Figure 4. Impaired induction of telomerase activity in CD3+ T-cells in MDS patients.

(A) Preliminary analyses were conducted with CD3+ T-cells from healthy donors to identify the optimal time for telomerase induction using the TRAP assay on day 0, day 1, day 2, and day 3. CD3+ T-cells were separated from peripheral blood by negative selection and unstimulated (day 0) and day 3 stimulated cells were analyzed. Stimulated cells were collated after incubation with anti-CD3/CD28-conjugated beads. (B) The case-control differences in telomerase activity are shown for controls, MDS, aplastic anemia (AA) and large granular lymphocyte leukemia (LGLL). CD3+ T-cells were negatively separated from peripheral blood of MDS patients (n=35) and healthy donor (n=42), AA patients (n=8) and from patients with LGL leukemia (n=17) to test for specificity. (C and D) Telomerase activity (TRAP assays) and CD69 expression were compared in cases and control grouped by age <65y and ≥65y. (D) To determine that the T-cells are generally responsive to stimulation, we examined the surface expression of CD69, which is a well-known early activation-associated antigen (25) by staining with PE-conjugated anti-human-CD69 and analysis by flow cytometry. (E) From MDS patients (n=8, gray box) and age-matched healthy controls (n=8, white box), telomerase activity using TRAP assays were examined in purified sorted populations of naïve and memory cells. The sorting method and telomere length are shown in Figure 3 for these cells. The change in telomerase activity, difference post-pre activation in T-cells is shown. hTERT activity data was normally distributed and case-control differences were compared using a T test. p-values for are shown at the top of each comparison. All tests were two-sided and associations were considered statistically significant at a significance level of p<0.05.