CYTOTOXIC T-LYMPHOCYTE RESPONSES AGAINST MUTATED P21 RAS PEPTIDES - AN ANALYSIS OF SPECIFIC T-CELL-RECEPTOR GENE USAGE

Generation of cytotoxic-T-lymphocyte (CTL) responses against mutated r as peptides from peripheral-blood mononuclear cells (PBMC) was attempt ed in a group of HLA-A2.I+ healthy donors. Bulk PBMC cultures were sti mulated in vitro with a mixture of peptides encompassing 12 Gly --> Va l, 61 Gin --> Lys or 61 Gln --> Leu ras mutations and displaying HLA-A 2.I binding motifs, selected by a computer program. A promiscuous teta nus toroid peptide was also added. Weekly thereafter, PBMC were re-sti mulated with peptide pulsed autologous Epstein-Barr-virus (EBV)-transf ormed B cells. After 8 rounds of re-stimulation, reproducible cytotoxi c activity against peptide-pulsed target cells was detectable in one d onor. The CTL line recognized 2 nonamers encompassing ras 61 Gln --> L eu mutation. Killing was mediated by CD8(+) T cells displaying alpha b eta TCR and was inhibited by anti-HLA-A2.1 monoclonal antibodies. No k illing of tumor cells expressing the specific mutation could be observ ed. More than 60 CTL clones were generated. Fine specificity studies r evealed effective, though differing cytotoxic activity against both 53 -LDILDTAGL-61 and 55-ILDTAGLEE-63, but not against 54-DILDTAGLE-62 mut ated peptides, in all but one of the clones. None was able to exert ef fective cytotoxic activity against tumor cells expressing the specific mutation. T-cell-receptor (TCR) usage was then analyzed phenotypicall y, by reverse-transcription-polymerase-chain-reaction (RT-PCR) and by sequence analysis. This study revealed the monoclonal nature of the CT L response against mutated nonamers, with TCR expressing V beta 14 gen e product in combination with, J beta 2.7 and C beta 2. (C) 1996 Wiley -Liss, Inc.