text: IL-10 Production by Th1 Cells Requires ERK1 and ERK2 Activation
  Our data showed that the maintenance of IL-10 induction in Th1 cells required stimulation with high antigen dose, which to some extent could be compensated for by the addition of IL-12. Signaling through the TCR with high doses of antigen induced stronger ERK1 and ERK2 activation than that induced by low antigen dose, not only in naive CD4+ Tcells (data not shown) as previously demonstrated (Jorritsma etal., 2003) but also in CD4+ Tcells restimulated with the same high and low antigen doses (Figure5A). Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure5B). 
  We then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure5C), a compound that blocks downstream ERK activation. To ensure that only Tcell signaling was being affected by U0126, we used an APC-free system in which the Tcells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12. As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-gamma after 1 week of culture (Figure5C). Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure5C). Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain etal., 2007; Mody etal., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain etal., 2007; Mody etal., 2001). PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure5D and FigureS5A). Upon addition of inhibitors to othersignaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3beta inhibitor, CT99021 (Bain etal., 2007), no effect on IL-10 production was observed (FigureS5B). Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3beta pathways. 
  