Mouse T cell clone 2C recognizes two different major histocompatibility (MHC) ligands, the self MHC K and the allogeneic MHC L. Two distinct peptides, SIY (SIYRYYGL) and QL9 (QLSPFPFDL), act as strong and specific agonists when bind to K and L, respectively. QL9 binding to MHC L is influenced by the majority of peptide side chains, distributed across the entire length of the peptide. Findings with both systems, but QL9-L in particular, suggest that many single-residue substitutions, introduced into peptides to improve their binding to MHC and thus their vaccine potential, could impair T cell reactivity due to their dual impact on TCR binding. T cell activation assays are performed to measure effects of peptide SIY and QL9 residues on T cell function.