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Keywords:

  • B lymphocyte;
  • telomere length;
  • telomerase;
  • immunosenescence;
  • flow-FISH

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Long telomeres in human B lymphocytes
  6. Telomere length asymmetry in peripheral blood B lymphocytes
  7. Telomere length dynamics and telomerase activity in activated B cells in vitro
  8. Discussion
  9. Acknowledgments
  10. References

Summary. Telomere shortening has been causally linked to replicative senescence in human cells. To characterize telomere-length heterogeneity in peripheral blood cells of normal individuals, we analysed the mean length of telomeric repeat sequences in subpopulations of peripheral blood leucocytes, using fluorescence in situ hybridization and flow cytometry (flow-FISH). Although the telomere length of most haematopoietic subsets was within the same range, the mean telomere length was found to be 15% higher in B compared with T lymphocytes in adult peripheral blood. Whereas telomere loss with ageing corresponded to 33 base pairs (bp) per year in T cells, telomere shortening was slower in B cells, corresponding to 15 bp per year. Separation of adult B-lymphocyte subpopulations based on CD27 expression revealed that telomere length was almost 2 kb longer in CD19+CD27+ (memory) compared with CD19+CD27 (naive) cells. Furthermore, peripheral blood B cells were activated in vitro. Whereas B-cell activation with Staphylococcus aureus Cowan strain (SAC) did not increase telomere length, a striking telomere elongation was observed when cells were stimulated with SAC and interleukin 2 to induce plasma cell differentiation. Our observations support the concept that telomere dynamics in B cells are distinct from other haematopoietic cell lineages and that telomere elongation may play an essential role in the generation of long-term B memory cells.

Age-related deterioration of the immune system is a well-documented phenomenon, and may have an impact on morbidity and mortality in the elderly (Miller, 1996). The decline of the immune function has been attributed to several alterations such as thymic involution, changes in the peripheral T-cell pool, qualitative deficiency of B cells and other factors (Malaguarnera et al, 2001). As repeated expansions of a limited number of antigen-specific lymphocytes are required for an effective immune response over a lifetime, the role of telomere-driven replicative senescence in age-related changes of the immune system is of particular interest (Effros & Pawelec, 1997). Indeed, the exhaustion of lymphocyte subpopulations such as CD8+ T cells has been suggested to contribute to immunodeficiency and chronic infections in ageing individuals (Effros et al, 1996; Fagnoni et al, 2000).

Human telomeres are the molecular caps of chromosomes and are composed of repetitive TTAGGG repeats and associated proteins (Collins, 2000). Telomeres stabilize and protect chromosome ends from end-to-end fusions, recombination and degradation. The maintenance of telomeres throughout many cycles of cell division requires the enzyme telomerase which consists at least of a constitutively expressed RNA subunit, hTR, containing the template and a catalytic protein subunit, hTERT (Mergny et al, 2002). In general, telomerase activity is absent in most somatic human cells, and shortening of telomeres has been observed in dividing cells and with ageing (Meyerson, 2000). Loss of telomere repeats beyond a certain threshold has been causally linked to cell cycle exit or replicative senescence in human cells (Allsopp & Harley, 1995; Bodnar et al, 1998; Martens et al, 2000). In contrast, the vast majority of tumour and germline cells circumvent replicative senescence by expression of high levels of telomerase activity, which leads to stabilization of telomeres and acquisition of an immortal phenotype (Kim et al, 1994; de Lange, 1994).

It has been hypothesized that the marked decline in reactivity of the immune system in the elderly may be partially due to telomere-driven exhaustion of lymphocyte proliferation. Studies in mice that are genetically deficient for telomerase activity have demonstrated that telomere instability with successive generations coincides with a decreased reactivity of the immune system (Lee et al, 1998; Herrera et al, 1999, 2000). In addition, recent findings indicate that Dyskeratosis congenita (DKC), which is characterized by anaemia, immune deficiency, skin and nail lesions, chromosomal instability and cancer, reflects a human phenotype with diminished telomerase activity and progressive telomere shortening, accumulated over development and adult life (Mitchell et al, 1999). Interestingly, the RNA component of telomerase, hTR, has been shown to contain mutations in affected individuals (Vulliamy et al, 2001).

