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 malig nancies and may encode unique, non-self epitopes for T cell-mediated antitu mor activity. In a BALB/c (H-2(d)) murine model, we have identified a singl e peptide sequence derived from the ras oncogenes that contained both CD8() and CD4(+) T cell. epitopes in a nested configuration. This peptide refle cted ras sequence 4-16, and contained the substitution of Gly to Val at pos ition 12 {i.e., 4-16(Val12)}. Mice immunized with this 13-mer peptide induc ed a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. I n contrast, mice inoculated with the wild-type ras sequence failed to gener ate a peptide-specific T cell. response, Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) c ytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic t umor 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 c orresponding ras codon 12 mutation, Analysis of the V alpha- and V beta -ch ains of the T cell receptor (TCR) expressed by these CTL revealed usage of the V alpha1 and V beta9 subunits, consistent with the TCR phenotype of ant i-ras Val12 CTL lines produced by in vivo immunization with the nominal pep tide 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 s ite, enhanced the production of the CD8(+) T cell response. Finally, immuni zation with plasmid DNA encoding the ras 4-16(Val12) sequence led to the in duction 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) lea ds to the generation of a quantitatively enhanced CD8(+) CTL response, like ly due to the intimate coexistence of CD4(+) help, which may have implicati ons in peptide- or DNA-based immunotherapies. (C) 2000 Academic Press.