Identification of a ras Oncogene Peptide That Contains Both CD4+ and CD8+ T Cell Epitopes in a Nested Configuration and Elicits Both T Cell Subset Responses by Peptide or DNA Immunization

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2d) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8+ and CD4+ T cell epitopes in a nested configuration. This peptide reflected ras sequence 4–16, and contained the substitution of Gly to Val at position 12 {i.e., 4–16(Val12)}. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4+ proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4–16(Val12) peptide concomitantly displayed an Ag-specific CD8+ cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide {i.e., 4–12(Val12)}, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Vα- and Vβ-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Vα1 and Vβ9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4+ T cell helper peptide {i.e., 5–17(Val12)} lacking the class I N-terminus anchor site, enhanced the production of the CD8+ T cell response. Finally, immunization with plasmid DNA encoding the ras 4–16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4+ and CD8+ T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8+ CTL response, likely due to the intimate coexistence of CD4+ help, which may have implications in peptide- or DNA-based immunotherapies.