Two days later, 30 IU/ml of human rIL-2 was added to the medium. After 5 days, the cultured cells were collected and used as CTL effector cells. To detect B16 melanoma-specific CTL activity, we used TRP-2-peptide-pulsed EL-4 target cells or EL-4 cells pulsed with lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP)34–41 peptide (H-2Kb-restricted peptide AVYNFATCGI; produced by Genenet) as a third-party control. To detect CT26-specific CTL activity, we used CT26 target cells or J558L target cells as a third-party control. The target cells were labelled with 100 μCi Na251CrO4 for 1.5 h, and the 51Cr release assay was performed as previously
described . The percentage of specific 51Cr release was calculated as follows:
% cytotoxicity = [(Cr release of experimental medium − culture medium background)/(maximum Cr release − culture medium background)] × 100. Each data point was obtained from triplicate wells. Statistical analysis. Bortezomib Tumour growth was analysed using two-way anova, and the significance was calculated using Bonferroni’s post hoc test. The number of tumour-specific IFN-γ-producing CD8+ T cells was analysed BMS-354825 molecular weight by one-way anova, and the significance was calculated using Bonferroni’s multiple comparison post hoc tests. Survival rates were analysed using a log-rank comparison test. A probability value of P < 0.05 was considered significant. All data were analysed using Graphpad Prism®4 software (version 4; GraphPad Software, Inc., San Diego, CA, USA). Our group and others previously reported that i.t. injection of syngeneic DC without pulsation with tumour lysates could induce efficient antitumour responses to various cancers with TAA-specific CTL responses in murine s.c. tumour models [14, 15]. In this study, we referred to this DC-based cancer immunotherapy as ITADT. We investigated whether allogeneic DC could be used for cancer immunotherapy Rebamipide in the setting of ITADT. First, we used a B16 melanoma model. C57BL/6 mice were subcutaneously injected
with B16.F1v cells, and an i.t. injection of DC was given 3 days later followed by two additional injections at 1- week intervals. Consistent with previous reports [14, 15], ITADT using syngeneic female C57BL/6 DC (BL6 F DC; H-2b) induced an efficient antitumour effect, resulting in significant suppression of tumour growth, with 2/10 tumours being totally eradicated. The BL6 F DC-treated mice also showed significantly improved survival rates compared with PBS-treated controls (Fig. 1A,B and supplementary Fig. S1A, P < 0.01). We then tested semi-allogeneic DC (C57BL/6 × DBA/2 F1: BDF1 DC; H-2b/d) or minor disparate allogeneic DC (male C57BL/6: BL6 M DC; H-2b) and found that ITADT using these DC could induce antitumour effects similar to ITADT using syngeneic DC (Fig. 1A,B). In 2/11 mice treated with BL6 M DC and 1/11 mice treated with BDF1 DC, the B16.F1v tumours were eradicated (supplementary Fig. S1A).