NKT TCR Recognition of CD1d-self Ag. (a) Antigen permissiveness by the NKT TCR. Staining of the hybridoma expressing the Vα14-Vβ8.2 TCR containing the CDR3β of 2A3-D with CD1d tetramers loaded with the indicated glycolipids or phospholipids (x axes). The MFI of CD1d tetramer staining was determined for a narrow TCR gate. Red dotted line, MFI with CD1d tetramer loaded with vehicle only (self-CD1d tetramer), blue dotted line, MFI with no addition of CD1d tetramer. Data represent the mean of two independent experiments. (b) Binding of the 2A3-D NKT TCR to mCD1d-PI as assessed by SPR. NKT TCR were injected over streptavidin immobilized mCD1d-PI and over an empty flow cell. Sensorgrams show the binding (response units, RU) of decreasing concentrations of TCR (22.5, 11.25, 5.625, 2.8125, 1.406 μM, 703, 351, 176, 87.9 43.9 nM) to the mCD1d-PI following subtraction of the control flow cell. Insets show saturation plots demonstrating equilibrium binding of NKT TCR to immobilized CD1d-PI. (c) Interactions between 2A3-D NKT TCR and PI (d) Interactions between Vβ8.2 NKT TCR and α-GC.