Numerous studies have documented that telomeric sequences are lost in fibroblasts and in peripheral blood cells in vitro (Harley et al, 1990; Engelhardt et al, 1997; Rufer et al, 1998; Martens et al, 2000) and in vivo (Harley et al, 1990; Vaziri et al, 1993; Slagboom et al, 1994; Allsopp et al, 1995). Although initial studies suggested that telomere decline appears to be gradual as a function of age, more recent analysis has revealed that telomere dynamics, particularly in blood cells, are more complex, which limits the interpretation of tracing the replicative history of cells based on their telomere length (Hodes, 1999). Despite the ability of lymphocyte and stem cell subpopulations to upregulate telomerase upon activation, the activity level appears to be insufficient to compensate for telomere shortening (Vaziri et al, 1994; Engelhardt et al, 1997; Rufer et al, 1998). Telomere loss seems to be accelerated in stem cells and memory T cells during early childhood (Frenck et al, 1998; Rufer et al, 1999). In T lymphocytes, telomere shortening occurs along with differentiation from naive to memory cells (Weng et al, 1995). In contrast, activated B cells appear to prevent telomere shortening by expressing high levels of telomerase (Norrback et al, 1996, 2001; Weng et al, 1997; Batliwalla et al, 2001). Furthermore, telomere loss with ageing is slower in B cells relative to T cells (Son et al, 2000). A recent study revealed that no significant telomere shortening occurred in antigen-specific T cells during acute infection and up to 1 year thereafter (Plunkett et al, 2001). However, at later timepoints, significant telomere shortening was observed, suggesting that transient telomerase activation may postpone but not completely prevent telomere shortening.

In this study, we showed that telomere length in peripheral blood B cells of adults is substantially longer than in T cells. Furthermore, using sensitive flow-FISH analysis, we showed telomere-length heterogeneity among peripheral B cells based on their differentiation state and, finally, we provided evidence that telomere length can increase upon activation in vitro.

Purification of lymphocyte subpopulations.  For this study, 30–40 ml EDTA blood was obtained from healthy volunteers after informed consent. Leftover buffy-coat preparations from the Department of Transfusion Medicine were used for the enrichment of B-lymphocyte subpopulations. Cord blood samples were provided from the Freiburg cord blood-banking programme scheduled for discard, according to procedures approved by the institutional review board of the Freiburg University Hospital, when the cell number was below a certain threshold. Leucocytes were obtained after osmotic lysis of red cells using 0·84% ammonium chloride (Merck, Darmstadt, Germany). Peripheral blood mononuclear cells (PBMC) were enriched following Ficoll–Hypaque (Biochrom, Berlin, Germany) density centrifugation from EDTA blood samples from healthy volunteers. To isolate cell subpopulations, magnetic-activated cell-sorting (MACS) cell enrichment kits (Miltenyi Biotec, Bergisch Gladbach, Germany) were used, according to the manufacturer's instructions, with following anti-beads: CD3, CD4, CD8, CD14, CD19 and CD56. Enriched B lymphocytes were further purified into CD27+ or CD27 fractions using a MoFlo® MLS flow cytometer (Cytomation, Fort Collins, CO, USA) equipped with a 480-nm Argon laser. Pre-enriched CD19+ cells were stained with CD27-PE monoclonal antibodies (mAb) (Becton Dickinson, Heidelberg, Germany). The purity of enriched cell populations was analysed using a FACSCalibur (Becton Dickinson) cytometer using the following mAb: CD3-phycoerythrin (PE), CD4-PE, CD8-PE, CD14-PE, CD15-PE, CD20-PE, BB4-fluorescein isothiocyanate (FITC), CD56-PE and CD38-PE (Becton Dickinson).

