PGE₂ inhibits upregulation of JAK3 in naive T cells. (A) Highly enriched naive T cells were cultured for 48 hours under the following conditions; medium alone; cross-linked anti-CD3 and IL-2 (1,000 U/mL) alone and anti-CD3/IL-2 plus PGE₂ with and without IBMX. Cell lysates were then subjected to immunoblotting using antibodies to JAK1, JAK3, p56^lck, and p59^fyn. In this and all experiments, equal amounts of protein were added to each lane. Densitometry readings of the immunoblot showed the following reduction of JAK3 by PGE₂alone and with IBMX: 0% 0.1 μmol/L PGE₂, 4% 1 μmol/L PGE₂, 40% 10 μmol/L PGE₂, 63% 0.1 μmol/L PGE₂/IBMX, 68% 1 μmol/L PGE₂/IBMX, and 76% 10 μmol/L PGE₂/IBMX. (B) Proliferation was assessed by the uptake of [³H] thymidine as described in Materials and Methods. (C) Effect of PGE₂ on cAMP production in resting T cells. Freshly isolated T cells were incubated in RPMI 1640 at 37°C in the presence of 100 μmol/L of the phosphodiesterase inhibitor IBMX for 30 minutes followed by incubation for 5 minutes with PGE₂ (10 μmol/L) before measuring cAMP levels. A significant reduction in JAK3 expression was observed in three experiments (P < .001).