Characterization of T cell epitopes restricted by HLA-DP9 in streptococcal M12 protein.

Interaction of the HLA-DP9 (DPA1*0201/DPB1*0901) molecule and M protein of serotype 12 (SS95/12) streptococci, a main component of the streptococcal cell wall Ag, has been investigated to decipher peptide-binding capacity and T cell activation in the context of the HLA-DP molecule. Seven antigenic peptides (amino acids 19-25) restricted by the HLA-DP9 molecule were identified in M12 protein, using M12 protein- or peptide-specific T cell lines from naturally exposed individuals. The binding affinity of each peptide to the HLA-DP9 molecule was measured by fluorescence intensity of biotinylated peptides bound to L cell transfectants expressing HLA-DP9, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to the HLA-DP9 molecule was inhibited by an excess amount of corresponding nonbiotinylated peptides and other nonbiotinylated peptides, indicating that the peptides were bound to the HLA-DP9 molecule at a single binding site. Seven synthetic peptides containing the T cell epitopes restricted by the HLA-DP9 molecule had high binding affinity to the HLA-DP9 molecule. Comparison of the amino acid sequences of truncated analogues that could bind to the HLA-DP9 molecule and/or activate T cells suggested an HLA-DP9-specific binding motif, composed of a positively charged residue (R or K) at position 1, a hydrophobic residue (A, G, or L) at position 6, and another hydrophobic residue (L or V) at position 9. Analysis of single amino acid-substituted analogues suggested that the positively charged amino acid in the motif served as a key anchor residue for binding to the HLA-DP9 molecule, which differs from the binding motif to the HLA-DR molecules.