Telomere length analysis.  The average length of telomere repeats at the chromosome ends of individual cells was measured by flow-FISH (Rufer et al, 1998) with minor modifications; in contrast to the original protocol, the denaturation temperature was increased to 85°C. Telomere length was expressed in telomere fluorescence units (TFUTRF) based on calibration experiments using TRF length analysis by Southern blotting (n = 10). Mean telomere fluorescence was shown to be highly proportional to TRF length (r = 0·84; P < 0·0001). The resulting slope in our fluorescence-activated cell sorter (FACS) setting was

  • image

indicating that a fluorescence intensity value of 0 corresponded to 3·78 kb in the Southern blot. This finding was similar to a previously reported calibration set-up (Hultdin et al, 1998) and may primarily reflect the fact that the used telomere PNA probe does not bind to subtelomeric repeat sequences (Martens et al, 1998), which appear to be in the range of 2–4 kb in length (de Lange, 1995). Intra-experimental variation of our flow-FISH procedure was found to be on average 2·1% (data not shown). To compare results between different experiments, aliquots of previously frozen mononuclear blood leucocytes were included in each experiment in duplicate. The average fluorescence intensity of these control cell samples was used as a correction factor for other samples. Thereby, interexperimental variation was reduced from 14% to 3·2% (data not shown).

Q-FISH analysis was performed as described previously (Lansdorp et al, 1996; Martens et al, 1998) with some modifications of the equipment. Digital images of metaphase spreads were recorded with a digital camera (Sensys; Photometrics, Tucson, AZ, USA) on an Axioplan II fluorescence microscope (Zeiss, Jena, Germany), using the Vysis work station QUIPS (Vysis, Downers Grove, IL, USA). Telomere profiles were analysed with tfl-telo software (Poon et al, 1999). Telomere length values were expressed in arbitrary units as IOD (Integrated Optical Density). Q-FISH analysis was performed on 10–15 metaphase spreads.

Mean telomeric restriction fragment (TRF) length was analysed by Southern analysis as described previously (Engelhardt et al, 2000).

Telomerase activity.  Telomerase activity was measured using the telomeric repeat amplification protocol enzyme-linked immunosorbent assay (TRAP-ELISAPlus Kit; Roche Diagnostics, Penzberg, Germany), according to the manufacturer's recommendations. Briefly, 2 × 105 cells per sample were lysed in 200 µl CHAPS-buffer {3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate}, and 0·5 µg of protein supernatant was used for polymerase chain reaction (PCR) in a total volume of 50 µl. Telomerase activity was described in a semiquantitative manner as ‘relative telomerase activity’ (RTA), relating the ELISA signal of the sample to the one obtained by a control template with a known number of telomeric repeats. Frozen aliquots of Phoenix ampho cells, which is a second-generation retrovirus producer line based on the 293T cell line (http://www.stanford.edu/group/nolan/retroviral_systems/phx.html), were analysed with each test as additional positive control. Telomerase activity was expressed as percentage of the RTA of B cells relative to the RTA of phoenix ampho cells.

Stimulation of B lymphocytes in vitro.  Freshly isolated CD19+ cells were suspended in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% fetal bovine serum (Gibco BRL Life Technologies, Germany). B cells were activated with 0·2 µg/ml of Staphylococcus aureus Cowan strain (SAC; Pansorbin; Calbiochem, Bad Soden, Germany) with or without 20 U/ml interleukin 2 (IL-2) (Chiron Behring, Marburg, Germany) at a density of 0·375–1 × 106 cells/ml. In order to specifically suppress proliferation of remaining T cells, enriched B cells were additionally cultured in the presence of 1 µg/ml Cyclosporin A (Novartis Pharma, Nurnberg, Germany).

Statistical analysis.  Data analysis was performed using microsoft excel and microcal origin software. Student's t-test was used to determine statistical significance.

