Identification of a Human CD8+ T Lymphocyte Neo-epitope Created by a ras Codon 12 Mutation Which Is Restricted by the HLA-A2 Allele

Author: Bergmann-Leitner E.S.   Kantor J.A.   Shupert W.L.   Schlom J.   Abrams S.I.  

Publisher: Academic Press

ISSN: 0008-8749

Source: Cellular Immunology, Vol.187, Iss.2, 1998-08, pp. : 103-116

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Abstract

Point mutations in the ras proto-oncogenes, notably at codon 12, are found in a high frequency of human malignancies and, thus, may be appropriate targets for the induction of tumor-specific T cell responses in cancer immunotherapy. In this study, we examined the mutant ras protein sequence reflecting the substitution of Gly to Val at position 12 as a putative point-mutated determinant for potential induction of an HLA-A2-reactive, CD8+ cytotoxic T lymphocyte (CTL) response. We identified the ras 4-12(Val12) sequence as a minimal 9-mer peptide, which displayed specific binding to HLA-A2 by T2 bioassays. Peptide binding to HLA-A2 on T2 cells was weak and required coincubation with exogenous &bgr;2-microglobulin to facilitate and enhance complex formation. In contrast, the wild-type ras 4-12(Gly12) peptide failed to bind to HLA-A2 even in the presence of &bgr;2-microglobulin, consistent with the hypothesis that the point mutation creates a C-terminus anchor residue. A CD8+ CTL line against the ras 4-12(Val12) peptide was derived in vitro from a normal HLA-A2+ donor using a model culture system consisting of T2 cells as antigen presenting cells pulsed with exogenous mutant ras peptide and &bgr;2-microglobulin plus cytokines (interleukin-2 and 12). Functional characterization of the CD8+ CTL line revealed: (1) peptide-specific and HLA-A2-restricted cytotoxicity against a panel of peptide-pulsed targets; (2) no specific lysis using the normal ras peptide sequence; (3) half-maximal lysis with exogenous peptide of ∼0.3 μM; (4) lysis of HLA-A2+ B cell lines infected with a recombinant vaccinia virus construct encoding the point-mutated human K-ras gene; and (5) specific lysis of the HLA-A2+ SW480 colon carcinoma cell line expressing the naturally occurring K-ras Val12 mutation. Maximal lysis of SW480 cells occurred following interferon (IFN)-&ggr; pretreatment, which correlated with enhanced HLA-A2 and ICAM-1 (CD54) expression. Specificity of lysis was revealed by the absence of lysis against a HLA-A2+ melanoma cell line (±IFN-&ggr;), which lacked the mutant Val12 mutation, and the inability of an irrelevant CD8+ CTL line to lyse SW480 (±IFN-&ggr;) unless the appropriate exogenous peptide was added. These findings demonstrated that tumor cells may endogenously process and express mutant ras epitopes, such as the 4-12(Val12) sequence, albeit in limiting amounts that may be potentiated by IFN-&ggr; treatment. These data support the biological relevance of this sequence and, thus, may have important implications for the generation of ras oncogene-specific CTL responses in clinical situations.

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