* Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada;
Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, Texas; and
Department of Medicine, University of British Columbia, Vancouver, Canada
Correspondence: Peter M. Lansdorp, Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada. E-mail: plansdor{at}bccancer.bc.ca
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Key Words: telomere length • replicative history • T lymphocytes • B lymphocytes • flow-FISH
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Hematopoiesis and immune responses must be well orchestrated to guarantee stable levels of mature blood cells and adequate defense mechanisms, especially in situations where the steady state is perturbed. How the hierarchy of primitive stem cells, progenitor cells, and mature blood cells is organized and how cell turnover and development of different precursors are regulated in vivo are only partially understood. Some of the pertinent questions are how many times do and can the different blood and immune cells and their precursors divide before terminal differentiation, and how often are limitations in cell proliferation responsible for cytopenia and immune failure. The latter could be especially critical in the elderly, where cell development and differentiation as well as repetitive immune responses have challenged the immune system over a lifetime. Unfortunately, there are no easy methods available to directly study cell turnover over many (e.g., more than 10) cell divisions or over a long period of time in vivo. However, it has become possible to study at least one indirect parameter, telomere length, which to some extent, will reflect cell turnover in somatic cells that express limiting levels of telomerase. Here, we show that by combining such measurements with immunophenotyping, novel insight in the organization of the immune system can be obtained.
Telomeres or the end of chromosomes in vertebrate cells consist of (TTAGGG)n repeats [7 ] and associated proteins [8 , 9 ]. Telomeres protect chromosomes from degradation and fusion [10 , 11 ]. With each cell division, telomeric repeats are lost as a result of the end replication problem [12 13 14 ] and other causes [15 ]. To compensate for the loss of telomeric DNA, selected cells express telomerase [16 , 17 ], a ribonucleoprotein enzyme complex that is able to add new telomeric repeats. Most somatic cells in humans appear to express limiting levels of telomerase and as a result, are unable to maintain telomere length upon multiple rounds of cells division [18 , 19 ]. Telomere shortening has been linked to replicative senescence by experiments showing that overexpression of telomerase can extend the replicative lifespan of somatic cells [20 , 21 ]. These observations, together with studies showing that reductions in telomerase levels in human cells have important consequences for the function of hematopoietic and immune cells [22 ], have greatly increased the interest in studies of telomere length regulation in relation to human aging.
We chose the baboon model to indirectly study the cell turnover of different subtypes of blood cells in vivo. So far, questions about (stem) cell turnover and telomeres have been mainly focused on the mouse. Several recent publications have highlighted differences between laboratory mice and humans in telomere biology [23 24 25 26 27 ] and in the turnover of the cells in the immune system [28 ]. Therefore, extreme caution should be used in extrapolating conclusions regarding cell turnover and telomere length regulation from murine studies to humans.
Baboons feature many similarities to humans, especially with respect to embryonic development [29 ], cell turnover [30 , 31 ], and development of the immune system [32 33 34 ]. Furthermore, telomere biology and cellular aging appear to be highly conserved among primates [35 ]. Together with the possibility of longitudinal and gene marking [36 ] studies, these similarities make nonhuman primates an attractive model system for studies regarding the role of telomeres in human biology and aging.
