title: Analysis of in vitro immunization: generation of cytotoxic T-lymphocytes against allogeneic melanoma cells with tumor lysate-loaded or tumor RNA-transfected antigen-presenting cells.
abstract: In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from naïve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.
authors.affiliation: Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201-1379, USA.
authors.forename: Alexey L
authors.initials: AL
authors.lastname: Glazyrin
authors.forename: June
authors.initials: J
authors.lastname: Kan-Mitchell
authors.forename: Malcolm L S
authors.initials: ML
authors.lastname: Mitchell
mesh_terms: D000938:Antigen-Presenting Cells; D017209:Apoptosis; D015153:Blotting, Western; D005434:Flow Cytometry; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D008545:Melanoma / Q000276:immunology* / Q000378:metabolism / Q000628:therapy; D014442:Monophenol Monooxygenase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D013601:T-Lymphocytes; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D010455:Peptides; D012313:RNA; D014442:Monophenol Monooxygenase