The dual-colour settings programme (AELVIS Technologies, Software-version 4.2 Reader, TEMA-Ricerca, Italy) allowed to count the spots separately for three different colours. After setting up the limits the spots were sorted into three groups: pure red (β-gal) or blue spots (IFN-γ) and violet spots (concomitant IFN-γ and ß-gal release). Wells with DHD-K12 target cells or PBMC cultured alone were considered

as controls and the corresponding spots were subtracted from the number of spots obtained in the co-cultures. Statistical analysis The results were analyzed by non parametric Mann Whitney t test, using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, http://​www.​graphpad.​com). Results Target cells Transfected buy I-BET151 tumour cells DHD-K12 showing β-gal expression ranged between 50% and 60% in different Protein Tyrosine Kinase inhibitor experiments (Figure 1). No background this website staining was observed in cells transfected with Lipofectamine 2000 without DNA, performed as negative control (not shown). IFN-γ release The specific T-cell recognition of the CSH-275 peptide antigen was evaluated in vitro through the analysis of the IFN-γ release. The stimulation of PBMC from DHD-K12-inoculated rats, using different concentration of

CSH-275 peptide, induced the production of IFN-γ in a dose-dependent manner. The response induced by concentrations of 4-10 μg/ml of the peptide Myosin antigen was even higher than that induced by the mitogen. PBMC from control rat did not respond to the CSH-275 peptide, while they had an IFN-γ response to mitogen similar to that observed in DHD-K12-inoculated rats. These findings confirmed that DHD-K12-inoculated rats develop a specific immune response against the CSH-275 peptide expressed on DHD-K12 cells [16], and that such response is measurable in vitro by the ELISpot assay for IFN-γ. In Figure 2 are reported the mean stimulation indexes obtained in three different experiments. Figure 2 IFN-γ release. IFN-γ-ELISpot results from

three different experiments, expressed as number of spots per well (mean ± SD), showed the immune-response of DHD-K12-inoculated rats (dark grey) against CSH-275 peptide. No effect was produced on PBMC from control rats (light grey). Increasing concentration of peptide yielded an increasing numbers of IFN-γ producing PBMC. Under each histogram there is the corresponding image illustrative of blue spots. As negative contros we showed the non stimulated PBMC (W/O). Cytotoxic activity DHD-K12-inoculated rats developed aspecific cytolytic T cell response towards tumor cells. In Figure 3A are depicted the histograms representing the number of spots corresponding to the release of β-gal from lysed target cells. In these experimental settings, 2 × 105/well PBMC were plated in the presence of different number of DHD-K12 β-gal transfected target cells.