CD8+ T cells were negatively selected after 72 hours post activation with anti-CD2/CD3/CD28 microbeads with or without PGE₂ or iso-PGE₂, and inhibitors of PKA pathway. (A) (Top Left) hTERTexpression was quantified at equivalent PDs by qPCR (n  =  5; *p  =  0.0312; **p  =  0.031 using one-sided T test because of decreased sample size for these conditions only; n  =  4). (Top Middle) Representative gel showing effects of PGE₂ (10⁻⁶ to 5⁻⁷M) and iso-PGE₂ (10⁻⁶ to 5⁻⁷M) on telomerase activity of CD8+ T cells. Band intensity per lane correlates with relative telomerase activity of 1,250 CD8+ T cells in each treatment group. (B) Telomerase activity was measured as described in (A) with PGE₂ or iso-PGE₂ in the presence of 1 µM of a PKA inhibitor, H89 dihydrochloride. (C) CD8+ T cell cultures were established and chronically activated as previously described in the presence of the immune modulators. Telomere lengths were evaluated by Real-Time PCR and expressed as a percentage of telomere length of a human tumor cell line, SAOS (∼23Kb). Data represent telomere lengths over the lifetime from 3 representative donor cultures. (D) (Top) The relative amount of intracellular ROS was determined by the mean fluorescence intensity of DCFDA–stained, live CD8+ T cells after 24 h of culture with media alone or in the presence of PGE₂, isoPGE₂, ox-LDL, or H₂O₂ (pos control). (Bottom) Representative flow cytometry profile of MitoSOX red oxidation in PGE₂- and iso-PGE₂-treated T cells.

https://doi.org/10.1371/journal.pone.0099432.g003