Self-reactive T cells respond to low peptide concentrations. (A) Proliferation of self-reactive and influenza-specific (HA:D7, BA-8) T cell clones. B cells were co-cultured with T cells for 72 h and peptides were added in serial dilutions in triplicates. Proliferation was measured by [³H]-thymidine incorporation. Graphs show the mean ± SD. Data are representative of at least two independent experiments. The peptide concentration required for half-maximal proliferation (EC₅₀) is indicated for each clone. (B) IL-2 secretion by self-reactive and influenza-specific T cells. T cells and B cells were co-cultured in the presence of 0.1 or 1 µM peptide. After 24 h, supernatants were subjected to IL-2 quantification. Graphs show the mean ± SD of triplicate measurements. Data are representative of at least two independent experiments. (C and D) TCR down-regulation and CD69 up-regulation on the cell surface. T cells were stimulated with peptide-pulsed B cells; at the indicated time points cells were stained with anti-CD3 and anti-CD69 antibodies and analyzed by FACS. MFI values were normalized to the MFI before stimulation. Graphs show the mean ± SD of at least two independent experiments.