Importance of T-Cell Proliferative Stress In Myelodysplastic Syndrome

Abstract 2939

During thymic development, T-cells are produced with a large array of genetically diverse aβ T-cell receptors (TCRs) primed for robust antigen recognition. In patients with myelodysplastic syndrome (MDS), the functional T-cell repertoire becomes contracted and the CD4/CD8 ratio reduced possibly reflecting antigen stimulation. Dynamics within the T-cell compartment are regulated by progressive telomere shortening that occurs with proliferation. Preservation of telomere length is mediated by the telomerase (hTERT) enzyme. In MDS, we previously showed that telomere length in peripheral blood mononuclear cells (PBMCs) was significantly reduced among cases compared to controls. We hypothesized that dysregulated telomere maintenance due to proliferative stress contributes to contraction of the T-cell compartment. First, we examined telomerase mRNA expression and telomerase activity in purified T-cells in a cohort of 32 healthy individuals. The age of these donors ranged from 17 to 82 years with a mean age of 58 years. CD3⁺ T-cells were separated from the peripheral blood by negative selection (Miltenyi Biotech Corp) and measurements were made in both unstimulated and stimulated cells or 3 days with CD3/CD28 T-cell expanders (Dynabeads®) to induce proliferation. hTERT expression was measured by quantitative RT-PCR (qRT-PCR) and telomerase enzymatic function was quantified by the Telomere Repeat Amplification Protocol (TRAP assay). We found that hTERT mRNA was low- to undetectable in unstimulated T-cells, but was significantly up-regulated by stimulation (1.71±1.45 vs 3.43±1.90, p<0.001). In stimulated T-cells, the amount of inducible hTERT transcription was inversely correlated with age (p=0.0086). Results of telomerase functional assays showed that the stimulated T-cells had dramatically increased telomerase activity compared with unstimulated cells (623.03±435.76 vs 3.71±6.37, p<0.001), but in contrast to hTERT, telomerase enzyme function did not changed with age (p=0.7). Individual purification and stimulation of CD4+ and CD8+ T-cells showed that CD4+ cells induced 4-fold greater increase in telomerase activity compared to CD8+ cells suggesting that the primary requirement for telomeric repair lies with highly proliferative CD4+ T-cells. Next, we measured DNA copy number of T-cell receptor excision circles (TRECs) in both unstimulated and stimulated T-cells as a function of age to determine the capacity of these cells to undergo proliferation in vitro. TRECs are episomal DNA fragments that do not transfer to daughter cells and are expressed exclusively in mature T-cells. After in vitro stimulation, TREC copy number decreased from a mean of 218 to 67 copies indicating that 2.9 population doublings (PD) had occurred within the 3-day incubation period. The number of PDs was maintained with age (p=0.26) suggesting that the proliferation capacity along with the telomerase function normally remains intact with age progression. These results varified the utility of applying TREC analysis to determine the proliferative history in vitro and in vivo. The proliferative history of peripheral T-cells in vivo was then determined in a separate cohort of healthy controls (n=63) age-matched to MDS patients (n=66) by comparing the change in the amount of TREC DNA in unstimulated cells. For every year increase in age, the log TREC values decreased by 0.05 DNA copies (p=0.0012) in healthy donors. In MDS patients, a 2-fold greater decline in TREC copy number occurred each year (0.099 unit decrease in TREC copies per year among cases, p<0.0001; 0.05 copies per year among controls, p=0.012) suggesting that T-cells in MDS patients have been exposed to more proliferative stress. Collectively, our results indicate that telomere maintenance is controlled normally in proliferating T-cells by inducing hTERT mRNA expression and by upregulating telomerase enzyme activity. In MDS cases, accelerated proliferative stress may contribute to repertoire contraction and CD4/CD8 ratio abnormalities.