© 2012 Landes Bioscience. Do not distribute. Figure 3. Vaccination with aGC-loaded EG-7 generates OVA-specific cytotoxic T cell response in vivo. (A and B) EG-7 cells were cocultured overnight with 1 mg/ml of aGC (EG-7/aGC) or vehicle (0.5% polysorbate, EG-7/veh) followed by irradiation (50 Gy). C57BL/6 mice (n = 3 per group) were vaccinated with EG-7/veh, EG-7/aGC or left untreated (Nil). (A) One week later, an in vivo CTL assay for SIINFEKL was performed. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. (B) One week after vaccination, CD8+ T cells were isolated and incubated with 1 mg/mL peptide-pulsed splenocytes that had been labeled with DDAO-SE for 2 h. The cells were fixed, permeabilized and stained with anti–cleaved caspase-3 mAb. The levels of cleaved caspase-3 in the DDAO-SE-positive cells were analyzed by flow cytometry. Data shown are representative FACS plots (upper panels) and mean ± SE (lower panel). *p , 0.05, in comparison with EG-7/veh group. (C) One week after the vaccination, splenocytes were isolated and restimulated with SIINFEKL for 5 h in the precence of Golgi-Plug before intracellular staining of granzyme B and IFNc. (D) Ovalbumin-specific CD8 T cells were isolated, labeled with 10 mmol/L CFSE and i.v. transferred into their syngenic mice as described in Figure 1. On the following day, mice were i.v. injected with irradiated EG-7 cells manipulated in vitro with indicated conditions. Five days later, lymphoid cells from the spleen of the recipient mice were restimulated with SIINFEKL for 5 h before intracellular staining of granzyme B and IFNc. Data are representative of at least two separate experiments. *p , 0.05, **p , 0.01, in comparison with non-treated (Nil) group. NK1.12CD82CD192CD11b2CD11c2Gr-12I-Ab2CD4+ cells We next asked whether T/aGC can induce antigen-specific from the lymphoid cells of C57BL/6 mice. These cells were virtually cytotoxicity when they were additionally pulsed with MHC class all CD3+CD1d+, indicating no contamination of professional APC I-restricted peptide. SIINFEKL peptide (1ng/mL) was added to (Fig. 4A). The sorted cells were loaded with aGC (T/aGC) or with T cells in the last one hour when they were incubated with aGC vehicle (T/veh) and were i.v. injected into syngenic naïve mice. When we analyzed splenocytes six hours after the injection, we observed that CD1d-tetramer+ cells in mice receiving T/aGC produced IFNc+ whereas the same population in mice receiving T/veh did not produce IFNc (Fig. 4B). Therefore CD4+ T cells efficiently triggered iNKT cell activation in vivo when they were manipulated to present aGC. or vehicle (T/aGC/pep and T/veh/pep, respectively). After extensive washing, C57BL/6 mice were immunized with T/veh/ pep or T/aGC/pep. One week later, we performed an in vivo CTL assay to determine the antigen-specific cytotoxicity against SIINFEKL-loaded syngenic splenocytes. Mice vaccinated with T/aGC/pep showed complete lysis of target cells while mice vaccinated with T/veh/pep did not (Fig. 4C). 144 OncoImmunology Volume 1 Issue 2