HLA binding characteristics and generation of cytotoxic lymphocytes against peptides derived from oncogenic proteins.
OBJECTIVE
Structurally altered proteins (derived from chromosomal translocations or gene mutations) can be considered tumor specific antigens and represent an attractive target for a T-cell mediated response. T lymphocytes recognize antigens in the form of peptides bound to HLA-molecules.


METHODS
Peptides derived from oncogenic proteins were screened for the presence of HLA binding motifs; actual binding were evaluated by HLA stabilization experiments using transfectants and specific anti-HLA antibodies. Specific lymphocytes were induced by in vitro peptide sensitization and screened by thymidine uptake or cellular cytotoxic assays.


RESULTS
We identified peptides derived from EWS/FLI-1 fusion protein and from mutated K-RAS protein (encompassing respectively the fusion point and the mutation at position 12) that showed binding motif for HLA-Cw*0702 and HLA-A3 respectively. The actual binding of these peptides was analysed in a stabilization assay. We detected binding for the EWS/FLI-1 peptide and for 5 RAS peptides (1 wild type and 4 mutated). The effect of temperature, beta 2-microglobulin (beta 2-m) and fetal calf serum (FCS) on the binding and the stability of the HLA/peptide complex was studied. A low temperature (26 degrees C) increased the binding both in HLA-A3 and HLA-Cw*0702, while FCS reduced it. beta 2-m increased the binding to the HLA-A3 molecule but did not influence the binding to the HLA-Cw*0702. The stability of already formed complexed was somewhat different in the HLA-A3 and HLA-Cw*0702 system: both were more stable at 26 degrees C than at 37 degrees C but while the beta 2-m and FCS did not influence the stability of the HLA-A3/peptide complex, they seemed to cause opposite effects in the HLA-Cw*0702 system (beta 2-m stabilized and FCS destabilized the complex). Finally, we were able to generate a specific CD8+ CTL line against a K-RAS mutated peptide.


CONCLUSIONS
Although binding motifs and actual HLA binding can be detected in several cases, the generation of a cellular response is infrequent, confirming that HLA binding is necessary but not sufficient to obtain an in vitro response. Further optimization of culture conditions, type of Antigen Presenting Cells (APC), peptides, use of stabilizers like beta 2-m are still needed.
Bertazzoli
C
C
Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy.
Marchesi
E
E
Dermime
S
S
Ravagnani
F
F
Parmiani
G
G
Gambacorti-Passerini
C
C
D003601:Cytotoxicity Tests, Immunologic; D019084:Fluorescent Antibody Technique, Indirect; D005805:Genes, MHC Class I / Q000276:immunology*; D006680:HLA Antigens / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D015514:Oncogene Proteins, Fusion / Q000276:immunology*; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism; D014157:Transcription Factors / Q000276:immunology*; D018631:ras Proteins / Q000276:immunology*
C106031:EWS-FLI fusion protein; D006680:HLA Antigens; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors; D018631:ras Proteins; D013936:Thymidine