Figure 1.

gp250 ligand induces sustained Ca²⁺ signals in AND DP thymocytes. (a) Equilibrium binding of soluble AND TCR to biotinylated gp250–I-E^k and MCC–I-E^k ligand at various concentrations of soluble AND TCR. Sample SPR sensorgrams were used to determine binding affinity of soluble AND TCR to gp250–I-E^k (K_D > 500 μM) or MCC–I-E^k (K_D = 13.03 μM). n = 2. (b) Tetramerized biotinylated gp250–I-E^k or MCC–I-E^k staining of AND.Rag1^−/−H-2^d DP thymocytes. Hb–I-E^k and hCLIP/I-A^b tetramer (Ctrl) were used as negative controls. n = 3. (c) Fura-2 AM ratios for AND.Rag1^−/−H-2^d DP thymocytes stimulated with gp250–I-E^k, MCC–I-E^k, or Hb–I-E^k. Traces represent Fura-2 AM ratio mean ± s.e.m (n = 50) as a function of time. Curves for each cell (Supplementary Fig. 1) were adjusted to time 0 being when the Ca²⁺ influx started. Black bar indicates the time from 7.5 to 15 min used for the summary of mean ratios in (d) of 250 cells in five experiments. *P < 0.0001 by two-tailed Mann-Whitney test. (e) Erk phosphorylation of AND.Rag1^−/−H-2^d DP thymocytes induced by gp250–I-E^k, MCC– I-E^k, Hb–I-E^k at 30 min and 60 min of stimulation time.