© 2012 Landes Bioscience. mice were vaccinated with Do T cells co-pulsed n with aoGCand t SIINFEKL distribute. (1 ! 106 per mouse). (B) T cells from WT or bm-1 mice were co-pulsed with aGC and Figure 6. Peptide and aGC on the same T cells are required for the optimal priming of CTL by iNKT-mediated T cell vaccine. (A) C57BL/6 (WT) or CD1d2/2 SIINFEKL before being i.v. injected into WT mice. (C) C57BL/6 mice were vaccinated with T cells co-pulsed with aGC and SIINFEKL (1 ! 106 per mouse) or ‘a combination of T cells pulsed with SIINFEKL and of T cells pulsed with aGC’ (1 ! 106 each) or T cells pulsed with SIINFEKL plus free form of aGC (i.p.). CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. Data are a representative of at least two separate experiments. signal to the immune system. To test this hypothesis, C57BL/6 mice were vaccinated with T/pep in combination with either soluble aGC (2mg, i.p.) or T/aGC. In mice vaccinated with the former combination, little peptide-specific CTL was generated (Fig. 6C, 13.4% vs. 97.2% lysis in T/aGC/pep-vaccinated mice). Moreover, mice vaccinated with T/aGC plus T/pep only showed a moderate peptide-specific CTL, significantly less efficient than that of T/aGC/pep (Fig. 6C, 47.2% lysis vs 97.2% in T/aGC/pep- vaccinated mice). Thus, these data demonstrate that the vaccinating T cells act as direct stimulators for both iNKT and CD8 T cells in vivo and that presentation of aGC and peptide by the same T cell is the most efficient regimen in generating antigen-specific CTL. Cytokine and costimulation requirements in the CTL response elicited by T/aGC/Pep. Upon activation, iNKT cells promptly produce a wide range of cytokines including IL-4 and IFNc. IL-4 produced by activated iNKT cells was shown to promote CD8 T cell proliferation.16 IFNc and IL-12 produced upon iNKT and DC interaction mediate anti-tumor activity.17,18 Recent studies showed that IL-21 is produced by iNKT cells upon TcR stimulation and regulates the activation and expansion of iNKT cells.19,20 Therefore, we asked whether these cytokines produced after iNKT cell activation had any role in the generation of CTL in our T cell vaccine model. We vaccinated IL-42/2, IFNc2/2, IL-12 p352/2 or IL-212/2 mice with T/aGC/pep and performed in vivo CTL assay. As depicted in Figure 7A, we observed a complete peptide-specific CTL activity in all of cytokine-deficient mice we tested. Thus, none of these cytokines is necessary in the generation of CTL activity by the T cell vaccine. Costimulatory signals are essential in regulating the activation and expansion of iNKT cells. Moreover, CD28 and ICOS signals are required for the anti-tumor activity of activated iNKT cells such as cytotoxicity and anti-metastatic activity.2,21-23 Activated T cells express CD80 and CD86 and may regulate immune response through T:T interaction.24,25 Therefore we asked whether these costimulatory factors were necessary for the induction of CTL in our model. T cells were isolated from C57BL/6(WT), B72/2 (B7.12/2B7.22/2), B7h2/2 or B7B7h2/2 and loaded with aGC and SIINFEKL ex vivo. The T/aGC/pep were injected into WT mice and in vivo CTL assay was performed. As depicted in Figure 7B, we observed a normal target cell lysis in mice vaccinated with costimulation-deficient T cells. Thus, the induction of peptide-specific cytotoxicity by the T cell vaccine does not require B7 and B7h costimulation. Discussion In the present study, we showed that vaccination with con- ventional CD4 T cells presenting both CD1d-aGC and MHC www.landesbioscience.com OncoImmunology 147