Long telomeres in human B lymphocytes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Long telomeres in human B lymphocytes
  6. Telomere length asymmetry in peripheral blood B lymphocytes
  7. Telomere length dynamics and telomerase activity in activated B cells in vitro
  8. Discussion
  9. Acknowledgments
  10. References

In order to assess telomere length heterogeneity in subsets of peripheral blood leucocytes, we analysed the mean telomere length in enriched subpopulations of mononuclear cells from a healthy 23-year-old individual, using flow-FISH. Telomere length was comparable in most subpopulations such as mononuclear (MNC), CD3+, CD4+, CD8+ cells and monocytes, respectively, and was slightly lower in natural killer (NK) cells (Fig 1A). However, in CD19+ B cells, telomere length was found to be strikingly increased by 2·1 kb compared with the MNC of this individual. Using Southern blotting, we analysed the mean telomere restriction fragment size (TRF) in mononuclear and B cells from a different individual (24 years old), which confirmed that the telomeres in B cells were substantially longer than in MNC (difference: 3·3 kb; Fig 1B). To survey this finding on more samples, we measured telomere length using flow-FISH in T and B cells from 36 healthy individuals with ages ranging from 12 to 92 years (median = 49 years) (Fig 1C). The telomere length of each individual was calculated by averaging TFUTRF of duplicate measurements. Mean telomere length was found to be on average 1·1 kb higher in the B cell compartment relative to T cells (mean ± SD: 8·5 ± 0·7 kb vs 7·4 ± 0·6 kb; P < 0·0001). We never observed shorter telomeres in B compared with T cells. However, this striking difference was not detected in lymphocytes from cord blood (n = 15). Here, telomeres were slightly longer in T cells, but without reaching statistical significance (mean ± SD: 9·4 ± 0·9 kb vs 9·1 ± 0·7 kb; P = 0·35).

image

Figure 1. Long telomeres in human B lymphocytes. (A) Mean telomere length analysis in leucocyte subpopulations from a healthy individual (23 years, ID 1) using flow-FISH. Telomere length was calculated by averaging TFUTRF values of duplicate measurements. MNC, 7·3 kb; T lymphocytes (CD3), 7·3 kb; helper lymphocytes (CD4), 7·3 kb; cytotoxic lymphocytes (CD8), 7·4 kb; monocytes (CD8), 7·3 kb; B lymphocytes (CD19), 9·4 kb; natural killer cells (CD56), 6·9 kb. (B) TRF size analysis of PBLs: (1) MNC from a healthy individual (24 years, ID 3), 7·9 kb; (2) MNC from a healthy individual (25 years, ID 4), 8·4 kb; (3) CD19+ B lymphocytes from donor ID 3, 11·2 kb. (C) Comparison of telomere length analysis in T and cells from adults (n = 36) and cord blood (n = 15) by flow-FISH.

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In order to determine telomere length heterogeneity in T and B lymphocytes as a function of age, a linear regression analysis was performed (Fig 2). The rate of telomere loss per year was 33 ± 3 bp in T cells (r =−0·80; P < 0·0001), whereas telomere shortening was clearly slower in B cells corresponding to 15 ± 3 bp per year (r =−0·55; P <0·0001).

image

Figure 2. Differential telomere shortening in T and B cells with ageing. The rates of telomere shortening are 33 ± 3 bp and 15 ± 3 bp/year for T (black diamond) and B (shaded circle) cells respectively (n = 51). Individual telomere length values represent mean values of duplicate measurements.

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Telomere length asymmetry in peripheral blood B lymphocytes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Long telomeres in human B lymphocytes
  6. Telomere length asymmetry in peripheral blood B lymphocytes
  7. Telomere length dynamics and telomerase activity in activated B cells in vitro
  8. Discussion
  9. Acknowledgments
  10. References

