Fig. 5

Ligand binding cooperativity depends on TCR nanoclusters adopting the Active conformation. a OT-1 T cells were incubated with 10 mM methyl-β-cyclodextrin (MβCD) or vehicle for 30 min at 37 °C, cooled to 0 °C and preincubated with 1 nM OVAp-APC tetramer for the indicated times, then incubated with 5 nM OVAp-PE for 5 additional min. Cells were fixed and stained with the anti-TCRβ antibody H57 to assess TCR levels. Left panel: TCR expression at each time point of preincubation with OVAp-APC for non-treated and MβCD-treated cells. Middle panel: OVAp-APC tetramer binding at different times of preincubation. Right panel: OVAp-PE tetramer binding during 5 min as a function of preincubation time with the OVAp-APC tetramer. b Cartoon of a TCR indicating the position of the Cys80 residue mutated to glycine in the extracellular domain of CD3ε. c CD8 + OT-1 T cells expressing wild type or C80G mutant CD3ε were incubated at 0 °C with the indicated concentrations of OVAp-PE tetramer for 5 min. Cells were fixed and stained with the anti-CD3 antibody 2C11 to assess TCR expression (left panel). Tetramer binding data (right panel) was obtained from WT and CD3ε C80G cell populations, and from a WT subpopulation gated for TCR expression similar to CD3ε C80G cells (WT_norm, left panel). d CD8 + OT-1 T cells expressing wild type or C80G CD3ε were preincubated at 0 °C with 1 nM OVAp-APC tetramer for the indicated times and with 5 nM OVAp-PE for 5 additional minutes. Cells were fixed and stained with the anti-TCRβ antibody H57 to normalise TCR levels. Left panel: OVAp-APC tetramer binding at different times of preincubation. Right panel: OVAp-PE tetramer binding during 5 min for all preincubation times in WT C80G OT-1 cells. OVAp-APC and OVAp-PE tetramer binding is compared between CD3ε C80G mutant cells and CD3ε WT cells normalised for equal TCR expression as in c. All data shown in Fig. 5 represent the mean ± s.d. of triplicate datasets; *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.00005 (2-tail unpaired t-test)