Telomere length dynamics and telomerase activity in human peripheral blood lymphocytes

The progressive shortening of telomeres the protective nucleoprotein caps at chromosome ends is one of the most consistent molecular markers of human aging, yet the pace and pattern of this shortening vary substantially across lymphocyte subsets. This research measured telomere length and telomerase activity in sorted peripheral blood lymphocyte populations from 144 healthy Saudi Arabian adults aged 20-78 years. Blood samples were collected at King Abdulaziz Medical City, Jeddah, between March 2021 and December 2022, and processed at the Red Sea Institute of Marine Biology. Telomere length was assessed by quantitative fluorescence in situ hybridization (Q-FISH) on metaphase spreads, while telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) assay. CD8+ T cells showed the fastest rate of telomere attrition with age, losing an average of 68 base pairs per year (R² = 0.74, p

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CD4+ T and CD8+ T and more: 20-29 yr vs 30-39 yr vs 40-49 yr vs 50-59 yr vs 60-69 yrClose ×

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CD4+ T and CD8+ T and more: Unstimulated vs PHA-stimulated (72h)Close ×

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Introduction

The immune system's ability to mount effective responses depends on the proliferative capacity of its lymphocyte populations and that capacity has a built-in expiration date. Each round of cell division shortens the telomeric repeats (TTAGGG)n at chromosome ends by 50-200 base pairs due to the end-replication problem [1] . Once telomeres reach a critical minimum length typically around 4-5 kb in human lymphocytes cells enter replicative senescence or undergo apoptosis [2] . This means that the most actively dividing lymphocyte populations are the ones most likely to exhaust their proliferative potential, with direct consequences for immune function in aging and chronic infection. Telomerase, a reverse transcriptase that adds telomeric repeats to chromosome ends, can partially offset this loss. Lymphocytes are among the few somatic cell types that upregulate telomerase upon activation, giving them a temporary buffer against replicative exhaustion [3] . But this upregulation is incomplete it slows telomere shortening without fully preventing it and the capacity to induce telomerase appears to diminish with age [4] . The balance between telomere attrition and telomerase compensation therefore shapes the functional lifespan of each lymphocyte compartment. Previous research has established that telomere length varies among lymphocyte subsets, with naive T cells generally maintaining longer telomeres than memory and effector populations [5] . CD8+ T cells, which undergo extensive clonal expansion during viral infections, tend to show shorter telomeres than CD4+ T cells at any given age [6] . B cells and NK cells have received less attention, and most published data come from European or East Asian populations. Whether these patterns hold in Middle Eastern populations where genetic background, environmental factors, and pathogen exposure history differ is not well established [7] . The practical relevance of telomere biology in lymphocytes extends beyond basic science. Shortened lymphocyte telomeres have been associated with increased susceptibility to infections, poor vaccine responses, and higher mortality from cardiovascular disease and certain cancers [8,9] . Understanding which lymphocyte subsets are most affected by age-related telomere loss and how telomerase responsiveness changes across the lifespan could inform strategies for immune monitoring and intervention. This research measured telomere length by Q-FISH and telomerase activity by TRAP assay across four sorted lymphocyte populations (CD4+ T, CD8+ T, B, and NK cells) in 144 healthy adults spanning six decades of age. The work was carried out at the Red Sea Institute of Marine Biology, Jeddah, Saudi Arabia, with the goal of mapping subset-specific telomere dynamics in a population that has been underrepresented in telomere research [10] .

Reagents and chemicals

Reagents and chemicals

Methods

PBMCs were stained with the fluorochrome-conjugated antibody panel and sorted into CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), B cells (CD19+), and NK cells (CD3-CD56+) on the FACSAria III. For Q-FISH, sorted cells were cultured briefly with PHA (10 μg/mL) and colcemid (0.1 μg/mL) to obtain metaphase spreads, then hybridized with the PNA telomere probe following the manufacturer's protocol. Telomere fluorescence units (TFU) were converted to kilobases using a regression calibration against cell lines with known telomere lengths (CCRF-CEM and 1301 cell lines) [11] . For TRAP assays, sorted cells were divided into unstimulated and PHA-stimulated (10 μg/mL, 72 hours) aliquots. Protein extracts (200 cells/μL equivalent) were assayed using the TRAPeze XL kit according to the manufacturer's instructions. Relative telomerase activity (RTA) was expressed in arbitrary units normalized to the positive control cell line included in the kit [12] . Linear regression was used to model the relationship between age and telomere length for each subset. ANOVA with Tukey's post-hoc test compared subsets within age groups. All statistics were computed in R version 4.2.2 (α = 0.05).

