Figure 5. Comparing contact duration-dependent calcium level with adhesion parameters.

A. Two- and single-stage patterns of P_a vs. t_c plots for OT1 T cells interacting with RBCs coated with 14.7 OVA:H-2K^b/μm² or 27.7 OVA:H-2K^bα3A2/μm², respectively. Nonspecific adhesion to biotinylated RCBs without ligand coating was negligibly low. So was binding to RBCs coated with 24 VSV:H-2K^b/μm², in agreement with our previous finding that without cooperation with the TCR, CD8 binding is undetectably low at such low pMHC density (25). For each t_c, mean P_a ± SEM was measured from 10-12 cell pairs each tested by repeated cycles for 10 min. B. Mean ± SEM of AUC is plotted vs. t_c for OT1 T cells tested by repeated contact cycles for 10 min with RBCs coated with H-2K^b or H-2K^bα3A2 bound with OVA at 37°C or 25°C. C. Numbers of bonds N_b formed during each contact of a given t_c and their lifetimes t_b were calculated using P_a of each cell pair and the previously measured off-rate according to Materials and Methods (cf. Eqs. 5b and 6b, respectively). Their respective accumulations, ΣN_b and Σt_b, over the entire 10-min experimental period were calculated from 10-12 cell pairs as mean ± SEM and plotted vs. t_c. D. Adhesion events were enumerated from 10-min repeated cycles of a given t_c for OT1 T cells contacting RBCs coated with OVA:H-2K^b or OVA:H-2K^bα3A2, respectively. Biotinylated RBCs coated with VSV:H-2K^b or without ligand coating serve as negative controls for nonspecific adhesion. The mean ± SEM values from 10-12 cell pairs were plotted vs. t_c.