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Telomere length measurement by fluorescence in situ hybridization and flow cytometry (flow-FISH)
Details of the isolation, in situ hybridization, and analysis on the flow cytometer were described by Baerlocher et al. [37
]. Briefly, peripheral blood nucleated cells were isolated by two steps of ammonium chloride (Cat. #07850, StemCell Technologies Inc., Vancouver, Canada) lysis and were resuspended in phosphate-buffered saline (PBS; Cat. #37350, Stem Cell Technologies Inc.)/0.1% bovine serum albumine (BSA; Cat. #12669, Calbiochem-Novabiochem Corp., San Diego, CA). In situ hybridization was performed on 3 x 105 cells/tube in 300 µl hybridization mixture containing 70% deionized formamide (Cat. #B 10326–80, BDH Inc., Toronto, Ontario, Canada), 20 mM Tris (Cat. #T-1503, Sigma-Aldrich Canada Ltd., Oakville, Ontario), pH 7.1, 1% BSA with no probe (unstained control), or 0.3 µg/ml telomere-specific fluorescent isothiocyanate (FITC)-conjugated (C3TA2)3 peptide nucleic acid (PNA) probe (kindly provided by Boston Probes, Bedford, MA) for stained samples. Denaturation was done at 87°C for 15 min, and hybridization was performed in the dark at room temperature for 90 min. Excess and nonspecifically bound telomere PNA probe was removed at room temperature by 4 x 1 ml washes with wash solution containing 70% formamide, 10 mM Tris, 0.1% BSA, and 0.1% Tween 20 (Cat. #R06435, BDH Inc.) followed by 1 x 1 ml wash at room temperature with a solution containing PBS, 0.1% BSA, and 0.1% Tween 20. For DNA counterstaining, cells were resuspended in a solution containing PBS/0.1% BSA/RNase A (Cat. #109 207, Boehringer Mannheim, Laval, CA) at 10 µg/ml and a subsaturating amount of (0.01 µg/ml) LDS 751 (Exciton Chemical Co. Inc., Dayton, OH) at least 20 min before analysis. The acquisition of telomere fluorescence was performed using a FacsCalibur (Becton Dickinson Biosciences, San Jose, CA). For each sample, duplicates of unstained and telomere-stained samples were tested, and the specific telomere fluorescence was determined as the difference between the fluorescence of a stained sample minus the (auto-) fluorescence of the corresponding unstained or control sample. Calibration beads (Cat. #824p-C, Bang Laboratories Inc., Fishers, IN) were run in each experiment to convert telomere fluorescence values into molecular equivalents of soluble fluorochrome (MESF) units and to perform comparisons from experiment to experiment. We calculated that for baboon cells, 1 kb telomere repeats per chromosome end measured by terminal restriction fragment Southern analysis corresponds to roughly two MESF x 103 (kMESF) units measured by flow cytometry. Gating on single cells and on a subpopulation of cells was done with Cell Quest (Becton Dickinson), WinMDI Version 2.8 (Windows Multiple Document Interface Flow Cytometry Application by Dr. Joseph Trotter, Salk Institute, La Jolla, CA), or FlowJo (Tree Star Inc., San Carlos, CA).
Telomerase assays
Telomerase activity was measured by the telomerase repeat amplification protocol (TRAP) assay as described [38
]. Peripheral blood mononuclear cells obtained by Ficoll Hypaque (Pharmacia, Uppsala, Sweden) density centrifugation were stimulated with 1.0 µg/mL phytohemagglutinin (PHA; Gibco, Grand Island, NY) and 100 U/ml recombinant interleukin-2 (IL-2; Roche, Nutley, NJ). Cell extracts from 2 x 103 viable cells, present 3 and 6 days after stimulation, as well as from a similar number of positive control cell line cells (K562) were used. Extension of the telomere substrate primer by telomerase in the TRAP assay was performed for 30 min at room temperature, and the products generated were amplified by 30 cycles of polymerase chain reaction at 95°C for 60 s, 50°C for 45 s, and 72°C for 60 s using the anchored return primer. Half of the amplified products was resolved on a 12% polyacrylamide gel and was visualized by a phosphoimaging system (Storm 820, Molecular Dynamics Inc., Sunnyvale, CA).
Immunostaining
The samples, which were stained for CD45RA-positive cells, CD45RA-negative cells, and CD20-positive cells, were resuspended in Hanks’ balanced salt solution (HBSS) modified with 10 mM Hepes (Cat. #37150, Stem Cell Technologies Inc.)/5% fetal calf serum (FCS; Hyclone FBC, Logan, UT)/0.1% sodium azide (Cat. #B30111, BDH Inc.) with 10 µg/ml phycoerythrin (PE)-labeled human anti-CD45RA antibody (8d2; ref. [39
]) and with 0.3 µg/ml PE-labeled human anti-CD20 antibody (L26, kindly provided by Dr. Andre van Agthoven, Beckman Coulter Immunotech, Fullerton, CA). Both antibodies were previously found to cross-react with cells from various monkeys [40
, 41
]. In our hands, these two antibody reagents reacted with similar and expected percentages of nucleated human and baboon leukocytes with low forward- and low side-scatter (SSC) properties. Furthermore, both antibodies reacted with similar numbers of cells before and after the flow-FISH protocol [unlike the large majority (>98%) of monoclonal antibodies specific for human cell-surface antigens]. Cells were incubated for 20 min on ice after the last wash step of the flow-FISH protocol. Cells were then washed twice with HBSS/5% FCS/0.1% sodium azide before DNA counterstaining. Analysis of telomere fluorescence on subsets of cells was performed by gating on the specific cell population. Little compensation was necessary among the detectors FL1, FL2, and FL3. However, the compensation between FL1–FL2 was most critical, as any fluorescence from the PE-labeled antibodies could interfere with the quantitation of the telomere–PNA–FITC fluorescence; each sample was therefore compensated individually. With the concentrations used for the telomere–PNA–FITC probe and the PE-labeled antibodies, compensation between FL1 and FL2 was in the range of 5–10%.
