Fig. 2: PE activates cells expressing the 4D4 TCR.
From: Direct recognition of an intact foreign protein by an αβ T cell receptor
A Plate bound or soluble versions of non-conjugated PE (from Prozyme) and various forms of SAV-conjugated PE (from different suppliers) at 50 or 100 μg/mL—respectively—were assessed for their ability to activate a BW58 cell line expressing the 4D4 TCR or a control NKT TCR (VII68). Anti-CD3 (10 μg/mL) mAb was included as a positive control for stimulation. After 16 h the supernatants were assayed for the presence of IL-2 by cytometric bead array. B Titrating amounts of plate bound SAV-PE, SAV-APC, SAV-FITC and non-conjugated PE were assessed for their ability to elicit IL-2 production by the 4D4 or the control A11B8.2 NKT TCR line. In order to investigate if PE induced activation could be prevented by a neutralising antibody or protein denaturation, the top concentration of each sample was pre-incubated with anti-PE mAb (20µg/ml), or incubated at 90 °C for 2 min. Data in A and B is representative of 2 independent experiments except for the heat-inactivated samples and the SAV-FITC samples in B which were one experiment. C After 16 h of stimulation with plate bound molecules as indicated, 4D4 (grey bar) or VII68 (white bar) cells were stained for CD44, CD69 CD25 or TCRβ surface expression. Mean Fluorescence Intensity fold variation relative to no activation, error bars represent ± SEM for each marker across 3 independent experiments, each represented by the individual dots. Single-molecule imaging of TCRβ (D), or pCD3ζ (E), clustering in BW58 thymoma cells transduced with the 4D4, 2C12 or VII68 TCRs following stimulation on a supported lipid bilayer decorated with either ICAM-1 only, ICAM-1 and SAV-PE or ICAM-1 and CD1d-α-GalCer. Inset bright-field images show thymoma cells used for single-molecule imaging. Scale bar: 5 µm. F Single-molecule data were analysed using DBSCAN across three experiments with a total number of cells (shown as dots) TCRβ /pCD3ζ for ICAM-1; 4D4 (40/42), 2C12 (40/42), VII68 (40/42), for PE; 4D4 (40/44), 2C12 (42/42), VII68 (42/40) and for CD1d-α-GalCer; 4D4 (40/45), 2C12 (39/44), VII68 (40/40). Data were expressed as mean ± SEM. One-way ANOVA was used for comparing stimulation against an ICAM-1 control. ns = not significant. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (exact P values shown in Supplementary Table 1). Source data provided as a Source Data File.
