recently showed that non-professional APCs, such as T and NK cells, can stimulate iNKT cell activation when they presented aGC in a cell-associated form.11,13 They also showed that tumors lacking costimulatory molecules, such as B16 melanoma and EL4 thymoma, can induce the activation of iNKT cells and NK cells in vivo when formulated to present aGC. These observations together raise an interesting question if iNKT ligand-loaded non- professional APC could act as an efficient APC in generating CD8 T cell response against the peptides loaded on their surface MHC class I. If this is true, it might be possible to use tumor cell itself or pathogen-infected cells to generate tumor- or pathogen-specific cytotoxic T cell immunity, which would be much easier, cost- effective and practical in clinical settings. Therefore in the present study, we tested whether conventional T cells- as a surrogate CD1d-expressing non-professional APC- can actively interact with iNKT cells and CD8 T cells in the presence of an iNKT cell ligand and MHC class I-restricted peptide. Our results showed an efficient activation of iNKT cells and antigen-specific CD8 T cells in mice vaccinated with T cells presenting both aGC and peptide on the same cells, which induced an effective immunity against microbial infection and tumor. Moreover, vaccination with aGC-pulsed thymoma generated a tumor-specific cytotoxic T cell response in vivo. Thus, in the presence of iNKT cell help, non-professional APC, such as T cells, can stimulate cytotoxic T cell function, providing an alternative means of cell vaccination method. © 2012 Landes Bio sResuclts ience. Vaccination with aGC-loaded EG-7 generates CD8+ T cell- dependent antitumor activity. We first asked whether vaccination with tumor cells of T cell origin can trigger anti-tumor activity, Do not disrespontses rmoire befficienutlyintviveo,we.employed ovalbumin- when manipulated to present aGC. To track tumor-specific T cell expressing EL4 thymoma (EG-7) as our tumor model. To address protective antitumor activity by vaccination with aGC-loaded EG-7, we vaccinated mice with irradiated EG-7 co-cultured with 1 mg/mL of aGC overnight. EG-7 cells incubated with the same volume of solvent (5 ml/mL of 0.5% polysorbate) were used as control (EG-7/veh). After extensive washing, these cells were i.v. injected into syngenic naïve mice. These mice were subcutaneously implanted with live EG-7 and the tumor volume and survival were monitored daily. Mice were sacrificed when tumor diameters reached 20 mm. As depicted in Figure 1A and B, the vaccination with EG-7/aGC significantly reduced the growth of solid tumors and induced prolonged survival in comparison to those of EG-7/veh group. Tumors affect myelopoiesis and induce the expansion of CD11b+Gr-1+ Figure 1. Vaccination with aGC-loaded EG-7 generated preventive antitumor activity. (A–D) EG-7 cells were co-cultured overnight with 1 mg/ml of aGC (EG-7/aGC) or vehicle followed by irradiation (50 Gy). C57BL/6 mice (n = 7 per group) were vaccinated with EG-7/veh, EG-7/aGC or untreated (Nil). One week later, all mice were subcutaneously injected with 1 ! 106 live EG-7 cells and the survival (A) and tumor size (B) were checked. (C) At the 3 weeks later, splenocytes were isolated from tumor bearing mice and analyzed by flow cytometer after staining with anti-CD11b, anti-Gr1, anti-CD19, anti-CD4 and anti-CD8 antibodies. (D) Splenocytes were restimulated with SIINFEKL in the presence of Golgi-Plug for 5 h before intracellular staining of IFNc on OVA specific T cells. *p , 0.05, **p , 0.005, p values were calculated with 2-way ANOVA (A), Kaplan-Meier method (B) or Student’s t-tests (C and D) in comparison with the EG-7/veh group. 142 OncoImmunology Volume 1 Issue 2