Moreover, although vaccinated T cells were found to directly Preparation of T and dendritic cells. Conventional CD4 stimulate iNKT and CD8 T cells, this action does not appear to T cells were purified from lymphoid cells of C57BL/6 mice by require costimulation. We recently found that dendritic cells sorting CD82 NK1.12 CD192 CD11b2 CD11c2 Gr-12 I-Ab2 loaded with aGC induced iNKT cell activation in the absence of but CD4+ cells with a FACS Aria1. These cells were . 99.5% B7 and B7h (data not shown). It is unclear at this stage whether CD3+CD4+ and CD1d+. In some experiments, CD4 T cells were there is any signal in addition to TcR that is required for iNKT isolated from CD1d2/2 or bm-1 mice. Bone marrow-derived DCs and CD8 T cell activation by our T cell vaccine. were generated by culturing bone-marrow cells in the presence of Nonetheless, T cells loaded with an iNKT ligand serves as GM-CSF and IL-4 for 6 d. Sorted T cells were co-cultured with a potent cellular vaccination approach. A mild suppression of aGC (1 mg/mL) or the same volume of solvent vehicle (5 ml/mL tumor growth and L. monocytogenes growth was observed in mice of 0.5% polysorbate) overnight. In some experiments, these cells vaccinated with T/aGC, indicating that an effective innate were further incubated with SIINFEKL peptide for 1 h. immunity was induced. However, vaccination with T/aGC/pep EG-7 cells were cultured in the presence of IFNc (100 ng/mL) was far more effective in eliciting CD8 T cell immunity and for 48 h including overnight culture with aGC or vehicle as specifically suppressing tumor growth and L. monocytogenes described above. These EG-7 cells were washed and irradiated growth, which was comparable to DC vaccination. Based on with Cs source (50 Gy) before i.v. injected into syngenic mice. our results, we propose that T cells from individuals infected with CFSE-labeled OT-I adoptive transfer. Ovalbumin-specific T cell-tropic pathogens—such as HIV—could be used as a source CD8 T cells were isolated from the lymphoid cells of OT-I mice of cellular vaccine for triggering pathogen-specific cytotoxic using anti-CD8 magnetic beads and AutoMacs (. 92% are response. Notably, i.v. injection of irradiated EG-7 thymoma Va2+). These cells were labeled with 10 mM of CFSE and i.v. ©OVA-spe2cific did not induce C0TLresp1onse. an OVA-specific 2Asa CTL resuLlt,vacacinationnwith response; however, daGCe-injsected injec- transferred wiBthCD4iT into C57BL/6 ocells or sirradciatediEGe-7cenllsformculateedex mice. In some experiment, Ly5.1 . tion of aGC-loaded, irradiated EG-7 efficiently generated an congenic mice were used as recipients. Recipient mice were i.v. loaded, irradiated EG-7 induced a potent anti-tumor activity vivo with indicated conditions. Two to five days later, lymphoid against challenge with live EG-7, which was CD8+ T cell- cells from the spleen of recipient mice were isolated and cultured dependent. Therefore we propose that vaccination of aGC-loaded with 0.1 mg/mL SIINFEKL in the presence of GolgiPlug T lymphoma may result in the generation of tumor-specific CTL (1 mL/mL) for 5 h. These cells were stained with phycoerythrin Our finding is novel Din thoatnon-pnrofesssoional AtPCs adrealsoisbeforetperrmeaibilibzedanudfurtther estaine.dwith APC-conjugated response. (PE)-conjugated anti-Va2 Ab or PE-conjugated anti-Ly5.2 Ab able to trigger cytotoxic T cell immunity with the help of iNKT anti-IFNc Ab or Alexa 647-conjugated anti-granzyme B Ab. cell. T cell is a major population in blood and can be easily Total 1 ! 106 events were collected and CFSE+ PE+ cells were expanded and cultured in vitro. Although resting human T cells gated and analyzed by FACS Calibur. express little CD1d, activated T cells as well as thymocytes in iNKT cell staining. For intracellular staining of IFNc in iNKT humans express CD1d on their surface.28 Moreover, cutaneous cells, splenocytes and liver mononuclear cells from mice injected T cell lymphomas in humans have been reported to express high with vehicle-pulsed T cells, aGC-loaded T cells were stained with levels of CD1D.29 These observations suggest that our findings PE-conjugated aGC-loaded/CD1d-tetramer together with FITC- can be directly applicable in clinical settings. Therefore, tumors conjugated anti-CD19 Ab (for splenocytes) or FITC-conjugated that are not professional APC in origin, especially thymoma and anti-TCRβ Ab. These cells were permeabilized and stained T lymphoma, could be used as efficient APC for cytotoxic T cell with APC-conjugated anti-IFNc Ab. CD192CD1d-tetramer+ or anti-tumor immunity when they are formulated to stimulate TCRβ+CD1d-tetramer+ cells were defined as iNKT cells and iNKT cells. Our T cell vaccine strategy will provide an alternative analyzed. cellular vaccine regimen that can induce the efficient activation of In vitro and vivo cytotoxic T lymphocytes response assay. The both innate and adaptive immunity in vivo. in vitro SIINFEKL- specific cytotoxic T lymphocytes response was measured using a caspase-3 cleavage assay.32 In brief, CD8 T Materials and Methods cells were isolated from the vaccinated mice by using anti-CD8 microbeads and magnetic activated cell sorting. Splenocytes from Mice. C57BL/6, OT-I, C57BL/6bm1 (bm-1), IL-42/2, IFNc2/2, syngenic mice were used as target cells after labeled with DDAO- IL-12p352/2, CD802/2CD862/2 (B72/2) mice were purchased SE and 1 mg/mL peptide. The sorted CD8 T cells were co- from The Jackson Laboratory (Bar Harbor, ME). B6.SJL cultured with the target splenocytes at a 1:1 ratio for 2 h at 37°C. mice (Ly5.1) were purchased from Taconic Farm. B7h2/2 and The cells were permeabilized and stained with PE-conjugated B7B7h2/2 mice were generated and backcrossed in our labora- anti-cleaved caspase-3 antibody. The percentage of cleaved tory.26,30 CD1d2/2 mice were bred in our animal facility. IL-212/2 caspase-3-positive cells among DDAO-SE-labeled target cells mice were previously described31 and bred in our animal facility. were analyzed by flow cytometry. The in vivo SIINFEKL-specific All mice were kept under specific pathogen-free conditions in cytolytic activity of CD8 T-cell responses was measured as our animal facility (MD Anderson Cancer Center). All animal described previously.33 Briefly, splenocytes were either loaded studies were approved by the Institutional Animal Care and Use with 1 mg/mL peptide or left untouched before being labeled with Committee of MD Anderson Cancer Center. CFSE at different concentrations (10 and 1 mmol/L, respectively). www.landesbioscience.com OncoImmunology 149