Fig. 1: PE binds to 4D4 TCR+ cells.
From: Direct recognition of an intact foreign protein by an αβ T cell receptor
A BW58 cells were transduced with the 4D4 TCR sequence which was identified amongst single cell BALB/c Jα18−/− thymocytes sorted as CD1d-α-GlcADAG-tetramer-(SAV-PE)+ CD1d-α-GalCer-tetramer (SAV-BV421)- (as per Almeida et al, 2019) and assessed for binding to α-GlcADAG-, α-GalCer-, sulfatide-loaded or unloaded CD1d tetramers (SAV-PE conjugated). Flow cytometry plots show TCRβ versus GFP expression on top and mean fluorescence intensity (MFI) of SAV-PE conjugated tetramers on TCRβ+GFP+ gated cells. CD1d-α-GlcADAG-reactive (A11B8.2 and A10B8.2, CD1d-sulfatide-reactive (XV19) and CD1d-α-GalCer-reactive VB8-STD TCR-expressing clones were included as controls. B Graded amounts of plate-bound CD1d loaded with α-GlcADAG, α-GalCer or vehicle (Veh) were assessed for their ability to activate the 4D4 clone compared to A11B8.2, A10B8.2 or VB8-STD control lines. Plots show flow cytometric detection of CD69 upregulation (mean of 2 experiments) or IL-2 secretion using a capture bead assay after 16 h. C α-GalCer-loaded CD1d was tetramerised using SAV-PE (top plot) or SAV-BV421 (bottom plot) and assessed for staining of the BW58 lines expressing the 4D4 TCR (red) or control A10B8.2 TCR (blue) by flow cytometry. D SAV-PE, SAV-PE-CY7, SAV-APC, SAV-APC-CY7, SAV-BV421, and SAV-PB (all from Becton Dickinson) were assessed for their ability to stain the 4D4 (red) or the control V1168 NKT (blue) TCR+ lines. E Two distinct isoforms of non-conjugated PE (from Prozyme and from Thermo Fisher Scientific) were assessed for their ability to stain 4D4 (red) or control A10B8.2 (blue) TCR+ cell lines, and analysed in a 10% SDS-PAGE gel (top). The ability of different PE conjugates (from the indicated suppliers) to stain the same cell lines was investigated by flow cytometry (bottom). F Anti-PE mAb (50µg/ml) or G soluble 4D4 TCR, and the control A11B8.2 NKT TCR or CD1a-restricted BK6 TCR were pre-incubated with SAV-PE or PE alone and assessed for impact on staining by FACS of the 4D4 cell line. Data in Fig. 1A–G are representative of 2 independent experiments, except for the gel in E, which was one of two runs (reducing and non-reducing) from one experiment, and the BK6 control in G which was used in one experiment. Source data are provided as a Source Data file.
