Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein

Abstract

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

Fig. 1

Generation of T cells specific for ras p5–14 (Val-12) in HLA-A2.1 transgenic mice. ⁵¹Cr-labelled P815-A2.1 cells (▪) and untransfected P815 cells (○) were added to 96-well plates containing the concentration of peptide ras p5–14 (Val-12) indicated. The cytotoxic T lymphocytes (CTL) were then added at an effector-to-target ratio of 10:1. After 4 h the ⁵¹Cr released was measured.

Fig. 2

Competitive inhibition of specific lysis by the HLA-A2.1 binding peptide MP_57–68. ⁵¹Cr-labelled P815-A2.1 cells were added to wells containing the competitor peptide at the concentrations shown. After 15 min at room temperature the antigenic peptide ras p5–14 (Val-12) was added to the wells at a concentration of 10 nm, followed 15 min later by cytotoxic T lymphocyte (CTL) clone 7 at an effector-to-target ratio of 3:1. After 4 h at 37°C the ⁵¹Cr released was measured.

Fig. 3

Recognition of ras p5–14 (Val-12) presented by human HLA-A2.1⁺ cells. The cell lines shown were used as targets in a standard 4-h ⁵¹Cr-release assay. A p5–14 (Val-12)-specific clone (clone 7) was added at an effector to target ratio of 10:1, and after 4 h the supernatants were harvested and counted.

Fig. 4

The ras p5–14 (Val-12)-specific T cells do not cross-react with the p21^ras wild-type sequence 5–14 (Gly-12). ⁵¹Cr-labelled P815-A2.1 cells were added to 96-well plates containing the concentration of ras p5–14 (Val-12) (▪) and ras p5–14 (Gly-12) (○) indicated. The cytotoxic T lymphocytes (CTL) were then added at an effector-to-target ratio of 10:1. The supernatants were harvested after 4 h and the ⁵¹Cr measured.

Fig. 5

Both the Lys at position 5 and the Val at position 14 are required for recognition of ras p5–14 (Val-12). ⁵¹Cr-labelled P815-A2.1 cells were incubated with the peptides for 1 h and then the cytotoxic T lymphocytes (CTL) were added at an effector-to-target ratio of 10:1. After 4 h the supernatants were harvested and the ⁵¹Cr measured.