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Table 1. Loss of Telomere Fluorescence in Blood Cells with Age
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Figure 1. (A) Decrease of telomere fluorescence (mean±STD, n=2) with age in granulocytes and lymphocytes from peripheral blood of 16 baboons. Note the more pronounced decline in telomere fluorescence early in life for both cell types and the overall higher rate of telomere loss in lymphocytes than in granulocytes. (B) Flow-FISH analysis for one representative baboon of each age group (young, middle-aged, old) at three different time points within a 1-year interval (t0, t2, t9=9 months after to). Shown are dot plots of SCC versus telomere fluorescence (horizontal axis: channel number). Note the changes in telomere fluorescence intensity and distribution in granulocytes (gray dots) and lymphocytes (black dots) with age and over time. The calculated telomere fluorescence for the indicated population is shown (in kMESF) in the panels. Note the heterogenous telomere fluorescence in lymphocytes.
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Figure 2. Telomerase activity in activated T cells decreases with donor age. For these experiments, mononuclear cells from three young (7–8 months) and three old (17–18 years) baboons were stimulated with PHA and IL-2 for 3 and 6 days, respectively, before measurement of telomerase activity by TRAP assay. Telomerase activity was measured in extracts from 2 x 103 viable cells present in the lymphocyte cultures at the indicated days of culture. K562 human erythroleukemic cell line cells (lysate also from 2x103 cells) served as a positive control. Note the much lower of telomerase levels in lysates from old animals at both time points that were analyzed. The bands on the left represent a 10 bp molecular weight ladder. Bands at the bottom of each lane correspond to the internal control of the TRAP assay.
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Figure 3. (A) Immunostaining for B cells, naïve T cells, and memory T cells. Dot plots of SSC versus FL2 show intense staining of B lymphocytes with a PE-labeled anti-CD20 (left plot), less intense staining of the majority of naïve T lymphocytes and B lymphocytes (CD45RA-positive) with a PE-labeled anti-CD45RA (middle plot), and three subsets of lymphocytes (B lymphocytes: dotted circle; naïve T lymphocytes: dashed circle; memory T lymphocytes: lower circle) with a combination of PE-labeled anti-CD20 and CD45RA antibodies (right plot). (B) Flow-FISH analysis of lymphocyte subsets. Dot plots of telomere fluorescence versus SSC (left panel) or FL2 (right panel). The distribution of telomere fluorescence in granulocytes (gray dots) and lymphocytes (black dots) is shown in the left panel. The telomere fluorescence in subsets of lymphocytes (described in Fig. 2A
) for three representative baboons (young, middle-aged, old) is shown in the right panel. Note the heterogeneity of telomere fluorescence in B lymphocytes.
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Figure 4. (A) The distribution of telomere fluorescence values in granulocytes, lymphocytes, and memory T lymphocytes varies with age. Histograms of telomere fluorescence from a representative baboon in each age group show a relative tight distribution for granulocytes (dotted line) and an increasingly skewed distribution for lymphocytes (black line). (B) Box plot histograms of telomere fluorescence values in granulocytes, T lymphocytes, and B lymphocytes. The horizontal line in the box represents the 50th percentile; the upper and lower margins of the box, the 25th and 75th percentile; and the vertical lines above and below the box, the 5th and 95th percentile. Note that the distribution is relatively symmetrical for granulocytes and T lymphocytes over the entire age range but markedly skewed for B lymphocytes in animals above 1.8 years.
