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

Abstract Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras 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 CD8 + 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 d ; 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 1 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 ras 4–12(V12) peptide, but not the normal ras 4–12(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 ras 4–12(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.