thus, telomere shortening is viewed as a mitotic clock that counts down the number of cell divisions (Harley et al., 1990). The cellular enzyme telomerase counteracts telomere shortening by RNA-templated addition of DNA nucleotides onto telomeres, thereby lengthening the telomeric DNA (Greider and Blackburn, 1989, Greider and Blackburn, 1987, Greider and Blackburn, 1985). In a cohort of otherwise healthy premenopausal women, lower telomerase activity, although not shorter telomere length, in resting peripheral mononuclear cells (PBMC) was associated with the major risk factors for cardiovascular disease (CVD) (Epel et al., 2006) and animal studies show that low telomerase is linked to diseased tissues (Serrano and Andres, 2004). Numerous clinical studies link shorter telomere length—and inferred accelerated telomere shortening rate—in leukocytes to aging and aging-related diseases (Cawthon et al., 2003, Samani et al., 2001, Brouilette et al., 2003, Benetos et al., 2004, von Zglinicki, 2000, Kurz et al., 2004, Jeanclos et al., 2000, Aviv et al., 2006, Valdes et al., 2005, Panossian et al., 2003, Zhang et al., 2003, Gardner et al., 2005, Zhai et al., 2006, Fitzpatrick et al., 2007, Collerton et al., 2007, Adaikalakoteswari et al., 2007, Starr et al., 2007, Harris et al., 2006). Furthermore, subsequent studies showed shorter LTL and more rapid leukocyte telomere shortening (Epel, 2009) predicts earlier mortality (Cawthon et al., 2003, Epel, 2009, Carrero et al., 2008, Honig et al., 2006, Kimura et al., 2008). Thus, low telomerase and short telomeres appear to be clinically important in human health and LTL is increasingly emerging as a biomarker for cellular aging in epidemiology studies. A better understanding of what telomerase activity and telomere length in immune subpopulations may reflect will provide mechanistic insights into how cellular aging contributes to poor health.
PBMCs are composed of subsets of lymphocytes (including T cells, B cells, and natural killer cells) and monocytes. Of these, only T and B cells have been reported to have detectable, although low, telomerase activity (Weng, 2008). Total PBMC telomerase activity would thus be predicted to be determined by the percentage of T and B cells as well as the activity per cell for each cell type. PBMC telomere length is determined by the percentage of all the cell types in the PBMC cell preparation as well as the telomere length of each cell type. Several factors contribute to telomere length: telomeres that were inherited (genetic), the level of telomerase activity, environmental and cellular factors that influence the rate of telomere attrition and telomerase activity, and number of cell divisions (history of division). While previous work has shown a decline in telomerase activity and shortening of telomere length as lymphocytes progress from naïve to memory cells, and in general with chronological age, no clinical study has directly examined telomerase activity and telomere length in lymphocyte subpopulations from the same donor.
Telomerase activity is highly regulated in the development and differentiation of T and B cells (Weng, 2008). When stimulated by their specific antigens, T and B cells are activated and undergo multiple rounds of cell division. While most of the activated cells die at the end of the immune response, memory cells remain in the body during the whole lifespan and become reactivated upon encounters with the same antigen. Telomere maintenance and telomerase regulation are closely linked to the activation and differentiation of T and B cells: telomerase activity in resting T and B cells decreases as they progress from naïve to memory cells, and is upregulated upon antigen stimulation (Weng, 2008). Concomitantly, telomeres are longer in naïve T lymphocytes than memory cells, supporting the notion that net telomere shortening occurs, despite the increased telomerase activity upon activation. Patients with an autosomal dominant form of the genetic disease dyskeratosis congenita, caused by mutations in the telomerase components leading to lower telomerase activity, die of bone marrow failure, demonstrating that sufficient telomerase activity is essential for proper immune function. Ectopic expression of the telomerase gene hTERT in human CD4+ and CD8+ T cells extends their lifespan in culture, underlining the importance of telomerase in T cell function (Luiten et al., 2003, Rufer et al., 2001, Roth et al., 2003, Dagarag et al., 2004). While telomere length reflects the cumulative effects of genetic and environmental factors, telomerase activity is dynamic and is likely an essential modifiable factor in mediating environmental and lifestyle factors and telomere length changes. Thus measuring both telomere length and telomerase activity in lymphocytes may provide key readouts for the effects of environmental factors and interventions on aging.
