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. 2011 Sep 19;6(9):e24931. doi: 10.1371/journal.pone.0024931

Figure 1. CD28-mediated costimulation relocalizes and augments Ras activation.

Figure 1

A. Splenic CD8+ T cells from 2C TCR transgenic mice were primed in vitro as described [15] to generate effector cells. They were then incubated with CMAC-labeled P815 or P815-B7.1 tumor cells (expressing a ligand for the 2C TCR). Conjugates were stained for RasGRP and Talin and analyzed by confocal microscopy (left). Statistical analysis of T cell RasGRP localization was done by measuring the ratio of the average pixel intensity of RasGRP staining at the T cell-APC interface to the average pixel intensity of RasGRP staining in the T cell cytoplasm. Analysis of at least 100 conjugates per experiment demonstrates a significant relocalization of RasGRP to the T cell-APC interface when CD28 costimulation is added to P815 stimulation (p<0.05, right). See materials and methods for details of image analysis and criteria used to definine T-cell APC conjugate, T cell-APC interface and T cell cytoplasm. Error bars indicate the standard deviation of the mean pixel intensity ratios (interface/cytoplasm) under the two indicated conditions from the same experiment. B. CAR Tg Th1 T cells were stimulated with beads coated with anti-CD3 antibody alone, or with anti-CD3 plus either anti-CD28 or B7.1-Ig fusion protein for 30 minutes. Cellular lysates were analyzed for Ras activity as described in Materials and Methods (top). Ras activation was quantified using densitometric analysis of western blots to determine the fold-increase in RasGTP-generation above unstimulated cells (bottom). Error bars represent the standard deviation in mean Ras activation for a given stimulation condition between replicate experiments. The p-value indicated in the figure demonstrates a significant difference in Ras-GTP generation when CD28 costimulation was added to CD3 stimulation. C. CAR Tg Th1 cells were stimulated as in A with increasing doses of anti-CD3 (0.01–1 µg/mL) with or without anti CD28 (2 µg/mL) and analyzed for phosphorylated AKT, JNK, and ERK by western blotting.