The coating buffer was removed from the plates, optimal concentrations of 2 × 105 responder cells in IMDM with 5% FCS were put into each well and 5 × 104 tumour target cells or lysates (equivalent of 2 × 104 cells), optimal concentrations of αGalCer (100 ng/ml, obtained from Dr H. Ovaa, the Netherlands Cancer Institute, Amsterdam, the Netherlands) or phorbol myristate acetate (PMA) (50 ng/ml) plus ionomycin (1 µg/ml) were HSP targets added and the plates were incubated overnight at 37°C. In experiments with NK T cell lines, optimal concentrations of responders were used at 5 × 102/well, targets at 2 × 103 cells/well and external antigen-presenting cells C1R-huCD1d or C1R

at 2 × 103 cells/well. After removal of the cell suspension, the plates were washed with PBS, developed according to the manufacturer’s instructions and read using the Bioreader 4000 pro-X ELISPOT reader (Bio-sys, Karben, Germany). Plasma samples were obtained at various time-points during IFN-α therapy,

preserved at −70°C and tested using ELISA according to the manufacturer’s instructions (human IL-7 Quantikine ELISA kit HS750, human IL-12 Quantikine ELISA kit D1200 and human IL-15 Quantikine ELISA kit D1500; R&D Systems, Abington, UK). PBMC subset analysis was performed as described previously [23]. Briefly, cells or cell lines were stained for MG-132 molecular weight 20 min at room temperature followed by washing steps in PBS containing 0·5% BSA with the following conjugated antibodies directed at: CD3-fluorescein isothiocyanate [fluoroscein isothiocyanate (FITC)/CD(16+56]-phycoerythrin (PE) (B&D Biosciences), CD8-FITC, CD56-PE cyanine5 (PC5),

CD19-PC5, CD69-PE Texas Red [electrochemical detection (ECD)], CD8-PC5, CD3-PE cyanine7 (PC7), CD45-FITC/CD14-PE, CD45RO-ECD and CD4-ECD (all from Beckman Coulter, Woerden, the Netherlands). For detection of NK T cells, staining with anti-TCR Vα24-FITC and Vβ11-PE in combination with anti-CD3-PC7 was used; in some experiments, NK T cells were measured using anti-TCR Vβ11-PE in combination with 6B11-FITC [24] (BD Biosciences Pharmingen, San Diego, CA, USA). Further NK T subset typing was performed using antibodies Bcl-w to CD4-ECD, CD8-PC5, CD56-PC5, CD69-ECD, CD45RO-ECD (all Beckman Coulter) and CD161-biotin (Ancell, Bayport, MN, USA). For enumeration of regulatory T cells (Treg), antibodies were used directed at: CD4-FITC, CD8-PE, CD45-ECD, CD25-PC5, CD3-PC7 (Beckman Coulter) and forkhead box P3 (FoxP3) (eBioscience kit; eBioscience, Inc. San Diego, CA, USA). In all experiments gates were set on viable [propidium iodide (PI)-negative] cells and fluorochrome-labelled isotype control antibodies were included in each assay to determine background staining. FACS analysis was performed with a Beckman Coulter flow cytometer FC500 and computer software Beckman Coulter program CXP.