© 2012 Landes Bioscience. Figure 7. Molecular requirements for efficient indication of CTL by iNKT-mediated T cell vaccine. (A) C57BL/6 mice (WT) or various cyokine-deficient mice with C57BL/6 background were vaccinated with T cells copulsed with aGC and SIINFEKL. (B) Pure CD4+ T cells were isolated from C57BL/6 (WT), B72/2, B7h2/2 or B7B7h2/2 as described in Figure 1. Isolated T cells were co-pulsed with aGC and SIINFEKL ex vivo before injected into WT recipient. A week later, an in vivo CTL assay was performed. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. Data are representative of three separate experiments. Do not distribute. 148 OncoImmunology Volume 1 Issue 2 class I-restricted peptide triggered iNKT cell activation and generated an antigen-specific cytotoxic T lymphocyte response in vivo. Mice receiving this novel T cell vaccine were resistant to L. monocytogenes infection as well as to tumor challenge in an antigen-dependent manner. Moreover, vaccination with aGC- pulsed T cell lymphoma also induced CTL response specific for an endogenous tumor antigen. Mechanistic analyses indicate that the vaccinated T cells directly stimulated peptide-specific cytotoxic T cells and that the CTL responses induced by these ‘T cell vaccines’ were independent of cytokines such as IFNc, IL-12 and IL-21. Therefore T cells as well as tumors of T cell origin can act as antigen-presenting cells that efficiently trigger cytotoxic T cell responses, when they are formulated to stimulate iNKT cells in vivo. Dendritic cells loaded with various iNKT ligands are potent inducer of T cell immunity. We recently showed that B cells co- pulsed with aGC and class I peptide also induce antigen-specific CTL response in vivo as efficiently as well.11 Moreover, our recent study showed that B lymphoma cells loaded with aGC elicited a strong protective immunity that is dependent on CD4 T cells.12 However, it has been unclear whether non-professional APC in the presence of iNKT cell help could induce T cell effector function. In the present study, we found that CD8 T cells were activated by antigens presented by CD4 T cells and proliferated extensively. However, they did not produce significant amounts of IFNc or Granzyme B that are associated with cytotoxic effector function, indicating that CD4 T cells are not an efficient APC. It is possible that T cells, like other non-professional APC, do not express high levels of costimulatory molecules, which is required for CD8 T cell effector function.26 We have also previously observed this type of non-productive activation in CD8 T cells reactive to a tissue antigen, which is mediated by PD-1-PDL1 interaction.27 In the presence of aGC and hence iNKT “help,” antigen-specific CD8 T cells were able to undergo effector differentiation by the CD4 T cell vaccine that results in protection against infection and tumor. Since iNKT ligands are either derived from infectious agents or induced during infection, their presence on non-professional APC may provide a “dangerous” signal to the immune system and, through iNKT cells, “license” a productive T cell activation and their effector differentiation. How iNKT cell help CD8 cell effector differentiation remains unclear at this stage. After activation, iNKT cells promptly produce a wide range of cytokines which can further activate themselves and other immune cells including NK, DC, T and B cells. IFNc and IL-12 are critical mediators for T cell immunity. IFNc produced by iNKT cells and NK cells mediates anti- angiogenic activity of aGC.18 IL-12 produced by DC after interaction with iNKT cells is critical for inducing IFNc in iNKT cells.17 However, in our T cell vaccine model, neither IFNc nor IL-12 was required for CTL generation. Recent studies suggest that IL-21 is a crucial cytokine for iNKT cell activation and function.19,20 However, an efficient induction of CTL response by our T cell vaccine in IL-21-deficient mice excludes the role of this cytokine in the generation of CTL response in our model.