Peptide-specific activation of cytolytic CD4+ T lymphocytes against tumor cells bearing mutated epitopes of K-ras p21

Alterations in the ras p21 protein have been associated with both rodent and human neoplasia. Thus, mutated ras p21 proteins may bear unique antigenic epitopes for immune recognition, such as by T cells, which have been implicated in host antitumor activity. Synthetic peptides that mimic segments of mutated ras p21 have been reported to be immunogenic in mice in vivo, although detailed functional analyses remains undefined. Here, in a murine model, we explored and characterized distinct effector properties of host-derived T lymphocytes reactive to mutated ras peptides, which was consistent with the CD4⁺ T helper type 1 (T_h1) subset. BALB/c mice (H-2^d) were immunized with a purified peptide, 13 amino acids in length, containing the substitution of Gly (G12) to Val (V12) at position 12, which is commonly found in human carcinomas. An αβ T cell receptor-positive, CD3⁺, CD4⁺, CD8^? T cell line was established, which expressed peptide-specific proliferation. Cytokine assays revealed the production of interleukin-2, interferon-γ, tumor necrosis factor and granulocytemacrophage colony-stimulating factor. Moreover, antigen-specific cytotoxicity was demonstrable against: (1) Ia^d-bearing A20 tumor cells incubated with exogenously bound V12 peptide; and (2) A20 tumor cells transduced with the K-ras p21 oncogene encoding the corresponding point mutation. CD4⁺-mediated cytotoxicity was major histocompatibility complex (MHC) class II-restricted, as revealed by the absence of lysis against MHC class II^? P815 targets, inhibition of A20 lysis with anti-Ia^d monoclonal antibodies, and induction of lysis against L cell targets transfected with EαAβ^d. Independent isolation of a second CD4⁺ V12 line revealed a very similar cytolytic and MHC class II-restricted profile. Overall, these data demonstrated that peptide immunization produced a CD4⁺ T_h1 response that specifically recognized tumor cells expressing endogenous activated K-ras epitopes, which may have implications for the development of peptide-based active immunotherapies.