For iNKT-cell identification 3–5 million mononuclear cells were stained. Human CD1d tetramers loaded with αGalCer were prepared as previously described , with staining performed according to standard protocols with the inclusion of a viability stain (Live/Dead® fixable aqua dye Invitrogen). Cells were washed twice with FACS buffer before resuspension
in BD FACS™ lysing solution. Isotype control antibodies in combination with fluorescence minus one staining protocols were used to establish gating. Samples were acquired on a three laser BD FACS Canto™ II flow cytometer using BD FACSDiva™ software version 6.1 collecting a minimum of 10 000 gated B cells or monocytes for CD1d expression or a minimum of 1 000 000 viable lymphocytes for iNKT-cell frequencies. Akt inhibitor Data were analysed using FlowJo software v8.6 (TreeStar Inc). B-cell
depleted mononuclear cells (B cells were collected for biochemical monitoring as part of a biomarker study) were washed three times in complete medium (RPMI) 1640 containing 5% human AB serum (Sigma-Aldrich, Poole, UK), 10 μM beta-mercaptoethanol, 20 μg/mL gentamycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 2 mM glutamine (all media from Invitrogen Paisley, UK)). The cells were counted and plated at 3–5 million cells in 2 mL complete medium with 100 ng/mL αGalCer. On day 4 recombinant human IL-2 was added at 20 U/mL (Peprotech EC, London, UK) and on day 6 IL-2 was increased to 500 U/mL. Cells were checked everyday and
split as required Raf inhibitor with fresh medium containing 500 U/mL IL-2 added every 2 days. Once expanded iNKT-cell frequency was checked by FACS using human CD1d/αGalCer tetramers and anti-CD3 as described above. When sufficient numbers of iNKT cells were present in the cultures iNKT cells were Histone demethylase sorted by staining with anti-CD3 antibody and CD1d/αGalCer tetramers using a MoFlo sorter (Dako Cytomation). The purified iNKT cells were then re-stimulated and expanded with 1 μg/mL PHA-P (Sigma-Aldrich) and irradiated feeder cells according to standard procedures. Purity of the cell lines was confirmed by FACS staining before use in stimulation assays and was greater than 98% tetramer positive. NPC1 genotypes of the donors used for the generation of the lines are line A not found, line B 3182T>C and 3562G>T, and line C I1061T, I1094T. Total blood lymphocytes (1–3 million cells) were washed twice with RPMI 1640 medium with 20 μg/mL gentamycin and then resuspended in 1 mL EBV containing supernatant in a T25 flask. After 24 h 9 mL of RPMI 1640 containing 15% fetal bovine serum (Biosera UK), 2 mM glutamine and 2 μg/mL cyclosporin A (New England Biolabs) was added and the cells were passaged as required once the transformed B cells started to grow out.