Fig. 3

Fine Ag-specificity of BW58 cell lines expressing CD1d–α-GlcADAG-reactive NKT TCRs. a BW58 cell lines expressing the A11B8.2 and A10B8.2 TCRs were tested by flow cytometry for their ability to bind a panel of CD1d tetramers loaded with: Mycobacteria smegmatis natural occurring α-GlcADAG species (R-C19:0/C16:0) and two synthetic analogue variants (C16:0/C19:0) and (C18:0/C16:0); Sphingomonas spp. α-GlcACer C14:0 (GSL-1); Sphingomonas spp. α-GalAcer C14:0 (GSL-1′); Streptococcus pneumoniae α-GlcDAG C16:0/C18:1; Borrelia burgdorferi α-GalDAG C17:1/C16:0; α-GlcCer C20:2; α-GalCer C:20:2; sulfatide C24:1 and GD3. CD1d-endogenous and vehicle-loaded CD1d tetramers were included as controls. The VB8-STD type I NKT TCR⁺ and the XV19 type II NKT TCR⁺ cell lines were also included as a control. Graphs represent the mean fluorescence intensity (MFI) of CD1d tetramer staining within cells with similar TCR expression from duplicate values of n = 2 (n = 3 for A11B8.2) independent experiments and SEM. b Synthetic versions of the naturally occurring forms of α-GlcADAG bearing a methyl group with R-enantiomer or S-enantiomer (R-C19:0/C16:0 and S-C19:0/16:0) or the oleic version containing a double bond between C9 and C10 (C18:1/C16:0) or the isooleic variant (C16:0/C18:1), were loaded into CD1d tetramers and assessed for their ability to stain the A11B8.2 type II NKT TCR⁺ cell line (left), or to induce IL-2 following 16 h culture on platebound CD1d-lipid (right). Variants in which the glucoronic headgroup (–GlcA) was substituted for a glycosydic headgroup (–Glc) were also tested. S. pneumoniae α-GlcDAG (SPN α-GlcDAG), α-GalCer, vehicle-loaded and endogenous-loaded CD1d tetramers were included as controls. Graph on left represents the MFI of CD1d tetramer fold increase over CD1d-endogenous staining within cells with similar TCR expression from four independent experiments ± SEM, and graph on right depicts the mean IL-2 production of three independent experiments ± SEM