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Cytotoxic T‐lymphocyte responses against mutated p21 ras peptides: An analysis of specific T‐cell‐receptor gene usage
Author(s) -
Juretic Antonio,
JürgensGöbel Jutta,
Schaefer Christoph,
Noppen Christoph,
Willimann Thomas E.,
Kocher Thomas,
Zuber Markus,
Harder Felix,
Heberer Michael,
Spagnoli Giulio C.
Publication year - 1996
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19961115)68:4<471::aid-ijc12>3.0.co;2-8
Subject(s) - cytotoxic t cell , ctl* , t cell receptor , biology , microbiology and biotechnology , cd8 , peripheral blood mononuclear cell , monoclonal antibody , antigen , t cell , virology , antibody , immunology , immune system , in vitro , biochemistry
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 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 Vβ14 gene product in combination with, Jβ2.7 and Cβ2. © 1996 Wiley‐Liss, Inc.