Determination of interaction kinetics between the T cell receptor and peptide-loaded MHC class II via single-molecule diffusion measurements

Abstract

The binding of peptide-loaded major histocompatibility complex (pMHC) to the T cell receptor (TCR) represents the central step in T cell antigen recognition. It proceeds in the cell contact area between a T cell and an antigen-presenting cell termed the immunological synapse. An important and unresolved issue is how T cells discriminate between potentially harmful and harmless antigens. One limitation has been the difficulty to measure interaction parameters directly, that is, as they occur in the immunological synapse. Here we present a single-molecule approach to determine pMHC-TCR interaction kinetics in situ based on diffusion analysis of dye-labeled pMHC. We find synaptic off-rates >10-fold accelerated when compared to the dissociation of purified proteins measured in vitro.

Figure 1

(A) Sketch of the method. (B) Diffusion was analyzed by plotting the mean-square displacement (MSD) as a function of the time-lag t_lag. It shows exemplary data obtained for (Cy5-MCC)MHC measured within (squares) or outside (circles) the interface of the bilayer with the T cell. A linear fit yielded D = 0.38 μm²/s ± 0.01 μm²/s and D_free = 0.68 μm²/s ± 0.01 μm²/s. Additionally the diffusion analysis of FRET events (shaded) yielded D_bound = 0.004 μm²/s ± 0.003 μm²/s. Data were recorded at 37°C with σ_{(Cy5−MCC)MHC} = 30 molecules/μm².

Figure 2

Mobility changes of pMHCs upon binding to the TCR. (A) The diffusion constants of (Cy5-p)MHCs were determined in the lipid bilayer outside (solid) and within the interface between the lipid bilayer and the T cell (shaded) for the peptides MCC, T102S, and β2m. σ_MCC = σ_T102S = 30 molecules/μm²; σ_β2m = 15 molecules/μm². (B) The diffusion constant of (Cy5-MCC)MHC was obtained in the IS for various surface densities (error bars smaller than marker size). (Dashed line) Fit to Eq. S1 in the Supporting Material, assuming D_free = 0.69 μm²/s, D_bound = 0 μm²/s, and σ_TCR-total = 40 molecules/μm², yielding K_D = 35 molecules/μm².

Figure 3

Binding probabilities and lifetimes for the interaction of (Cy5-MCC)MHC with the TCR. The distribution of the single-molecule diffusion constants $\overline{D}$ is shown for experiments at 28°C (A) and 37°C (C). (Shaded and solid lines) Data recorded outside and within the IS, respectively. Experiments were performed at σ = 30 molecules/μm². The data sets obtained at 28°C (B) and 37°C (D) were compared to comprehensive Monte Carlo simulations using the following fixed parameters for the simulations: D_bound = 0 μm²/s, D_free = 0.52 μm²/s (28°C), and D_free = 0.69 μm²/s (37°C). (Solid line) The 5% significance contour.