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 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-2Kd; 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-2Kd was demonstrated with the mutant ras4-12(V12) peptide, but not the normal ras4-12(G12) peptide, which specifically inhibited an H-2Kd-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-2d) mice injected with ras4-12 (V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2Kd 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 ras4-12(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.
Abrams
S I
SI
Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, MD 20892-1750, USA.
Stanziale
S F
SF
Lunin
S D
SD
Zaremba
S
S
Schlom
J
J
D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation / Q000235:genetics; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects*; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D000939:Epitopes / Q000235:genetics* / Q000494:pharmacology*; D005260:Female; D005807:Genes, Overlapping / Q000276:immunology; D011905:Genes, ras / Q000276:immunology*; D006183:H-2 Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000145:classification / Q000187:drug effects*; D018631:ras Proteins / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*
D000939:Epitopes; D006183:H-2 Antigens; D018631:ras Proteins