Suppressing the mTOR pathway inhibits the costimulatory effects of TLR2 in CD8 T cells. (A) CD8 T cells were activated as described in Figure 2. Briefly, purified T cells were activated in each of the absence or presence of TLR2 ligand for 72 hours. Various concentrations of rapamycin were added for the last 6 hours of culture. Alternatively, rottlerin and AKT inhibitor were added with or without the TLR2 ligand for the initial 18 hours of activation. After this time point, inhibitors and TLR2 ligand were washed off and T cells were cultured for an additional 54 hours. After 72 hours, the levels of IFN-γ and granzyme B were determined by ELISA. The top panels show the amount of IFN-γ and granzyme B, whereas the bottom panels show the percentage inhibition after normalizing levels to cultures that did not receive inhibitors. (B) Alternatively, T-bet protein levels were determined in the presence of rapamycin, rottlerin, or Akt inhibitor by Western blot. The fold changes in T-bet expression levels in TLR2-stimulated over non–TLR-stimulated T cells are shown.