1B). In addition the CD4 and CD8 status of the iNKT cells was investigated and there was no difference between the groups (Fig. 1C). It has previously been reported that the expression of CD1d on peripheral blood monocytes is increased in Gaucher disease and this was suggested to be due to lysosomal glycosphingolipid storage [20]. We analysed the expression of CD1d on monocytes (CD14+) and B cells (CD19+) (gating strategy, Supporting Information Fig. 2) and found no differences between the groups (Fig. 2) suggesting that in NPC1 patients and heterozygote carriers there is no alteration in cell surface CD1d expression. In

order to test the function of iNKT cells derived from NPC1 patients we generated iNKT cells lines from three patients that were co-cultured with human CD1d expressing THP1 cells that had been pulsed with three different exogenous antigens or treated with the

TLR 7/8 ligand Wnt activation Angiogenesis inhibitor R848 [22]. The response of the iNKT cells was determined by measuring IFN-γ, IL-4 and GM-CSF production in the supernatant. The three NPC1 iNKT-cell lines responded to both exogenous and endogenous ligands and produced comparable levels of the cytokines compared to a control iNKT-cell line (Fig. 3A). Finally, we investigated the ability of antigen presenting cells derived from NPC1 patients and heterozygotes to stimulate iNKT-cell lines by generating EBV transformed B-cell lines. Once established these B-cell lines down-regulated endogenous CD1d, and we therefore transduced them with a lentiviral human or mouse CD1d construct before use in antigen presentation assays. Expression of human or mouse CD1d was comparable between NPC1 and heterozygote EBV-B-cell lines but slightly lower than that of C1R, an EBV-B-cell line used

as a control (Supporting Information Fig. 3). Using the intensity of Etomidate LysoTracker green staining, which accumulates in acidic intracellular vesicles, as a measure of lysosomal storage, we confirmed that the enhanced lysosomal storage characteristic of NPC1 peripheral blood B cells [23] was retained in the NPC1 EBV-B-cell lines (Fig. 3D). We used three different iNKT-cell ligands that have been reported to require different conditions for loading onto CD1d. αGalCer loading has been reported to require access to a functional lysosomal compartment [9], Gal(α1-2)GalCer requires cleavage of the terminal galactose residue by lysosomal α-galactosidase before it can stimulate iNKT cells [15] and C20:2 can be loaded at the cell surface [24]. We found all three iNKT-cell ligands could be presented by the NPC1 and heterozygote EBV-B-cell lines transduced with human CD1d and resulted in similar or greater iNKT-cell activation compared with control C1R cells as determined by IFN-γ in the supernatant (Fig. 3B).