The advantage of telomere length analysis using flow-FISH was the ability to detect major intercellular telomere length differences based on the histogram shape of analysed cells. Interestingly, there was a homogeneous histogram pattern of telomere fluorescence in T cells, whereas telomere fluorescence distribution in B cells was asymmetrical, suggesting an additional subpopulation of B cells with longer telomeres (Fig 3). We detected this phenomenon only in the CD19+ cells of adult PBLs, but never in B cells of cord blood. As this striking pattern was found consistently and the purity of enriched cells was high (mean purity ± SD in T cells: 94% ± 13; B cells: 88% ± 18), it is unlikely to be the result of cell contamination or an artefact due to the FACS measurement. In order to unravel this intercellular telomere length heterogeneity, we enriched for naive and memory B cells based on the expression of the CD27 surface marker (Agematsu et al, 2000). The purity of total CD19+, CD19+27+ (memory) and CD19+27 (naive) cells (n = 3) was (mean ± SD): 99 ± 0%, 85 ± 5·6% and 99 ± 0·4% respectively. Interestingly, a marked difference in telomere length was found for the naive and memory B-cell population, reaching almost 2 kb (Table I, Fig 4A). Furthermore, we found that the individual histograms of CD27 subpopulations reflected the asymmetric fluorescence pattern of adult B lymphocytes (Fig 4B); the narrow peak represented the CD27-negative cells, whereas CD27-positive cells were displayed as a minor fraction with longer telomeres corresponding to the tail of histograms in CD19+ B cells. Typically, about 40% of peripheral blood B cells were compromised of memory B cells expressing CD27 which appeared to be in accordance with our observed heterogeneous telomere length distribution (Klein et al, 1998). In contrast, CD27+ B cells have been reported to be almost absent in cord blood, which is devoid of memory B cells (Agematsu et al, 1997).

image

Figure 3. Asymmetrical telomere profile in adult peripheral B lymphocytes. Representative telomere fluorescence histograms of CD3+ T and CD19+ B cells measured by flow-FISH are shown which demonstrate the asymmetrical telomere histogram pattern observed only in adult B cells indicative for a minor B-cell subpopulation with longer telomeres. Fluorescence intensity was expressed in original FACS channels.

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Table I.  Telomere length analysis of B lymphocyte subsets.
Donor numberAgeMNCCD3+CD19+CD19+27+CD19+27
  • *

    Individual telomere length values represent mean values of duplicate measurements.

  • †Values represent mean TFUTRF and standard deviation.

  • ‡The difference between CD27+ and CD27 B cells was significant (P < 0·0001, Student's t-test).

ID10237·6*7·69·310·28·4
ID11207·77·58·6 9·98·0
ID12277·17·38·4 9·87·8
Mean ± SD 7·5 ± 0·37·5 ± 0·18·8 ± 0·4 9·9 ± 0·28·1 ± 0·3
image

Figure 4. Telomere length heterogeneity in peripheral B-cell subpopulations. Telomere length analysis of CD27+ (memory) and CD27 (naive) B cells. (A) Summary of TFUTRF values from three buffy-coat samples (ID10, 11 and 12 respectively) showing mean and standard deviation (see also Table I). Individual telomere length values represent mean values of duplicate measurements. (B) Telomere fluorescence histogram of one individual sample for CD19+, CD19+27+ and CD19+27 cells respectively.

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Telomere length dynamics and telomerase activity in activated B cells in vitro

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Long telomeres in human B lymphocytes
  6. Telomere length asymmetry in peripheral blood B lymphocytes
  7. Telomere length dynamics and telomerase activity in activated B cells in vitro
  8. Discussion
  9. Acknowledgments
  10. References

In order to analyse telomere length dynamics in B cells after activation in vitro, freshly isolated CD19+ cells from five buffy-coat samples (ID 55–59) were set up in a short-term culture (5–10 d), using SAC treatment, which has been shown to be a potent polyclonal antigenic stimulator of B cells. Alternatively, we stimulated B cells with a combination of SAC and IL-2, which has also been reported to induce differentiation towards the plasma cell lineage (Falkoff et al, 1982; Nakagawa et al, 1985). Telomere length was analysed by d 0 and d 5 of the cell culture. As indicated in Table II, telomere length was increased by 520 bp upon stimulation with SAC alone (mean ± SD TFUTRF d 0: 8·9 ± 0·3 kb, d 5: 9·5 ± 0·5 kb respectively; P = 0·064). When the same cells were stimulated with SAC and IL-2, a considerable increase in mean telomere length of 1220 bp could be observed (mean ± SD TFUTRF d 0: 8·9 ± 0·4 kb, d 5: 10·2 ± 1·0 kb respectively; P = 0·022). In order to control that the observed difference in telomere length kinetics was not due to any technical limitations related to the flow-FISH method, we also determined telomere length on selected samples by alternative techniques such as Southern blot analysis (n = 2) and Q-FISH (n = 1) when enough cells were available. Consistent with flow-FISH measurements, TRF and Q-FISH analysis revealed that telomere elongation was more pronounced in B cells stimulated with IL-2 and SAC relative to SAC alone (data not shown).

