Variable binding affinities of listeriolysin O peptides for the H-2Kd class I molecule

Previously we used the peptide-binding motif for the murine class I major histocompatibility complex molecule H-2K^d to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H-2K^d. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91-99, proved to be a CTL target. Using peptide binding competition assays with H-2K^d-restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H-2K^d; 2 of 11 peptides have approximately 10-fold lower affinity, while the remaining 6 peptides have no or very low affinity for H-2K^d. Single residue changes were made in the LLO 91-99 peptide and two other LLO peptides to identify non-anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H-2K^d to attempt to restimulate a secondary CTL response from L. monocytogenes-primed spleen cells. Only LLO 91-99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H-2K^d class I molecule, and only a fraction of these serve as CTL epitopes.