Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4(+) and CD8(+)T cell responses

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous rats and. thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here. we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CDS cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4-12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2K(1): specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8(+) CTL: and finally. the mutant sequence overlapped with a newly characterized anti-ras CD4(+) T helper type I epitope. which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2K(d) was demonstrated with the mutant ras4-12(V12) peptide. but not the normal ras4-12(G12) peptide. which specifically inhibited an H-2K(d)-restricted. anti-nucleoprotein NP147-155 CTL response in a dose-dependent fashion. An anti-ras CD8(+) T cell line was then established from immune splenocytes of BALB/c (H-2(d)) mice injected with ras4-12(V12) in adjuvant. which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2K(d) and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene. suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12 (Gly --> Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule: the ability of the mutant. but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue: and the ras 4-12(V12) peptide was immunogenic for the production of antigen-specific CD8(+) CTL. which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.