Human T Cell Differentiation Negatively Regulates Telomerase Expression Resulting in Reduced Activation-Induced Proliferation and Survival

Abstract

Maintenance of telomeres is essential for preserving T cell proliferative responses yet the precise role of telomerase in human T cell differentiation, function, and aging is not fully understood. Here we analyzed human telomerase reverse transcriptase (hTERT) expression and telomerase activity in six T cell subsets from 111 human adults and found that levels of hTERT mRNA and telomerase activity had an ordered decrease from naïve (T_N) to central memory (T_CM) to effector memory (T_EM) cells and were higher in CD4⁺ than their corresponding CD8⁺ subsets. This differentiation-related reduction of hTERT mRNA and telomerase activity was preserved after activation. Furthermore, the levels of hTERT mRNA and telomerase activity were positively correlated with the degree of activation-induced proliferation and survival of T cells in vitro. Partial knockdown of hTERT by an anti-sense oligo in naïve CD4⁺ cells led to a modest but significant reduction of cell proliferation. Finally, we found that activation-induced levels of telomerase activity in CD4⁺ T_N and T_CM cells were significantly lower in old than in young subjects. These findings reveal that hTERT/telomerase expression progressively declines during T cell differentiation and age-associated reduction of activation-induced expression of hTERT/telomerase mainly affects naïve CD4⁺ T cells and suggest that enhancing telomerase activity could be a strategy to improve T cell function in the elderly.

Keywords: T cell subsets; T lymphocytes; aging; alternative splicing; differentiation; hTERT; proliferation; telomerase.

Figure 1

hTERT mRNA expression and telomerase activity decrease with T cell differentiation. (A) A schematic of experimental design and a representative FACS plot shows the sorting strategy for isolation of CD4⁺ and CD8⁺ T cell subsets. Subsets of CD4⁺ or CD8⁺ T lymphocytes were sorted from healthy donors as follows: CD62L⁺CD45RA⁺ (naive, T_N), CD62L⁺CD45RA⁻ (central memory, T_CM), CD62L⁻CD45RA⁻ (effector memory, T_EM). (B) Total, FL, and βΔ ASP of hTERT mRNA were measured by RT-qPCR in resting T cell subsets and presented from left to right. Unpaired Student's t-tests were performed followed by the Benjamini–Hochberg correction. The FDR adjusted p-values are presented in the table above the figure and significant differences are marked in red. Each dot represents one individual from a total of 98 individuals and the values of Log₁₀ transformed -ΔCT are presented with addition of 5 to make all values positive. (C) Total, FL, and βΔ ASP of hTERT mRNA was measured by RT-qPCR in activated T cell subsets. Sorted T cell subsets were stimulated anti-CD3/CD28 antibody for 48 h and total, FL and βΔ ASP of hTERT were quantified by RT-PCR. (D) A representative gel image shows telomerase activity measured in one donor by TRAP assay. NC, no template control; Jk, Jurkat positive control. At right, telomerase activity was quantified as the ratio of telomerase products to internal control (I.C.). (E) Correlation between the levels of hTERT mRNA and levels of telomerase activity. Stimulated T cell subsets were measured for both hTERT mRNA and telomerase activity (n = 118). The relative hTERT and telomerase activity were plotted. Pearson's correlation (r = 0.77, p < 0.001). Values represent the mean ± SEM (n = 45). *≤0.05, **≤0.01, ***≤0.001, and ****≤0.0001 are used for all figures.

Figure 2

Enhanced cell proliferation and viability are enabled by high levels of hTERT mRNA and telomerase activity. (A–D) The indicated T cell subsets were stimulated with anti-CD3/CD28 conjugated microbubbles at day 0 and cultured for the indicated times. (A) The average number of accumulated cell divisions (n = 9) are shown. Average accumulated cell divisions were calculated from the sum of the log2-transformed fold-increases in number of viable cells harvested over the number of viable cells harvested at the prior time point. (B) Averaged percentages of live cells are shown (n = 7). Live cells were defined as those negative for AnnexinV and GhostDye as determined by flow cytometry. (C) Kaplan-Meier survival analysis of cell subsets from seven subjects during 15-days of culture. CD4⁺ and CD8⁺ T cell subsets are shown in the left and right panels, respectively. Death of the cell culture was defined as when the sum of all gates for AnnexinV⁺ and GhostDye positive cells reached ≥50%. (n = 7) (D) Total hTERT mRNA measured by RT-qPCR (top panels) and TRAP measurement of telomerase activity (bottom panels) during the 15-days of culture. p-values are derived by two-way ANOVA test where *as p ≤ 0.05, **as p ≤ 0.01, and ***as p ≤ 0.001.

Figure 3

Knockdown of hTERT mRNA leads to increased cell death in activated CD4⁺ T_N cells. (A–E) CD4⁺ T_N cells were activated with anti-CD3/CD28 antibody in the presence of scrambled control [Ctrl (SO)] or hTERT-specific antisense oligonucleotide (hTERT-AS). After 72 h, total hTERT mRNA expression was measured by qPCR (A), telomerase activity was measured by TRAP (B), and viable cells were counted by trypan blue exclusion (C). Lines connect the mean value of triplicate wells treated with Ctrl or hTERT-AS. Data are from 17 individual donors from 12 independent experiments. (D) A representative graph shows CFSE-labeled CD4⁺ T_N cells after 72 h of activation in the presence of Ctrl or hTERT-AS antisense oligonucleotide. Freshly isolated CD4⁺ T_N cells cultured for 72 h in the presence of 20 U/ml IL-2 [Ctrl (no stimulation)] are shown as an undivided control. Change in proliferation index is shown at right. Data are from 8 individual donors from 8 independent experiments. (E) Following 72 h of activation in the presence of oligonucleotide, cells were analyzed for live (AnnexinV⁻,7AAD⁻), early apoptosis (AnnexinV⁺,7AAD⁻), late apoptosis (AnnexinV⁺7AAD⁺), and necrosis (AnnexinV⁻,7AAD⁺). Mean percentages of populations are quantified at right. Data are representative of 16 individual donors. Paired Student's t-tests were performed between hTERT-AS and control. **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 4

Age-associated alterations in hTERT mRNA and telomerase activity in resting and activated T cell subsets. Comparison of total hTERT mRNA in CD4⁺ and CD8⁺ freshly isolated (A) and activated by in vitro stimulation with anti-CD3/CD28 antibody for 48 hrs. (B) T cell subsets from young (Y, closed symbol) (age ranges 17–40, n = 25–37) and old (O, open symbol) (age ranges 68–85, n = 16–27) donors. (C) Comparison of activation-induced telomerase activity in T cell subsets of young (n = 13–20) and old (n = 9–16) subjects. Each dot represents one individual. (D) Cell recovery after in vitro stimulation of six CD4⁺ and CD8⁺ T cell subsets from young and old subjects (Y, n = 16; O, n = 28). Percentages (mean and SEM) of seeded cells are presented. Values represent the mean ± SEM and p-values are derived by unpaired Student's t-test. *p ≤ 0.05.