FIGURE 6. Ca²⁺ signals in 1G4 CTL upon engagement with C1R cells loaded with ESO 9V and ESO 9L peptides.

[Ca²⁺]_i changes were monitored in fluo-3-loaded 1G4 CTL using a confocal microscope. A2 (A–E) or CD8-null 227/8 KA A2 (F–J) C1R cells were loaded with ESO 9V or ESO 9L peptide (5 nM or 100 nM) and added to the chamber (‘cell addition’). (A, F) Uncalibrated [Ca²⁺]_i traces are from representative single cells normalized to initial fluorescence (F/F₀) using 100 nM of the indicated peptide and have been aligned to the time of engagement (TCR–pMHC interaction). Time on the x-axis is represented to the scale shown. Basal (initial) and stimulated (final) images of fluo-3 fluorescence of a field of cells depict basal and stimulated [Ca²⁺]_I, and are aligned with the appropriate trace. (B and G) Maximal peak fluorescence changes were determined as the difference between the basal and the maximum fluorescence, ΔF/F₀ (calculated as shown in the inset schematic). (C and H) The mean elevated ratio (MER) is the mean value of fluorescence throughout the post-engagement period, as normalized to F₀. (D, E, I, J) % of total 1G4 T cells stimulated with either the ESO 9V or ESO 9L peptide with the shown MER value. The MER range is incremented with the maximum value for each range indicated on the x axis. Data are plotted as the mean ± SEM of 59–104 cells (A2 C1R cells) or 54–260 cells (A2 227/8 AK C1R cells).