1/5.2-specific antibody. We found that the amount of peptide required for detectable TCR internalization was reduced in the high (−9MCTL) compared with the low (−5MCTL) avidity cells (Fig. 2a). This result suggested the possibility that TCR signalling differed in the high versus low avidity cells

at a given level of pMHC. To further address the response of the lines to TCR engagement we analysed the production of IFN-γ following stimulation with immobilized anti-CD3 antibody (Fig. 2b). We found that the high and low avidity lines exhibited significant differences in the amount of anti-CD3 required to produce IFN-γ. Hence, high and low avidity cells differ in their requirement for pMHC and in their sensitivity to activation by anti-CD3 antibody. These data suggest that the sensitivity to peptide antigen may be the result of differences in the signalling that results from TCR engagement. Following initiation of TCR signalling, the AG-014699 ic50 Selleckchem BTK inhibitor cascade bifurcates,

with distinct pathways leading to increases in cytoplasmic calcium levels and phosphorylation of ERK.34,35 Both of these signals have been shown to be critical for TCR activation.35,36 We first determined whether high versus low avidity cells differed in their ability to signal for calcium uptake when cells were stimulated with titrated amounts of peptide antigen. The CTL were loaded with the calcium-sensitive dye Fluo3 AM and basal readings were obtained for 60 seconds. Antigen-presenting cells pulsed with 10−6, 10−9, or 10−12 m peptide were then added. Calcium levels were measured for an additional 240 seconds to allow CTL–APC interaction, followed by addition of extracellular Ca2+ to assess uptake from the extracellular environment. As shown in Fig. 3, high avidity CTL had detectable increases in Fluo3 at all the peptide concentrations assessed, with the levels increasing in a dose-dependent fashion. In contrast, low-avidity CTL exhibited only a minimal increase in

Fluo3 fluorescence at 10−9 m. Stimulation with APC bearing 10−6 m peptide was required to achieve calcium levels similar to those observed when high avidity cells were activated with 10−12 m peptide. EL4 cells alone failed to induce any calcium response (data not shown). However, of note, when the optimal activating peptide concentration for Sitaxentan each line was used (as defined by the lowest concentration that resulted in maximum IFN-γ levels) the two lines exhibited similar levels of calcium flux. We next assessed the kinetics and magnitude of MAPK-ERK1/2 phosphorylation over time following stimulation with a range of peptide concentrations. At the early time-point of 10 min, increases in phospho-ERK1/2 were apparent only in high avidity CTL (Fig. 4). Phosphorylation in this population was detectable at this time with all peptide concentrations, although there was a clear dose-dependent increase. Low avidity CTL exhibited a detectable increase in phospho-ERK1/2 at 30 min (Fig. 4).