Inefficient clustering of tyrosine-phosphorylated proteins at the immunological synapse in response to an antagonist peptide.

Interactions of T cells with MHC plus peptide in the peripheral lymphoid system are important for their survival. In this study we investigated further the molecular consequences of such interactions using F5 TCR transgenic mice and peptides previously shown to induce either negative or positive selection in the thymus. Following TCR ligation with the negatively selecting agonist peptide, mature CD8(+) cells proliferated and up-regulated the activation marker CD69. Interestingly, ligation of this TCR with MHC molecules loaded with high concentrations of the positively selecting peptide also resulted in the aforementioned changes, but with slower kinetics. Analysis of the biochemical changes that occur following stimulation with these peptides showed that phosphorylation of key signaling molecules, such as ZAP-70, CD3zeta, Vav, SLP-76, LAT, and ERK-1 and 2, could be detected after exposure to agonist but not antagonist peptide. Confocal microscopy, however, revealed infrequent phosphorylation 'patches' at the site of contact between T cells and APC presenting the antagonist peptide. Our data suggest that peptides capable of inducing positive selection in the thymus can be recognized by mature T cells and cause proliferation, up-regulation of CD69 and accumulation of phosphorylated proteins at the immunological synapse with low efficiency; however no phosphorylation of signaling molecules can be detected using conventional biochemical assays.