Figure 2.

Kinetics of peptide/L^d binding by soluble, high-affinity TCR m6. (A) Representative SPR sensorgrams of soluble m6 TCR binding to QL9/L^d. Experiments were performed by immobilizing QL9/L^d/Ig by capture with rabbit anti–mouse IgG followed by injections of purified m6 TCR (900 nM, 400 nM, 200 nM, PBS blank; duplicate runs shown from top to bottom, respectively). TCR binding to the null peptide, MCMV/L^d/Ig, immobilized on the chip was undetectable (data not shown). (B) Soluble m6 TCR dissociation from four different cell-bound peptide/L^d complexes. Peptide-loaded T2-L^d cells were incubated with full length m6 TCR for 30 min. After washing, cells were resuspended at room temperature in buffer containing excess competing Ab (F23.2 at 300 μg/ml). At various times, cell aliquots were washed and remaining cell bound m6 TCR was detected by flow cytometry with F23.1–biotin followed by SAv:PE. Data points were fit with an exponential equation to determine dissociation rate constants. Representative data is shown for the peptides QL9 (•), QL9-Y5 (▪), p2Ca (▴), and QL9-M5 (▾) (inset). The dissociation rate constants (k_d) determined for the m6 TCR and pMHC ligands were: QL9/L^d, 8.5 × 10⁻⁴ s⁻¹ (n = 3); QL9-Y5/L^d, 5.9 × 10⁻⁴ s⁻¹ (n = 2); p2Ca/L^d, 1.2 × 10⁻³ s⁻¹ (n = 3); QL9-M5/L^d, 5.0 × 10⁻³ s⁻¹ (n = 2). The t_1/2 values calculated from these data were: QL9/L^d, 830 s; QL9-Y5/L^d, 1,180 s; p2Ca/L^d, 580 s; QL9-M5/L^d, 140 s. MFU, mean fluorescence units.