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The main findings in this study can be summarized as follows: The overall pattern of telomere length dynamics in leukocytes from baboons is strikingly similar to what has been described previously in humans [46 ]. First, the telomere length in granulocytes and T cells varies considerably between individuals of the same age. Second, the telomere length in these cell types shows a highly significant, overall decline with age that is most notable early in life. Third, the decline in telomere length in lymphocytes is more pronounced in T cells than in B cells, and memory T cells with (very) short telomeres accumulate in old baboons. Fourth, telomerase levels in activated T cells declined with age. Finally, B cells with (very) long telomeres appear in the circulation after the age of 1 year.
Studies in various organisms, including yeast, Tetrahymena, mice, and man have established that many factors and genes have effects on telomere length and telomere function. For example, in yeast, at least 50 genes are known to be involved in telomere length regulation [53 ]. Through these studies, it has become clear that the average telomere length of chromosome ends in a cell is only indirectly related to the overall function of all the telomeres in that cell. Indeed, it has been suggested that in the absence of telomerase, the shortest, not the average, length is more important for cell function [54 ]. It is striking that in baboons, just as in humans, the average telomere length in leukocytes at any given age shows marked variation. Most likely, the average length is only indirectly related to the function of individual telomeres and replicative senescence.
Our limited longitudinal data suggest that differences in telomere length in various leukocytes are relatively stable within individual baboons, in that animals with the longest telomeres in any age group at the onset of the study still had the longest telomeres upon retesting about 1 year later. These findings are compatible with a model, where clonal variations in telomere length between hematopoietic and immune precursor cells are small or evened out by polyclonal production of circulating cells. Further evidence in support of this model is the observation that different cell types display different telomere length kinetics with age but that such differences are similar for individual baboons and resemble the kinetics seen in humans. Assuming a more or less constant rate of telomere loss per cell division [46 ], the pronounced decline in telomere length for all cell types in the first few years of life most likely points to a higher number of cell divisions early in childhood compatible with the development and expansion of the hematopoietic and the immune system. The accelerated telomere shortening in lymphocytes, compared with granulocytes in very young individuals, could reflect a high cell turnover, not only in lymphocyte progenitors but in antigen-specific lymphocytes as well. As was reported in humans [46 ], we observed a higher percentage of naive T cells (with long telomeres) than memory T cells (with shorter telomeres) in young animals and a shift in this ratio in older animals.
In old baboons, memory T cells with a telomere fluorescence value 12–15 kMESF (corresponding to 6–7 kb) seemed to accumulate, and a symmetrical distribution in telomere fluorescence values was no longer observed. These results could indicate that cells with telomeres shorter than 6–7 kb do not exist or do not circulate in baboons. It is possible that at this average telomere length, the shortest telomeres are unable to form a T loop structure, which has been suggested to be essential for telomere end-capping and could be required to prevent activation of cell-cycle exit and apoptosis signals [10 ].
Naïve T cells and memory T cells cannot be distinguished on the basis of a single cell-surface marker, such as CD45RA [55 ]. Nevertheless, the overall median telomere length in CD45RA-positive and CD20-negative cells, which consists mainly of naive T cells but most likely also includes natural killer (NK)/NK–T cells and some CD8+ memory and cytotoxic T cells, was longer than in CD45RA-negative and CD20-negative cells, which consist mainly of memory T cells. The telomere length kinetics in B cells also appear similar to what has been reported in humans [47 , 48 ]. The higher telomere fluorescence values in subsets of B cells in older animals and the constant values in middle age and old baboons are suggestive of telomere elongation and maintenance in vivo. If telomere elongation by telomerase is indeed the mechanism by which B cells avoid replicative exhaustion, it is of interest why similar mechanisms do not extend the lifespan of T cells. The increase in telomere length in B cells of baboons appears to take place during the second year of life. Possibly, this is the time period in baboons when secondary germinal centers in lymphatic tissues develop. In humans, secondary germinal centers seem to exist very soon after birth [56 , 57 ], and we could observe a few human B cells with long telomeres in blood samples from babies in their first year of life (unpublished).
This study highlights, on the one hand, the differences in telomere length regulation in different subsets of hematopoietic cells and conversely describes the similarities in telomere length kinetics in subsets of cells among individuals and primate species. In general, the observations point to cell type-specific differences in telomere maintenance that may impact on immune function and tumor development.
Received July 19, 2002; revised November 5, 2002; accepted November 19, 2002.
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