Active Ras prevents CD4⁺ T cells from acquiring a Th1 or Th2 effector cytokine profile.

<p><b>A.</b> Purified splenic CARTg CD4⁺ T cells were transduced with H-Ras61L, primed under Th1 or Th2 skewing conditions (for 4 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112831#s2" target="_blank">Materials and Methods</a>), and then re-stimulated with anti-CD3/anti-CD28-coated beads. Cytokine production was measured by ELISA. <b>B.</b> Western analysis for H-Ras, phosphorylated and total ERK confirmed the presence of functional active Ras in cells after 4 days of priming. <b>C.</b> Purified splenic CARTg CD4⁺ T cells were primed under Th1 and Th2 skewing conditions for 4 days. This was followed by transduction with constitutively active Ras or vector control. Cells were re-stimulated with anti-CD3 +/− anti-CD28-coated beads overnight, and supernatants were analyzed for cytokine production by ELISA. <b>D.</b> Cells were purified, transduced, primed and re-stimulated as in A. IL-2 production was measured by ELISA. A significant difference was noted between EV and Ras61L transduced cells in IFN-γ production under Th1 priming conditions, IL-4 under Th2 priming conditions (A and C) and IL-2 production under both priming conditions (D) (p<0.05 for all). All data contained in this figure is reflective of a single experiment that has been replicated at least 5 times with similar results.