Table II.  Telomere length and telomerase activity in activated B lymphocytes in vitro.
Donor numberDay 0SAC (day 5)SAC + IL-2 (day 5)
Telomere length (kb)Telomere lengthening (kb)Telomere length (kb)Telomerase activity (%)*Telomere lengthening (kb)Telomere length (kb)Telomerase activity (%)*
  • *Relative telomerase activity (RTA) was measured using the TRAP-ELISAplus kit, containing a control template with a known number of telomeric repeats. As additional positive control, the phoenix ampho cell line was used which is a derivative of the 293T cell line. Telomerase activity was expressed as percentage of the RTA of B cells relative to the RTA of phoenix ampho cells. Telomerase activity was typically absent in non-activated B cells and was upregulated by d 5 irrespective of the activators used.

  • The difference in telomere length between d 0 and d 5 was not significant (Student's t-test: P = 0·067).

  • The difference in telomere length between d 0 and d 5 was significant (Student's t-test: P = 0·0022).

ID 559·1+ 1·110·2N.D.+ 2·011·1N.D.
ID 568·5+ 0·6 9·114+ 1·3 9·817
ID 579·2+ 0·4 9·618+ 2·111·317
ID 589·1+ 0·2 9·319+ 0·4 9·527
ID 598·9+ 0·3 9·138+ 0·3 9·225
Mean ± SD8·9 ± 0·3+ 0·5 ± 0·4 9·5 ± 0·522·3 ± 10·7+ 1·2 ± 0·910·2 + 1·021·5 ± 5·3

Unstimulated B cells from peripheral blood and tonsils have been reported to express low or undetectable levels of telomerase (Broccoli et al, 1995; Hiyama et al, 1995; Norrback et al, 2001). Similarly, we also found that telomerase activity was absent in unstimulated B cells from peripheral blood (data not shown). In order to investigate whether telomere lengthening in SAC and SAC plus IL-2-activated B cells was accompanied by telomerase activation, we measured telomerase activity at d 5 of the short-term culture. Upon stimulation, we observed moderate telomerase activity, which was in the range of 20% relative to a transformed control cell line (Table II). Interestingly, the level of telomerase activity was not significantly different in SAC and SAC plus IL-2-activated B cells, although telomere elongation was more pronounced upon the latter treatment.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Long telomeres in human B lymphocytes
  6. Telomere length asymmetry in peripheral blood B lymphocytes
  7. Telomere length dynamics and telomerase activity in activated B cells in vitro
  8. Discussion
  9. Acknowledgments
  10. References

Loss of telomeric sequences has been shown to be a general phenomenon in dividing human somatic cells. In this study, we have shown that there are fundamental differences in the telomere dynamics of B cells compared with other haematopoietic cells. First, we found that telomeres in B cells were about 1 kb longer compared with those in T cells from adult peripheral blood. Secondly, the telomere profile in adult peripheral B cells is strikingly heterogeneous based on the differentiation state. Thirdly, telomere length appears to be increased during B-cell activation and differentiation in vitro.

