Fig. 3. Space, time, and kinetics of pMHC-TCR molecular binding events contribute to activation.

(A) The location, time, and duration of a subset of the pMHC-TCR binding events (color tracks) experienced by a cell (white outline) relative to NFAT activation. Scale bar, 3 μm. (B) Dwell time of all pMHC binding inputs experienced by a single T cell from initial contact (t = 0) until end of acquisition. Each line represents a single pMHC-TCR trajectory. Time of NFAT activation indicated by a purple dashed line. Data in (A) and (B) are representative of >20 independent experiments. (C) Dwell time of T102S-Atto647N-MHC (blue) and MCC-Atto647N-MHC (red) using long exposure and low-power intensity microscopy after 3- and 10-s time lapses, respectively. The mean dwell time (<τ_off>) was determined after data fitting (colored lines) and corrected for fluorophore photobleaching (gray). The probability distributions were determined from the analysis of >3000 trajectories from at least 25 cells. (D) Density-dependent change in NFAT activation for a population of AND TCR T cells on T102S pMHC (blue) and MCC (red) bilayers. Peptide densities that approach a half-maximal NFAT response were designated as threshold values [gray dashed lines, σ(MCC) = 0.12 μm⁻², σ(T102S) = 0.90 μm⁻²]. Data are from >30 cells per condition from five independent experiments.