Fig. 6.

Ligand binding induces changes in the proximity or orientation of TCRs within nanoclusters. a Schematic of the experimental set-up of Fig. 6b. FRET changes were detected upon simultaneous binding of PE- and APC-labelled OVAp tetramers. b CD8 + OT-1 T cells were incubated on ice with 5 nM each of OVAp-PE and OVAp-APC tetramers for the indicated times and fixed. FRET intensity and efficiency (left and right panels) were measured by flow cytometry. For FRET controls, OT-1 T cells were incubated with 5 nM of the OVAp-PE tetramer for the indicated times and, after fixation, cells were further incubated for 50 min with either APC-labelled anti-CD3 (positive control, C + ) or APC-labelled anti-CD27 (negative control, C-) at saturating concentrations. Arrows indicate the time points at which an upward tendency of the FRET slope was noticed. c Schematic of the experimental set-up in d–f. FRET changes were detected in CD8 + OT-1 T cells that bound PE-labelled OVAp tetramer and APC-labelled anti-Vβ5 antibody. d–f CD8 + OT-1 or OT-1xHY T cells pretreated or not with 10 mM MβCD for 30 min at 37 °C and subsequently were incubated at 0 °C with 1 nM unlabelled OVAp tetramer for the indicated times. After fixation, they were incubated for 50 min with anti-Vα2-PE and anti-Vβ5-APC (d), anti-Vβ5-PE and anti-Vβ5-APC (e) or anti-T3.70-PE and Vα2-APC (f). For all antibody combinations, a negative control is included using the donor antibody and anti-CD27-APC (C-). FRET efficiency is calculated as described in the Methods section and represented versus time of preincubation with tetramer. All data shown in Fig. 6 represent the mean ± s.d. of triplicate datasets; *p < 0.05 (2-tail unpaired t-test)