A number of studies have addressed telomere length analysis in subsets of the haematopoietic system in order to trace the replication history of cell lineages (Vaziri et al, 1994; Weng et al, 1995, 1997; Yamada et al, 1995; Monteiro et al, 1996; Rufer et al, 1998, 1999; Burns et al, 2000). A recently published study, surveying telomere length with flow-FISH in CD4+, CD8+ and CD19+ cells of a large number of individuals, reported that telomeres were longest in CD4+ T cells (7·6 kb), followed by CD19+ B cells (7·1 kb) with CD8+ T cells being the shortest (6·3 kb) (Son et al, 2000). These findings are in contrast to our observations, which show that adult B cells have substantially longer (∼1 kb) telomeres than adult T cells. Although the study by Son et al (2000) was done on a larger number of individuals (n = 121) reflecting a broader age range, subanalysis of their data comparing the same age groups did not reveal strikingly longer telomeres in B cells relative to pan T cells, as our study did. We believe that variation in performance of the flow-FISH method is responsible for the discrepancy of results, because we used a higher denaturation temperature (85°C instead of 80°C), which in our hands appeared to be critical for optimal hybridization performance. A similar observation has been published recently in a technical report (Baerlocher et al, 2002). In addition, we assessed telomere length also in a proportion of samples by TRF analysis and Q-FISH, which confirmed that telomeres are substantially longer in B relative to T cells. Interestingly, the difference in telomere length of B and T cells was not found in cord blood samples. The telomere length of cord blood T cells was on average much higher than of those in adult T cells (difference: 1·9 kb; P < 0·0001), indicating that there is a decline in telomere length in T lymphocytes relative to B cells, depending on the ontogenetic state. Similarily, we found that telomere shortening occurred differentially with ageing. While T-cell telomere loss was found to be on average 33 bp/year, the decline of telomeres in B cells was less than half that number at 15 bp/year. These results are in accordance with other studies correlating telomere length with age in normal individuals (Vaziri et al, 1993, 1994; Rufer et al, 1999; Son et al, 2000). Importantly, we could confirm the slow rate of telomere loss in B cells found by Son et al (2000), which supports the concept that telomere dynamics is distinct in B cells compared with other lineages.

We observed a marked heterogeneity in the telomere length in adult peripheral B cells. Although the results of the flow-FISH technique are very sensitive to minor changes in experimental conditions and require careful control experiments, this finding demonstrates one of the advantages of this technique with regard to the analysis of haematopoietic cells. Specifically, telomere length heterogenity in adult B cells had only been detected using Southern blotting when B-cell subpopulations were analysed but not on bulk B-cell populations. Preselection of naive and memory B cells revealed that the observed asymmetric telomere profile derives from telomere length heterogeneity within these subpopulations. Ideally, the combination of immunfluorescence and flow-FISH could circumvent preselection of cells, but this approach adds another layer of technical difficulty to the procedure and has been demonstrated in only a few reports (Batliwalla et al, 2001; Norrback et al, 2001; Plunkett et al, 2001).

The difference in telomere length between naive and memory B cells was strikingly high, at an average 1·8 kb. In contrast to T lymphocytes, in which telomere shortening occurs in the process of somatic cell differentiation and cell division, telomere lengthening was reported during differentiation of B cells in germinal centres (GC) of lymphoid organs (Weng et al, 1997; Norrback et al, 2001). Although a significant transient increase of 800 bp was reported for GC cells, no significant difference was found between telomere length of naive and memory B cells, suggesting that a significant shortening occurred in the transition from GC to memory B cells. The obvious difference to our results might be due to the following aspects: first, the origin of B cells was different. Weng et al (1997) used lymphoid organs (tonsils), whereas our study focused on B cells from peripheral blood cells. However, to our knowledge, there are no major phenotypic differences between circulating and homing memory B cells. Thus, it appears unlikely that such a striking difference would be observed in telomere length between tonsil and peripheral B cells. Second, the enrichment strategy using monoclonal antibodies was different. Whereas Weng et al (1997) enriched memory B cells with a combination of IgD, CD38 and CD44, we used the CD27 antibody. The CD27 antigen has been reported to be a useful marker for detection of somatically mutated (memory) B cells which compromise about 40% of peripheral blood B cells (Agematsu et al, 1997; Klein et al, 1998). Within the CD27+ B-cell pool, further heterogenity of subsets has been described which is based on the expression of immunoglobulin surface markers. Possibly, the relatively broad telomere histogram of CD19+CD27+ cells may reflect additional telomere length heterogeneity within the CD27+ memory B-cell pool. Finally, the difference in telomere length estimation between memory and naive B cells might be due to the different techniques that were used [flow-FISH in our study, Southern blotting by Weng et al (1997)]. Interestingly, in a recent report, a significant telomere lengthening in the range of 4 kb was observed in purified tonsil B-cell subsets during the GC reaction (Norrback et al, 2001).

