Diversity of the fine specificity displayed by HLA-A*0201-restricted CTL specific for the immunodominant Melan-A/MART-1 antigenic peptide

HLA-A*0201 melanoma patients often develop a CTL response to an immunodomin ant peptide derived from the melanocyte lineage-specific protein Melan-A/MA RT-1, We have shown previously that the antigenic peptide most often involv ed is the decapeptide Melan-A(26-35) (EAAGIGILTV). We also observed some cl onal diversity in the fine specificity of Melan-A-specific CTL, To substant iate this observation, we have now tested a series of Melan-A(26-35) varian t peptides containing single alanine substitutions for binding to HLA-A*020 1 and recognition by polyclonal and monoclonal Melan-A-specific CTL, Substi tution of several residues with alanine reduced peptide binding activity by >10-fold, In contrast, substitution of E26 with alanine (AAAGIGILTV) resul ted in a 5-fold higher binding activity as well as in stronger stability of the corresponding HLA-A*0201/peptide complexes. Interestingly, the peptide variant AAAGIGILTV was recognized more efficiently than the natural decape ptide by hort term cultured, tumor-infiltrated lymph node cell cultures and a number of Melan-A-specific CTL clones derived from different individuals , Moreover, this analysis revealed that the fine specificity of the CTL res ponse to the Melan-A immunodominant epitope is quite diverse at the clonal level, At least three distinct patterns of fine specificity were identified . This diversity appears to reflect the diversity of the TCR repertoire ava ilable for this Ag, since similar results were obtained with a panel of Mel an-A-specific CTL clones derived from a single melanoma patient, These find ings have important implications For the formulation of Melan-A peptide-bas ed vaccines as well as For the monitoring of Melan-A-specific CTL responses in melanoma patients.