In vivo cytotoxic T lymphocyte assay

The in vivo cytotoxicity was assessed as described elsewhere [25]. Briefly, syngeneic splenocytes were divided into equal numbers and either loaded with 1 μg/mL of cytotoxic T lymphocyte (CTL) epitope peptide (human HER2/neu p63 [TYLPTNASL]) or left unpulsed. Peptide-pulsed cells (CFSE^high) were labeled with 20 μM chloromethyl fluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA) and unpulsed cells were labeled with 2 μM CFSE (CFSE^low). Equal numbers of CFSE^high and CFSE^low cells were mixed and injected intravenously (i.v.) into groups of mice. After 24 h, the splenocytes from treated mice were analyzed using flow cytometry to assess antigenic peptide-specific target lysis. The specific lysis was calculated as follows: r (ratio) = (% CFSE ^high/CFSE ^low) and % lysis= [1-(r _unpulsed/r _pulsed)]x100.