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. 1999 Jul 19;190(2):157-67.
doi: 10.1084/jem.190.2.157.

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood

N Rufer  1 , T H Brümmendorf, S Kolvraa, C Bischoff, K Christensen, L Wadsworth, M Schulzer, P M Lansdorp
Affiliations

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood

N Rufer et al. J Exp Med. .

Abstract

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

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Figures

Figure 1
Figure 1
Flow FISH analysis of normal human peripheral blood cells. Nucleated cells after lysis of red blood cells from a healthy 32-yr-old donor were analyzed after hybridization with or without FITC-labeled (C3TA2)3 peptide nucleic acid (shaded and open histograms, respectively, in C, D, G, and H). The cells were gated on region 1 (R1) on the basis of propidium iodide (PI) fluorescence and forward light scatter (FSC) as is shown in A. Regions 2 and 3 (R2 and R3) were selected within R1 from forward versus side scatter (SCC) dot plot histograms as is shown in B, E, and F. Results with purified lymphocytes (E and G) and granulocytes (F and H) confirmed the validity of selected light scatter gates R2 and R3 for analysis of unseparated blood cells (B). All parameters were collected on a linear scale. Green fluorescence is expressed in MESF units using calibration beads analyzed at identical instrument settings at the beginning and end of each experiment (data not shown). Note that (i) the fluorescence of gated and purified cells is similar, (ii) granulocytes have a higher autofluorescence than lymphocytes, and (iii) the telomere fluorescence of lymphocytes (C and G) is more heterogeneous than that of granulocytes (D and H), reflecting a more diverse replicative history 22. The telomere fluorescence of cells was calculated by subtracting the mean background fluorescence from the mean fluorescence obtained with the telomere probe as is shown in C, D, G, and H.
Figure 2
Figure 2
Loss of telomere fluorescence in lymphocytes and granulocytes from peripheral blood measured by flow FISH. The specific telomere fluorescence of lymphocytes and granulocytes was analyzed after gating on these cells as is shown in Fig. 1. Note the heterogeneity in telomere fluorescence values, the overall decline in telomere fluorescence in both cell types and the higher rate of telomere decline in lymphocytes. Insets show the results of bisegmented fit analysis for cells in the lymphocyte (top) and granulocyte (bottom) gate. Arrows indicate the optimal intersection of calculated regression lines (see also text and Table ).
Figure 3
Figure 3
Examples of the changes in cell surface phenotype of CD4+ (top) and CD8+ (bottom) T lymphocytes that occur with age. Note that in early childhood the majority of CD4+ T cells have a CD4+ CD45RA+CD45RO− naive phenotype, whereas in older adults most CD4+ T cells have a CD45RA−CD45RO+ memory phenotype. Similar changes occur in CD8+ T cells. Boxed areas indicate windows that were used to sort cells with the indicated phenotype as well as the gates that were used to calculate the percentage of cells with a given phenotype shown in Table .
Figure 4
Figure 4
Linear regression analysis of telomere fluorescence and age in subpopulations of peripheral blood T lymphocytes. Subsets of CD4+ and CD8+ T lymphocytes were purified from donors of the indicated age using the sort windows described in Fig. 3. Note the relatively constant difference in telomere fluorescence between naive and memory CD4+ T lymphocytes after the first few years and the increasing difference in telomere fluorescence between CD8+ naive and memory T lymphocytes.
Figure 5
Figure 5
Rapid loss of telomere fluorescence in T cell subsets in early childhood. The data over the whole age range shown in Fig. 4 were subjected to bisegmented fit analysis. Arrows indicate the age that resulted in a significantly better fit for most cell types than was obtained by linear regression. Note the similar telomere fluorescence in naive and memory CD4+ and CD8+ T lymphocytes at birth and the relative rapid loss of telomere fluorescence particularly in CD4+ memory T lymphocytes.
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