Naturally processed HLA class II peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-MHC interactions

MHC class II heterodimers bind peptides 12-20 aa in length. The peptide fla nking residues (PFRs) of these ligands extend from a central binding core c onsisting of nine amino acids. Increasing evidence suggests that the PFRs c an alter the immunogenicity of T cell epitopes. We have previously noted th at eluted peptide pool sequence data derived from an MHC class II Ag reflec t patterns of enrichment not only in the core binding region but also in th e PFRs. We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis -associated allele HLA-DR2, pool sequence data from naturally processed lig ands were compared with the patterns of enrichment obtained by binding semi combinatorial peptide libraries to empty HLA-DR2 molecules. Naturally proce ssed ligands revealed patterns of enrichment reflecting both the binding mo tif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, po lar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed sim ilar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class It ligands. There is evidence that addition of C-terminal basi c PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope Identification and vaccine design. Th e Journal of Immunology, 2001.