Telomere length of in vivo expanded CD4+CD25+ regulatory T-cells is preserved in cancer patients

Purpose: CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the upregulation of telomerase activity, whose regulation also remains unknown for Treg. Experimental Design: Treg and CD4+CD25− T-cells were isolated from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17) and analyzed for their content of T-cell receptor excision circles (TREC) and for telomere length using flow-FISH, real-time PCR and Southern blotting. The in vitro regulation of telomerase of Treg was studied using PCR-ELISA in bulk cultures as well as in isolated proliferating and non-proliferating Treg. Results: Treg isolated from peripheral blood of cancer patients exhibit significantly decreased levels of TREC when compared to Treg from healthy controls. Despite their in vivo proliferation, telomere length is not further shortened in Treg from cancer patients. Accordingly, telomerase activity of Treg was readily inducible in vitro. Notably, sorting of in vitro proliferating Treg revealed a significant telomere shortening in Treg with high-proliferative capacity. The latter are characterized by shortened telomeres despite high telomerase activity. Conclusions: Increased frequencies of Treg in peripheral blood of cancer patients are due to active proliferation rather than due to redistribution from other compartments (i.e., secondary lymphoid organs or bone marrow). In vivo expansion does not further shorten telomere length, probably due to induction of telomerase activity. In contrast, under conditions of strong in vitro stimulation telomerase induction seems to be insufficient to avoid progressive telomere shortening.