COX-2 activity and EP4 expression increases in T cells during chronic activation.

<p>(A) (Top left) <i>COX-2</i> transcripts were quantified by qPCR in <i>ex vivo</i> and activated (anti-CD2/CD3/CD28) CD8+ T cell samples from healthy donors. Each sample was tested in triplicate and normalized to the housekeeping gene <i>36B4</i> (n = 5; *p = 0.031). Supernatants from early and late cultures from 3 donors were collected 72 h post activation with Ab-coated microbeads and their average PGE₂ concentration was calculated from triplicate wells, *p = 0.05. (Top Right) <i>COX-2</i> and <i>EP4</i> transcripts were quantified 72 h post each round of activation in four healthy donors. Each sample was tested in triplicate and normalized to the housekeeping gene, <i>36B4</i>. In addition, COX-2 activity was determined in CD8+ T cells at early and late time points 72 h post activation and at quiescence (15–17 d post stimulation) in three healthy donors using the COX Activity Assay (Cayman Chemical). (B) CD8+ T cells were pre-incubated with a highly specific COX-2 inhibitor CAY10404 (1 µM) (Cayman Chemical) or diluent (DMSO) for 30 min and then activated with Ab-coated microbeads as described. 1×10⁶ CD8+ T cells were collected 24h post activation during early and late PDs and intracellular cAMP was measured in triplicate using a direct cAMP ELISA kit (Enzo Life Sciences) (n = 4; *p = 0.05). (C) CD8+ T cells were cultured in the presence of 1 µM CAY10404 or DMSO for 30 min, and then activated with Ab-coated microbeads. 72 h after each round of activation, cells were collected and <i>IL-2</i> and <i>CD28</i> transcripts were quantified by qPCR. Each sample was tested in triplicate and normalized to the housekeeping gene <i>36B4</i> (n = 5; *p = 0.031).