PEPTIDE-SPECIFIC ACTIVATION OF CYTOLYTIC CD4(-LYMPHOCYTES AGAINST TUMOR-CELLS BEARING MUTATED EPITOPES OF K-RAS P21() T)

Alterations in the ms p21 protein have been associated with both roden t and human neoplasia. Thus, mutated I ds 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 th at mimic segments of mutated ras p21 have been reported to be immunoge nic in mice in vivo, although detailed functional analyses remains und efined. Here, in a murine model, we explored and characterized distinc t effector properties of host-derived T lymphocytes reactive to mutate d ras peptides, which was consistent with the CD4(+) T helper type 1 ( T(h)1) subset. BALB/c mice (H-2(d)) were immunized with a purified pep tide, 13 amino acids in length, containing the substitution of Gly (G1 2) to Val (V12) at position 12, which is commonly found in human carci nomas. An alpha beta T cell receptor-positive, CD3(+), CD4(+), CD8(-) T cell line was established, which expressed peptide-specific prolifer ation. Cytokine assays revealed the production of interleukin-2, inter feron-gamma, tumor necrosis factor and granulocyte-macrophage colony-s timulating factor. Moreover, antigen-specific cytotoxicity was demonst rable against: (1) Ia(d)-bearing A20 tumor cells incubated with exogen ously bound V12 peptide; and (2) A20 tumor cells transduced with the K -vas p21 oncogene encoding the corresponding point mutation. CD4(+)-me diated cytotoxicity was major histocompatibility complex (MHC) class I I-restricted, as revealed by the absence of lysis against MHC class II - P815 targets, inhibition of A20 lysis with anti-Ia(d) monoclonal ant ibodies, and induction of lysis against L cell targets transfected wit h E alpha A beta(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(h)1 response that specifically recognized tumor cells expre ssing endogenous activated K-ras epitopes, which may have implications for the development of peptide-based active immunotherapies.