Henning Walczak (Tumor Immunology Unit, Division of Medicine, Imperial College, www.selleckchem.com/products/Bortezomib.html London, UK). Concanamycin A (CMA) and mevastatin were purchased from Sigma, while zoledronate was from Novartis Pharma, Basel, Switzerland. Generation of Polyclonal V��9V��2 T Cell Lines Polyclonal V��9V��2 T cell lines were generated by first enriching PBMC using a �æ� T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by sorting single V��9V��2 T cells through a FACSAria (BD Biosciences) with specific mAbs. Cells (2��103) were then cultured into each well of round-bottom, 96-well plates containing 2��104 irradiated (40 Gy) allogeneic PBMC, 2��103 irradiated (70 Gy) EBV-transformed allogeneic B cells, 0.5 ��g/ml PHA (Sigma), and 200 U/ml recombinant interleukin 2 (Proleukin, Novartis Pharma).

Growing lines were expanded in 200 U/ml IL-2 and restimulated every 2 weeks. Usually, cells were collected after 4�C6 weeks of culture to be used for functional assays in vitro. Cytotoxic Assay Target colon CIC (105 cellsml) were pre-treated with 5-FU (2.5�C250 ��g/ml), DXR (0.025�C2.5 ��M) or zoledronate (0.5 ��M) for 24, 48 or 72 hrs. Cells were extensively washed in PBS and stained with CFSE (Merck, Milano, Italy) as follows: 50 ��l of CFSE were added to 1 ml of target sphere cell suspension (5��105 cells/ml) in PBS to obtain the final concentration of 2.5 ��M CFSE. The cells were incubated for 10 minutes at 37��C and gently mixed every 5 min. At the end of incubation, 1 ml of FBS was added to the cell suspension to stop the staining reaction and the cells were centrifuged at 600 g for 5 min at room temperature, washed twice with cold PBS and resuspended in serum-free medium.

V��9V��2 T cell lines were resuspended at the final concentrations of 106 and 2.5��106 cells/ml, were added to CFSE-stained target colon CICs (1��105) and co-cultures were maintained for 6 hrs a 37��C in presence of 5% of CO2. At the end of the incubation period, the cells were washed with PBS Dacomitinib and stained with 20 ��l of Propidium Iodide (PI, Sigma, 1 ��g/ml) for 10�C15 min in ice. Finally 100 ��l of cold PBS were added before acquisition on a FACSCalibur cytometer (BD Biosciences). The calculation of cytolytic activity was based on the degree of reduction of viable target cells with the ability to retain CFSE and exclude PI (CFSEhigh PI?), according to reference [27]. Blocking agents were used to evaluate the mechanisms of V��9V��2 T cell-mediated cytotoxicity of colon CICs. To evaluate the contribution of mevalonate metabolites tumor target cells were treated with mevastatin (25 ��M for 2 h) a selective upstream inhibitor of the mevalonate pathway.