We describe here methods for simultaneously sorting total PBMCs into three T cell subtypes: CD4+, CD8+CD28+ and CD8+CD28− cells as well as B cells, and for quantitatively measuring telomere length and telomerase activity in these cell types directly ex vivo. We also report findings from our methodology studies testing effects of collection methods (time of day, latency from draw to assay, and tube type). We found that, on a per cell basis, B cells have the highest telomerase activity and longest mean telomere length; CD4+ T cells have slightly higher telomerase activity than CD8+CD28+ T cells, but similar telomere length. Consistent with earlier reports that CD8+CD28− T cells are the most differentiated cell type and hence predicted to have undergone the most cell divisions, they had the shortest telomere length among the four groups and, interestingly, the lowest telomerase activity. In addition, having a high percentage of CD8+CD28− T cells correlated with shorter mean telomere length measured in total PBMCs (r = − 0.26, p = 0.05), consistent with the notion that both measurements serve as indicators of cellular aging. A novel observation was that telomerase activity in all four cell types from the same individual was correlated, with CD4+ and CD8+CD28+ T cell telomerase activity levels most strongly correlated (r = 0.55, r < 0.001). These findings indicate that telomerase activity in these cell types might be controlled by common pathways. However, we did not find correlations between telomerase activity level and telomere length for any specific cell type, reflecting the fact that telomere length is determined not only by the level of telomerase activity, but also other factors including genetic, cellular and environmental factors.
These are the first quantitative data we are aware of in humans examining telomere length and basal levels of telomerase in subsets of PBMCs from the same individuals. This work will facilitate further understanding of leukocyte aging and human health.
Section snippets
Blood draw and staining protocol
Fasting blood (50 ml) was drawn from healthy participants of an ongoing clinical study. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll–Hypaque (Sigma-Aldrich, St. Louis, MO) gradient centrifugation, washed in PBS (Dulbecco's Phosphate Buffered Saline, PBS without Mg++ and Ca++, Invitrogen-Biosource, Carlsbad, CA) and then incubated for 15 min at 4 °C with fluorescent-conjugated monoclonal antibodies: anti-CD3-PB, anti-CD4-FITC, anti-CD28-PE, anti-CD19 APC-Cy7 (all from BD
Modified gel-TRAP assay to quantitatively measure telomerase activity in resting immune cells
The telomere repeat amplification protocol (TRAP), developed by Kim and Wu (1997), is the most commonly used method to measure human cell telomerase activity. In this method, an oligonucleotide (“oligo”) is used as the substrate for telomere addition by telomerase. This oligo, called TS primer, is first end-labeled by T4 polynucleotide kinase (PNK) to add radioactive 32P onto its 5′ end for signal detection. The labeled primer is then incubated with the extract and dNTPs to allow addition of
Discussion
The study described here is a systematic examination of telomere length and telomerase activity in the four cell types from the adaptive immune system from the same donor, analyzing natural (unstimulated) cells. While a rich body of evidence has linked leukocyte telomere length to aging and aging-related diseases, nearly all studies have used PBMCs or leukocytes for the measurement. A limitation of such studies is therefore that the measurements from PBMCs do not offer any mechanistic
Acknowledgements
We thank the study participants for their support and generous contribution of time. We thank Dr. Richard Cawthon for his technical advice on the telomere length measurement method. Jue Lin and Joshua Cheon were supported by the Bernard and Barbro Foundation. The Gladstone flow core and the Core Immunology Lab were supported by The UCSF-GIVI Center for AIDS Research P30AI027763.
The Core Immunology Lab was also supported by NIH/NCRR UCSF-CTSI grant number UL1 RR024131-01. The contents of this
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