The mechanism of active Ras-mediated IL-2 costimulation mimics the mechanism of CD28-mediated costimulation.

<p><b>A.</b> CAR Tg Th1 T cells were transduced and stimulated for four hours with antibody-coated beads as indicated. IL-2 mRNA production was assessed by real-time RT-PCR analysis of trizol lysates. <b>B.</b> CARTg/Luc Tg Th1 T cells expressing an IL-2 promoted-driven luciferase transgene were transduced and stimulated in triplicate overnight with antibody-coated beads as indicated. Luciferase activity was measured as a marker for IL-2 transcription as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024931#s4" target="_blank">Materials and Methods</a>. P values shown indicate a significant increase in luciferase activity when active Ras is added to CD3 stimulation and no significant difference in luciferase activity between Ras-transduced cells stimulated through CD3 alone and empty vector transduced cells stimulated through CD3 and CD28. <b>C.</b> CAR Tg Th1 T cells were transduced and stimulated as in A. Actinomycin D was added after 4 hours to stop further transcription. IL-2 mRNA levels were assessed at the time points indicated by real-time RT-PCR. Degradation curves were generated by linear regression modeling of real-time PCR data. Error bars shown indicate standard deviation of the mean between triplicate samples from the same experiment.