B7-H3/pMXC and B7-H3/pMXs-neo were used for SCCVII, EL4, E.G7, B16 cells and J558L cells, respectively. Tumour MK1775 cells were retrovirally transduced with B7-H3.28 For infecting EL4, SCCVII and B16 cells, pVSV-G was co-transfected

to generate pan-tropic retrovirus. After drug selection, transfectants expressing high levels of B7-H3 were sorted by flow cytometry as described previously.31 The TLT-2 complementary DNA was inserted into pMXs-IG, and control IRES-GFP (pMXs-IG) or TLT-2/pMXs-IG was retrovirally transduced into OT-I CD8+ T cells stimulated with OVA peptide (SIINFEKL).28 GFP+ cells were sorted by flow cytometry and used as mock- or TLT-2-transduced OT-I CD8+ T cells. CD4+ and CD8+ T cells from BALB/c mice were isolated by negative selection, as described previously.28 The purity of the CD4+ and CD8+ T cells was over 95% and 90%, respectively, as confirmed by flow cytometry. For the anti-CD3 mAb-induced co-stimulation assay, isolated T cells (2 × 105/well) were co-cultured with mitomycin C-treated parental P815 or B7-H3-transduced P815 (B7-H3/P815) cells at the indicated responder : stimulator ratios, in the presence of anti-CD3 mAb (145-2C11, 0·2 μg/ml in CD4+ T cells and 1·0 μg/ml in CD8+ T cells). The proliferative responses for the final 18 hr of the 3-day culture and IFN-γ production in the culture

supernatants at 72 hr were then measured.32 Anti-CD3 PI3K inhibitor mAb-induced redirected cytotoxicity against P815 and B7-H3/P815 cells was measured by the 6-hr JAM test.33,34 Splenocytes from OT-1 mice were cocultured with mitomycin C-treated E.G7 cells for 3 days for in vitro sensitization.

The cells were harvested, separated into CD8+ T cells, and used as in vitro-sensitized pentoxifylline OT-I CD8+ T cells. Cytotoxicity against E.G7 and B7-H3/E.G7 was measured by a 6-hr JAM test. For the in vivo cytotoxicity assay, E.G7 and B7-H3/E.G7 cells were labelled with CellTracker Orange [5-(and-6)-(((4-chloromethyl)benzoyl)amino)] tetramethylrhodamine (CMTMR; 10 μm, Invitrogen, Carlsbad, CA) and/or carboxyfluorescein diacetate succinimidyl ester (CFSE; 10 μm, Invitrogen). The CMTMR-labelled cells (2 × 106) were mixed with a twofold number of CFSE-labelled parental E.G7 (A-mix) or B7-H3/E.G7 (B-mix) cells (4 × 106) and then the mixed cells were injected intraperitoneally (i.p.) into OT-I mice. Peritoneal exudate cells (PEC) were analysed by flow cytometry after 24 hr. B6 mice were sensitized in vivo by peritoneal injection with DBA/2-originated allogeneic P815 or B7-H3/P815 cells (2 × 107 cells) to evaluate CTL against the alloantigen. After 8 days, PEC were collected and cytotoxicity against P815 and B7-H3/P815 was measured as described above. The OT-I mice received a peritoneal injection of mitomycin C-treated OVA-expressing EL4 (E.G7 or B7-H3/E.G7) cells (2 × 107) to induce OVA-specific CTL. Three days later, PEC were harvested and cytotoxicity against E.G7 and B7-H3/E.G7 was assessed as described above.