CD8 T cell stimulation for IFN-c production. For intracellular IFNc detection, splenic lymphocytes were isolated and then incubated with IE-1 ( 168 YPHFMPTNL 176 ) or m164 peptide ( 257 AGPPRYSRI 265 ) (10 À7 M) pulsed P815.B7 cells at a 10:1 ratio for 6 h with brefeldin A (Sigma-Aldrich, http://www.sigmaaldrich.com/) added for the last 3 h of culture. Cells were then harvested and analyzed for CD8a, CD43, and intracellular IFN-c protein by three-color staining followed by flow cytometry.