Increase of intracellular cAMP in primary human T cells inhibits proliferation in response to TCR/CD3 plus CD28 but not in response to phorbol ester plus CD28.
(A) Kinetics of intracellular increase of cAMP. Purified primary human T cells were cultured for the indicated time intervals with forskolin and IBMX as described in “Materials and methods” and levels of endogenous intracellular cAMP were examined. Results are representative of 3 independent experiments. (B) Purified primary human T cells were cultured with either anti-CD3 and anti-CD28 mAbs or with phorbol ester and anti-CD28 mAb as described in “Materials and methods,” either in media or with the indicated concentrations of forskolin and IBMX. Incorporation of 3H-thymidine was examined at the final 6 hours of a total 72-hour culture interval. Results are representative of 5 independent experiments. Although there was variability in the responses among different donors, the same trend was observed in all tested samples. Error bars indicate range of responses. (C) Increase of intracellular cAMP does not induce apoptosis. Primary human T cells were cultured under the indicated conditions in the presence or absence of forskolin and IBMX and the percentage of apoptotic cells was examined by annexin and propidium iodide. Cells were analyzed at various time intervals (24, 48, 72 hours after culture), and results representing the 72 hours of culture are shown. The results are representative of 2 experiments.