Results

Results

Discussion

The telomere attrition rate of 68 bp/yr observed for CD8+ T cells in this Saudi cohort is consistent with published values from European studies, which generally report 50-80 bp/yr for this subset [6,13] . The slightly higher rate compared to some Western reports may reflect differences in chronic antigen exposure particularly viral burden or could be a sampling artifact given the relatively modest cohort size. That said, the internal ranking of attrition rates (CD8+ T > NK > CD4+ T > B cells) matches the expected pattern based on proliferative demand [5] .

The telomerase data add a mechanistic layer to the telomere length observations. B cells showed the highest stimulated activity (22.6 RTA units) but didn't have the slowest attrition rate suggesting that telomerase upregulation during activation partially but incompletely compensates for the proliferative expansion that B cells undergo during humoral immune responses [14] . NK cells, with the lowest stimulated activity (8.9 units), may rely more on turnover from progenitor populations than on self-renewal of mature cells [15] . The age-related decline in telomerase responsiveness across all subsets is perhaps the most clinically relevant finding, as it indicates that older adults lose not only telomere length but also the compensatory mechanism that slows its loss. It's worth noting that Q-FISH measures telomere length at the single-chromosome level on metaphase spreads, which requires cells capable of reaching mitosis. This may introduce a subtle selection bias toward cells with longer telomeres the very shortest-telomere cells may be senescent and unable to divide, thus escaping detection. Southern blot or qPCR-based methods avoid this bias but lack the singlecell resolution of Q-FISH [16] . Future work could combine both approaches for a more complete picture. A practical implication of these findings is that CD8+ T cell telomere length may serve as a useful biomarker for immunological aging in clinical settings it declines predictably, shows the widest inter-age-group separation, and reflects the functional capacity of a clinically important immune compartment [17] .

Conclusion

This research provides a detailed map of telomere dynamics across four sorted lymphocyte populations in 144 healthy Saudi adults spanning six decades of age. CD8+ T cells showed the steepest telomere attrition rate (68 bp/yr), the shortest absolute telomere lengths at every age, and lower telomerase upregulation capacity than B cells or CD4+ T cells. These characteristics position CD8+ T cells as the lymphocyte subset most susceptible to replicative senescence a finding with direct implications for understanding age-related immune decline. B cells maintained the longest telomeres and the highest telomerase response to mitogenic stimulation, consistent with their need for extensive clonal expansion during antibody responses. NK cells occupied an intermediate position, with moderate attrition rates and the weakest telomerase induction among the subsets examined. Across all subsets, telomerase responsiveness declined with age, suggesting that the compensatory machinery for telomere maintenance gradually fails in parallel with the telomere shortening it is meant to counteract. The data from this Saudi cohort align well with findings from European and East Asian populations, supporting the universality of lymphocyte telomere biology across ethnic backgrounds while providing the first large-scale dataset from a Middle Eastern adult population. CD8+ T cell telomere length emerges as a candidate biomarker for immunological aging that warrants further evaluation in longitudinal and clinical settings.

Figures and tables from the paper

Age Group	CD4+ T	CD8+ T	B Cells	NK Cells	Total PBL
20-29 yr	9.82 ± 0.71	9.41 ± 0.68	10.13 ± 0.83	9.67 ± 0.74	9.76 ± 0.69
30-39 yr	8.94 ± 0.63	8.37 ± 0.59	9.28 ± 0.72	8.91 ± 0.66	8.88 ± 0.61
40-49 yr	7.83 ± 0.58	7.14 ± 0.54	8.42 ± 0.67	7.69 ± 0.61	7.77 ± 0.57
50-59 yr	6.91 ± 0.52	6.23 ± 0.48	7.38 ± 0.59	6.74 ± 0.53	6.82 ± 0.51
60-69 yr	6.12 ± 0.47	5.47 ± 0.42	6.71 ± 0.54	5.93 ± 0.46	6.05 ± 0.44
70+ yr	5.38 ± 0.41	4.71 ± 0.39	5.94 ± 0.48	5.21 ± 0.43	5.31 ± 0.40

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Table 1Close ×

Mean telomere length (kb) by age group and lymphocyte subset

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Condition	CD4+ T	CD8+ T	B Cells	NK Cells
Unstimulated	2.3 ± 0.8	1.9 ± 0.7	3.1 ± 1.0	1.4 ± 0.5
PHA-stimulated (72h)	18.7 ± 4.2	14.3 ± 3.6	22.6 ± 5.1	8.9 ± 2.4
Fold increase	8.1×	7.5×	7.3×	6.4×
p-value (stim vs unstim)	<0.001	<0.001	<0.001	<0.001

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Table 2Close ×

Telomerase activity (relative TRAP units) by lymphocyte subset before and after PHA stimulation

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Figure 1Close ×

Heatmap of mean telomere length (kb) across six age groups and five lymphocyte categories. Darker orange tones indicate shorter

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Telomerase activity (relative TRAP units) in unstimulated and PHA-stimulated lymphocyte subsets averaged across all age groups. B

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