The trial was approved by the local ethical committee and closed prematurely after the clinical implementation of tyrosine kinase inhibitors. IFN-α therapy consisted of subcutaneously applied escalating doses of a 2-month induction regimen of IFN-α2b (Roferon®, Hoffman-LaRoche, Nutley, NJ, USA): 2 weeks 5 × 3 × 106; 2 weeks 5 × 6 × 106; 2 weeks 5 × 9 × 106; and 2 weeks 3 × 9 × 106 IU/week). Tumour and lymph node tissues were obtained at nephrectomy. Peripheral blood mononuclear cells (PBMC) were harvested at regular time-points pre-, during and

post-therapy by Ficoll-Hypaque, washed and resuspended in phosphate-buffered saline (PBS) complemented with 0·5% bovine serum albumin (BSA; Sigma Aldrich, Zwijndrecht, the Netherlands) and cryopreserved in liquid nitrogen for later analysis. RCC tumour cell lines were established from Raf inhibitor fresh tumour (patient

B2) or tumour-involved lymph node (patient B7) after digestion with collagenase type 4 (1 mg/ml; Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands) and expressed the epidermal growth factor receptor (EGFR) and clear cell RCC-associated G250 antigen. Established Epstein–Barr virus (EBV)-transformed B cell lines used were JY, C1R and C1R-huCD1d, the latter transduced with human CD1d (C1R and C1R-huCD1d [20], kindly provided by Dr V. Cerundolo, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK). All cell lines were cultured in RPMI-1640 (Invitrogen Life Sciences/Gibco, Invitrogen Corporation Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) Crizotinib purchase (inactivated; Greiner Bio-one GmbH, Frickenhausen, Germany), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Roche Diagnostics,

Mannheim, Germany) and were refreshed twice a week. NK T cell lines from patients B2 and B7 were established by fluorescence activated cell sorting (FACS) of cells labelled with anti-TCR Vα24 plus Vβ11 antibodies (Beckman Coulter, Woerden, the Netherlands), cultured for 1–3 weeks in serum-free Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen Life Sciences/Gibco) supplemented with 2% normal human serum (Invitrogen, Brown Deer, WI, USA), penicillin/streptomycin Pregnenolone and IJssel’s supplements [21] in the presence of IL-2 (100 U/ml; Eurocetus, Amsterdam, the Netherlands) and IL-15 (5 ng/ml, Peprotech, London, UK) and were refreshed twice a week. Tumour cell lysates were prepared from tumour cell lines or tumour-involved lymph node tissues which were suspended in 250 µl PBS, followed by snap-freezing three times and sonification on ice. IFN-γ and IL-4 ELISPOT assays were carried out according to the manufacturer’s instructions (U-cytech Biosciences, Utrecht, the Netherlands), as described previously [22]. Briefly, flat-bottomed 96-well plates (Costar 3799) were incubated with coating antibody (U-cytech) overnight at 37°C, washed with PBS and incubated with coating buffer for 2 h.