Cytotoxic T-lymphocyte responses against mutated P21 ras peptides: An analysis of specific T-cell receptor gene usage

Abstract

Generation of cytotoxic-T-lymphocyte (CTL) responses against mutated ras peptides from peripheral-blood mononuclear cells (PBMC) was attempted in a group of HLA-A2.1+ healthy donors. Bulk PBMC cultures were stimulated in vitro with a mixture of peptides encompassing 12 Gly → Val, 61 Gln → Lys or 61 Gln → Leu ras mutations and displaying HLA-A2.1 binding motifs, selected by a computer program. A promiscuous tetanus oxoid peptide was also added. Weekly thereafter, PBMC were re-stimulated with peptide pulsed autologous Epstein-Barr-virus (EBV)-transformed B cells. After 8 rounds of re-stimulation, reproducible cytotoxic activity against peptide-pulsed target cells was detectable in one donor. The CTL line recognized 2 nonamers encompassing ras 61 Gln → Leu mutation. Killing was mediated by CD8+ T cells displaying αβ TCR and was inhibited by anti-HLA-A2.1 monoclonal antibodies. No killing of tumor cells expressing the specific mutation could be observed. More than 60 CTL clones were generated. Fine specificity studies revealed effective, though differing cytotoxic activity against both 53 LDILDTAGL-61 and 55-ILDTAGLEE-63, but not against 54-DILDTAGLE 62 mutated peptides, in all but one of the clones. None was able to exert effective cytotoxic activity against tumor cells expressing the specific mutation. T-cell-receptor (TCR) usage was then analyzed phenotypically, by reverse-transcription polymerase-chain-reaction (RT-PCR) and by sequence analysis. This study revealed the monoclonal nature of the CTL response against mutated nonamers, with TCR expressing Vβ 14 gene product in combination with, Jβ2.7 and Cβ2.