Figure 1. Recruitment of CD8 and imaging of the induction of TCR-CD8 FRET by agonist.

(a) OT-I hybridomas expressing CD8α wild-type, CD3ζ-CFP, and CD8β-YFP (OT-I.ZC.8βY) were incubated with Cy5-stained CHO cells expressing tetracycline-induced wild-type single-chain H2-K^bOVA (left) or a mutant that cannot bind to CD8 (right). The image of each panel shows mid-cell sections of fluorescence images (CD8β-YFP in yellow and Cy5 in purple). Scale bar 5 μm. The bottom graph shows the quantitation of CD8β-YFP recruitment to the immune synapse when scH2-K^b-OVA (black) or scH2-K^b- CD8_mut-OVA (grey) was expressed on CHO cells surface. Mean fluorescence intensity ± s.e.m., n=25 conjugates. Unpaired t-test * p<0.0001. (b) Top row shows a representative image of OT-I.ZC.8βY cells forming the immune synapse formation (CD3ζ-CFP in cyan, CD8β-YFP in yellow and CHO APC membrane labeled with Cy3.5 in red). Bottom left plot shows live FRET efficiency of OT-I.ZC.8βY cells incubated with scH2-K^b-OVA (closed circles) or –VSV (open triangles) expressing CHO cells. Data represent average FRET efficiency at the synapse. Bottom right panel shows Ca²⁺ flux from a Fluo-4 loaded OT-I cell expressing CD8α/CD8β during interaction with a CHO cell expressing scH2-K^b-OVA (circles) or –VSV (triangles). The results are representative of at least three independent experiments.