Cytotoxic T-lymphocyte responses against mutated p21 ras peptides: an analysis of specific T-cell-receptor gene usage

International Journal of Cancer. Journal International Du Cancer
A JureticG C Spagnoli

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 toxoid 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 alphabeta 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 t...Continue Reading

References

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