In vivo cytotoxicity assay. Antigen-specific CD8 T cell-mediated cytotoxicity was assayed as described in [74] . Briefly, splenocytes from naive mice were costained with PKH26 (Sigma-Aldrich) and either 1 lM, 100 nM, or 1 nM CFSE (Molecular Probes, http://probes. invitrogen.com/). Labeled cells were then pulsed with the indicated peptides, mixed in equal ratios, then transferred i.v. (5 3 10 6 total cells) into the indicated groups of mice. Lymphocytes were isolated from the spleens of recipient mice 4 h post-transfer and analyzed for PKH26 (all transferred cells) and level of CFSE expression (unpulsed or peptide-pulsed cells). Percent killing within the PKH26 þ gate was calculated by: 100 À ([(% peptide-pulsed in infected/% unpulsed in infected)/(% peptide-pulsed in uninfected/% unpulsed in uninfected)] 3 100).