myeloid-derived suppressor cell (MDSC) in the bone marrow, whereas EG-7/veh did not (Fig. 3A; -0.37% and 72.8% lysis, blood, spleen.14 Of note, we also observed a great decrease of respectively). To examine if CD8+ T cells in the vaccinated mice CD11b+Gr-1+ population in the spleen of EG-7/aGC vaccinated directly kill target cells in an antigen-specific way, we performed an mice (, 4%) compared with that of EG-7/veh group (. 20%) in vitro CTL assay with purified CD8+ T cells from the vaccinated (Fig. 1C), another indicative of antitumor activity induced by mice by analyzing caspase-3 activation in the target cells. As the vaccination. Importantly, mice vaccinated with EG-7/aGC depicted in Figure 3B, the CD8+ T cells from the EG-7/aGC showed higher numbers of CD8+ T cells producing IFNc upon vaccinated mice induced a significantly higher percentage of SIINFEKL stimulation (Fig. 1D). These results overall indicate caspase-3 cleavage in the target cells than those from the EG-7/veh the induction of anti-tumor activity as well as tumor-specific vaccinated mice. In addition, the effective target lysis in EG-7/ CD8+ T cell responses upon the vaccination with EG-7/aGC aGC-vaccinated mice was associated with increased antigen- in vivo. specific IFNc+ granzyme B+ CD8 T cells in the spleen (Fig. 3C). Therefore, we next examined if the observed anti-tumor To further characterize the antigen-specific CD8 T cells, we activity by vaccination with EG-7/aGC depends on CD8+ T cells. adoptively transferred CFSE-labeled OT-I T cells (CD45.2) into We injected anti-CD8 depleting antibody into the vaccinated congenic mice (CD45.1). The recipients were vaccinated with mice before we transplanted live EG-7 cells into them. Depletion either EG-7/veh or EG-7/aGC and CD45.2+ cells were analyzed of CD8+ cells greatly decreased the protective anti-tumor activity five days later. OT-I T cells in mice vaccinated with EG-7/veh by vaccination with EG-7/aGC (Fig. 2A) and the survival of underwent an extensive proliferation, but expressed little IFNc vaccinated mice (Fig. 2B). Taken together, these results indicate (, 10%) or granzyme B (, 3%). By contrast, significantly higher that antitumor immunity generated by aGC-loaded EG-7 is, at population of OT-I T cells in EG-7/aGC-vaccinated mice ©cytotoxic least in part, 2respo0nses. mediated 1Our by CD8 E2G-7 T cells. tumLormaodels n(Fig. d1)andethsymoma expressed iBnduced IFNc (. itumoor-spescific 50%) and granzyme CcD8+ iTceells B (. to ndifferecntiate 8%) (Fig. einto 3D). . Vaccination with aGC-loaded EG-7 induces ova-specific Taken together, these data demonstrate that aGC-loaded EG-7 CD8 depletion experiment (Fig. 2) raised a hypothesis that tumor cytotoxic effector T cells expressing high levels of IFNc and cells of T cell origin, when they present iNKT ligand, are able to granzyme B in vivo. induce cytotoxic T cell response specific for endogenous tumor aGC induces cytotoxic T cell response to T cell-associated antigens. To test our hypothesis, we co-cultured EG-7 cells with antigens. Our study thus far showed that, with help of aGC, tumors veh, respectively). To Dincrease othe expnression oofpetptide/MdHC-IisTheretfore rwe inext basked uif cotnveentiona.lT cells can also act as aGC or vehicle before being irradiated (EG-7/aGC and EG-7/ of T cell origin can trigger tumor-specific cytotoxic T cell responses. complex, EG-7 cells were first pre-treated with IFNc. One week antigen presenting cells and trigger cytotoxic T cell responses in vivo, after vaccination, we performed an in vivo CTL assay to deter- when formulated to present aGC. A previous study by Shimizu et al. mine the antigen-specific cytotoxicity against SIINFEKL-loaded showed that T cells, purified by negative selection using magnetic syngenic splenocytes11. When injected into naïve syngenic mice, beads and when loaded with aGC, stimulate iNKT cells in vivo.13 EG-7/aGC resulted in an evident peptide-loaded target cell lysis To further assess iNKT cell activation by T cells, we first sorted Figure 2. CD8 T cells play a critical role in mediating antitumor activity. (A and B) C57BL/6 mice were vaccinated with EG-7/veh or EG-7/aGC on day -7 (n = 5 per group). EG-7/aGC vaccinated mice were intraperitoneally injected with CD8 depleting Ab (563.8) on day-3 and -1. On day 0, all mice were subcutaneously injected with 1 ! 106 live EG-7 cells and the tumor volume (A) and the survival (B) were checked. *p , 0.01, **p , 0.005, p values were calculated with 2-way ANOVA (A) or Kaplan-Meier method (B) in comparison with EG-7/veh and EG-7/aGC+rIgG or EG-7/aGC+rIgG and EG-7/aGC+anti-CD8 group. www.landesbioscience.com OncoImmunology 143