Risk of cross-reactivity by affinity-matured TCR. Engineered TCRs with artificially high affinity require high peptide concentration to stimulate T-cell responses (black titration curve indicates stimulation with cognate peptide). This is because of the long half-life of binding, which prevents sequential engagement of several TCRs with a single HLA/peptide ligand. Cross-reactive peptides are expected bind the same TCR with reduced affinity and binding half-life and may fall into the optimal range for sequential TCR engagement that is required for T-cell activation at low peptide concentration. Hence, the red titration curve indicates that the affinity-matured TCR is triggered by a lower concentration of cross-reactive peptide compared with cognate peptide (black curve).