Figure 4. Active Ras can recapitulate CD28-mediated cell survival and increased glucose uptake.

A. CAR Tg Th1 cells were transduced with empty vector or Ras61L and stimulated with antibody-coated beads in triplicate as indicated. Cells were analyzed for survival after 48 hours by staining with FITC-annexinV and propidium iodide followed by flow cytometry. P value indicates significantly increased AnnexinV/PI negative (live) cells when active Ras is added to CD3 stimulation. B. CAR Tg Th1 cells were transduced as in A and left unstimulated or stimulated in triplicate overnight (20 h) as indicated. Cells were pre-incubated in glucose uptake buffer and then exposed to ³H-2-deoxy-glucose (³H-2-DOG) for 2 minutes. ³H-2-DOG uptake was measured in a ß-scintillation counter. P value indicates no significant difference in glucose uptake by unstimulated Ras-transduced cells and empty vector-transduced cells stimulated with anti-CD3 and anti-CD28 antibodies. For all data shown in this figure, error bars represent standard deviation between triplicates samples from the same experiment.