An interesting aspect of our studies was the finding of telomere elongation upon B-cell activation. Although we cannot rule out that selection of B cells with initially long telomeres may occur upon proliferation, there was no evidence that CD27-positive cells with particularly long telomeres were enriched during in vitro proliferation (data not shown). The humoral immune response which occurs predominantly within GC includes a variety of changes in response to antigen-driven activation, many of which contribute to the ability to develop a more potent and specific response to antigenic rechallenge (Rudin & Thompson, 1998). It has been suggested that telomerase elongates telomeres during GC formation, facilitating the clonal expansion that occurs in the GC to form the memory B-cell pool (Weng et al, 1997; Norrback et al, 2001). In addition, studies in mTR–/– mice, which lack the mouse telomerase RNA, have supported the concept that telomere maintenance in B cells is mediated by telomerase activation (Herrera et al, 2000). In humans, telomerase activity is absent or low in the majority of resting B cells (Hiyama et al, 1995; Hideya & Sakaguchi, 1997; Hu et al, 1997; Weng et al, 1997; Son et al, 2000). Different activators have been described which upregulate telomerase upon an immune response in vitro. Specifically, SAC alone and in combination with IL-2 have been reported to be the most potent stimulators of telomerase activity and cell proliferation (Hu et al, 1997; Weng et al, 1997). In our study, the levels of telomerase activity observed in activated B cells were similar irrespective of the stimulators used, although the outcome of telomere elongation was different. Therefore, it is possible that additional factors influence telomerase-induced telomere elongation in B cells which might help to recruit telomerase to the telomere or increase the ability of the enzyme to facilitate the extensive lengthening (Grandin et al, 2000; Baumann & Cech, 2001; Peterson et al, 2001). Interestingly, the DNA end-binding protein, ku, which is involved in telomere length homeostasis and DNA double-strand-break (DSB) repair in yeast (Boulton & Jackson, 1998), has also been reported to be required for immunoglobulin class switching during an immune response (Casellas et al, 1998). In fact, class switch recombination appears to be induced upon activation of naive B cells from peripheral blood in vitro (Nagumo et al, 2002). Therefore, it is tempting to speculate that an overlap between the molecular machinery that mediates DNA DSB repair, telomere maintenance and isotype switching could be responsible for the distinct telomere kinetics observed in human B lymphocytes.

In summary, this study extends our knowledge about telomere length homeostasis in human B lymphocytes, which is different compared with T lymphocytes and other haematopoietic cell lineages. Telomere maintenance in B cells might be a necessary step to generate long-term memory B cells (Weng et al, 1998) which appear to persist over extended periods of time independent of persisting immunizing antigen (Maruyama et al, 2000). Finally, the distinct kinetics of telomeres in B cells may help to unravel the mechanisms involved in telomere length regulation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Long telomeres in human B lymphocytes
  6. Telomere length asymmetry in peripheral blood B lymphocytes
  7. Telomere length dynamics and telomerase activity in activated B cells in vitro
  8. Discussion
  9. Acknowledgments
  10. References

We gratefully acknowledge the excellent technical assistance from I. Skatulla. We also thank Dr A. Dwenger and Professor Ch. Niemeyer for providing (cord) blood samples, and Drs M. Schlesier, D. Behringer, F. Gärtner and M. Ohlke for their advice in experimental procedures and for helpful comments. Explicitly, we thank Professor R. Mertelsmann for his continuous support. This work was supported by grants from the Deutsche José Carreras Leukaemie-Stiftung e.V, the Deutsche Forschungsgemeinschaft (SFB 364) and from the European Union (QLG1-CT-1999-01341).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Long telomeres in human B lymphocytes
  6. Telomere length asymmetry in peripheral blood B lymphocytes
  7. Telomere length dynamics and telomerase activity in activated B cells in vitro
  8. Discussion
  9. Acknowledgments
  10. References
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