To investigate the functional consequences of expressing active Ras in primary T cells, we utilized primary CD4+ T cells from Coxsackie/Adenovirus Receptor Transgenic mice. These mice express a truncated human adenovirus receptor on all peripheral T cells, and transduction of T cells from these mice with adenoviral constructs allows for genetic manipulation of naı¨ve T cells without the need for cell stimulation or proliferation. Using this model, we observed that introduction of active Ras into primary CD4+ T cells augmented acute activation of both freshly isolated and primed effector cells. However, when active Ras was introduced prior to priming, the differentiation of these cells into Th1 and Th2 effector cytokine-producing cells was severely impaired. Impaired effector cytokine production was associated with impaired demethylation of the IL-4 gene, suggesting that the presence of active Ras during priming alters certain differentiation-related epigenetic modifications that occur at effector cytokine gene loci. Previous work from our laboratory and from others demonstrating the ability of active Ras to augment and maintain T cell activation and responsiveness have made this signaling pathway an attractive target for immunotherapeutic interventions aimed at potentiating T cell-dependent immune responses. However, our current data suggest that Ras signaling in naı¨ve T cells may be more complex than previously appreciated and that blanket therapies attempting to potentiate Ras signaling may have paradoxical effects. Our results PB 203580 indicate that introduction of active Ras impairs the ability of T cells to acquire effector cytokine production during Th1/Th2 differentiation, at least in part due to a failure in epigenetic modification. It is of interest that deregulated Ras signaling did not lead to a complete failure of the process of differentiation, as several other aspects of Th1/Th2 differentiation, including responsiveness to IL-12 and IL-4 cytokine signaling and upregulated expression of T-bet and GATA-3, proceeded normally. While active Ras did induce a partial anti-proliferative effect, this could not fully explain the effect of Ras on impaired cytokine production. The inability of Ras61L-transduced cells to properly remodel the IL-4 locus suggests that epigenetic modification during T cell differentiation requires properly regulated Ras signaling. Ras61L-transduced cells not only failed to upregulate effector cytokines, but also failed to modulate IL-2 production in a lineage-specific way, thus maintaining a mor cytokine profile. This observation suggests a global effect on cytokine regulation that may be consistent with a failure of epigenetic modifications. A model of this process is shown pictorially in Figure 7. In contrast to the IL-4 locus, we were unable to find a site in the IFN-c gene whose methylation was differentially regulated comparing naive, Th1, and Th2 cells in our own experiments. As such, analysis of a potential role for active Ras in preventing IFN-c locus remodeling was not possible.