© 2012 Landes Bioscience. Do not distribute. Figure 5. Vaccination with T cell-based vaccine generates protective immunity against L. monocytogenes infection and tumor challenge. C57BL/6 mice (n = 3 mice per group) were vaccinated with the indicated cellular vaccine (day 0) before they were challenged with 5 ! 104 live L. monocytogenes expressing OVA (day 10). Three days after the bacterial challenge, bacterial burden in spleen and liver was measured (A). Data are a representative of three separate experiments. (B) C57BL/6 mice (n = 3 mice per group) were vaccinated with the indicated cellular vaccine (day 0). Ten days later, recipients were intravenously challenged with live 2 ! 105 B16-OVA. Two weeks after the tumor challenge, tumor foci in the lung were measured. (C) PBMC was isolated in mice vaccinated with the indicated cellular vaccine and restimulated with SIINFEKL in the presence of Golgi-Plug for 5 h before intracellular staining of IFNc. Data are mean ± SE *p , 0.05, **p , 0.01, ***p , 0.001 in comparison with non-treated (Nil) group. Data are representative of two separate experiments. To assess if the vaccinated mice were also resistant to tumor growth, we i.v. injected OVA-expressing B16 melanoma cells into the vaccinated mice. Fourteen days later, we counted tumor foci in the lung of recipients. Compared with non-vaccinated mice, mice vaccinated with T/aGC had less tumor foci (Fig. 5B). On the other hand, fewer tumor foci were found in mice vaccinated with T/aGC/pep or DC/pep (Fig. 5B). Intracellular staining of peripheral blood mononuclear cells after peptide restimulation revealed that both DC/pep and T/aGC/pep vaccinations efficiently induced peptide-specific IFNc-producing CD8 T cells (Fig. 5C), which correlated well with anti-Listeria and anti- metastatic activity in the vaccinated mice. Collectively, these data demonstrate that vaccination with T/aGC/pep established protective immunity against intracellular bacteria and tumor in a peptide-specific manner. T cells simultaneously presenting iNKT and class I-restricted ligands directly induce antigen-specific cytotoxicity. We next sought to elucidate the mode of action in the efficient induction of peptide-specific cytotoxicity during T/aGC/pep vaccination. When we vaccinated CD1d-deficient mice with T/aGC/pep, we did not observe peptide-specific cytotoxicity in our in vivo CTL assay (Fig. 6A). Therefore, the antigen-specific cytotoxicity elicited by T/aGC/pep requires iNKT cells in vivo. Next we asked if vaccinated T cells directly stimulate CD8 T cells or require host APC. We utilized bm-1 mouse whose cells are able to load SIINFEKL onto their MHC I, but the resulting complex cannot be recognized by OT-I TCR due to a mutation in the H-2K region.15 In this experiment, vaccination with T/aGC/pep using T cells from bm-1 mice failed in induction of antigen-specific target cell lysis (Fig. 6B), indicating that appropriate MHC/peptide complex on the vaccinating T cells is essential and that they probably directly primed OT-I cells. Since aGC and class I peptide are both presented by the same T cells, this suggests that iNKT ligand may provide a “dangerous” 146 OncoImmunology Volume 1 Issue 2