Biochemical identification of a mutated human melanoma antigen recognized by CD4(+) T cells

CD4(+) T cells play a critical role in generating and maintaining immune re sponses against pathogens and alloantigens, and evidence suggests an import ant role for them in antitumor immunity as well. Although major histocompat ibility complex class II-restricted human CD4(+) T cells with specific anti tumor reactivities have been described, no standard method exists for cloni ng the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spec trometry sequencing techniques and molecular cloning to isolate a unique me lanoma Ag recognized by a CD4+ tumor-infiltrating lymphocyte (TIL) line. Th e HLA-DR beta 1*0101-restricted Ag was determined to be a mutated glycolyti c enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detec ted in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity wa s enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutat ed amino acid residue was a T cell receptor contact. Defining human tumor A g recognized by T helper cells may provide important dues to designing more effective immunotherapies for cancer.