© 2012 Landes Bioscience. Figure 4. Injection of conventional CD4 T cells coated with SIINFEKL and aGC induces a functional cytotoxic T cell response. (A) Lymphoid cells from spleen and lymph nodes of C57BL/6 mice were stained with FITC-conjugated anti-CD19, anti-NK1.1, anti-Gr-1, anti-CD11b, anti-CD11c, anti-CD8a, anti-I- conjugated anti-CD3 or D anti-CD1d oAb. Filled n histogoramis istotype control. dis (B) Sorted CtD4+ r T celliswebreco-cultured ut with eaGC (1 . mg/ml). Cells were washed Ab antibodies together with APC-conjugated CD4 Ab. Lineage negative and CD4+ cells were sorted by FACSAria. The sorted cells were stained with PE- and intravenously injected into syngenic mice. Six hours later, lymphoid cells from spleen were stained with CD1d-tetramer and CD19 before intracellular IFNc staining. CD1d-tetramer+CD19- cells were gated and analyzed. (C) The sorted CD4+ T cells were co-cultured with aGC (1 mg/ml) or vehicle (0.5% polysorbate) for 16 h including 1 h pulse with SIINFEKL. Cells were washed and intravenously injected into syngenic mice. One week later, syngenic lymphocytes were either loaded with 1 mmol/L peptides or left untouched before being labeled with CFSE at different concentrations (10 and 1 mmol/L, respectively). Equal numbers of the two populations were mixed and injected i.v. into mice. Eighteen to 24 h later, lymphoid cells from spleen and lymph nodes were analyzed to assess peptide-specific killing. (D) OT-I T cells were isolated using anti-CD8 microbeads and AutoMacs. These cells were labeled with 10 mmol/L CFSE and i.v. transferred into their syngenic mice. On the following day, mice were i.v. injected with T cells manipulated in vitro with indicated conditions. Forty-eight hours later, lymphoid cells from the spleen of the recipient mice were stained with phycoerythrin (PE)-conjugated anti- Va2 antibody and then analyzed by flow cytometry. For intracellular Granzyme B and IFNc staining, cells were restimulated with SIINFEKL for 5 h before intracellular staining of these molecules according to manufacturer’s instruction. Data are representative of at least two independent experiments. To analyze whether T/veh/pep vaccination results in cytotoxic function of antigen-specific CD8 T cells, we isolated CD8+ T cells from OT-I mice, labeled them with 10 mM CFSE before transferring into C57BL/6 mice, which were then vaccinated with T/veh/pep or T/aGC/pep. When OT-I cells in the spleen of recipients were analyzed 48 hours after vaccination, both types of T cell vaccination induced the proliferation of OT-I T cells in vivo, although the proportion of T cells that had undergone over five times of division was higher in mice receiving T/aGC/pep vaccination than those with T/veh/pep (Fig. 4D, upper panels). More importantly, OT-I T cells in mice vaccinated with T/aGC/ pep displayed remarkably higher expression of granzyme B and IFNc compared with those in T/veh/pep-vaccinated mice (Fig. 4D, middle and lower panels). These data demonstrate that in response to an antigen on CD4 T cells as a non-professional APC, although antigen-specific CD8 T cells could proliferate, their functional differentiation into cytotoxic cells could only occurr in the presence of iNKT cell ‘help’. T/aGC/Pep vaccination confers protection against Listeria monocytogenes and melanoma. We next tested whether the cytotoxic T cell response generated by T/aGC/pep vaccination is effective enough in suppressing the growth of intracellular bacteria and tumor in an antigen-specific manner. We first employed L. monocytogenes infection model since clearance of this bacterium is largely dependent on CD8 T cell response. Mice were vaccin- ated with T/aGC, T/aGC/pep or peptide-pulsed dendritic cells (DC/pep) as a control. Ten days later, the vaccinated mice were i.v. injected with L. monocytogenes expressing OVA and the bacterial burden in the spleen and liver was measured. As expected, mice vaccinated with DC/pep showed significantly lower bacterial burden in both spleen and liver compared with non-vaccinated mice (Fig. 5A). Compared with non-vaccinated group, mice vaccinated with T/aGC showed slightly lower bacterial burden, especially in the liver. In contrast, mice vaccinated with T/aGC/pep also showed significantly lower bacterial burden in both organs, which is comparable to those of DC/pep-vaccinated mice (Fig. 5A). www.landesbioscience.com OncoImmunology 145