Isolation of human leukocyte antigen (HLA)-associated peptide(s) in the absence of HLA-restricted specific cytolytic T lymphocytes. 2002

N Torabi-Pour, and A M E Nouri, and R Saffie, and W J W Morrow, and R T D Oliver
Uro/Oncology Research Unit, Department of Medical Oncology and Urology, The Royal London Trust, UK. n.torabi-pour@mds.qmw.ac.uk

METHODS In this study, immunobead purification, dot-blot, immunocytochemical staining, and SDS-PAGE techniques in combination with high-performance liquid chromatography were used to isolate human leukocyte antigen (HLA) class I antigens and associated peptides from a bladder tumour cell line (Fen) before and after gene transfection. RESULTS The results showed that: (1) Transfection of the class I negative Fen cell line with normal beta-microglobulin (beta(2)-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the beta(2)-m gene transfected cells using Sepharose CNBr-W6/32 beads. (3) Dissociation of class I antigens from beads and analysis by the SDS-PAGE showed the presence of both free heavy and light chains of class I antigens. (4) More than 22 class I-associated peptides with a molecular weight of 700-3,000 daltons could be isolated from W6/32-loaded beads but only from lysate of HLA-positive Fen cell line. The data also showed that 1 x 10(6) of positive Fen cells contained about 200 microg total protein of which about 0.10 microg was class I and about 2 ng was class I-associated peptides. CONCLUSIONS These findings demonstrated that the gene transfection approach could be used to restore missing class I antigens on an otherwise class I negative bladder tumour cell line. The results also showed the feasibility of using above techniques for isolation of HLA-associated peptides. These approaches may provide a realistic possibility for identification of putative tumour-specific peptide(s) from tumour specimens with the long-term aim to use such peptide(s) for immunotherapy in cancer patients.

UI MeSH Term Description Entries
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D001749 Urinary Bladder Neoplasms Tumors or cancer of the URINARY BLADDER. Bladder Cancer,Bladder Neoplasms,Cancer of Bladder,Bladder Tumors,Cancer of the Bladder,Malignant Tumor of Urinary Bladder,Neoplasms, Bladder,Urinary Bladder Cancer,Bladder Cancers,Bladder Neoplasm,Bladder Tumor,Cancer, Bladder,Cancer, Urinary Bladder,Neoplasm, Bladder,Neoplasm, Urinary Bladder,Tumor, Bladder,Tumors, Bladder,Urinary Bladder Neoplasm
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D013601 T-Lymphocytes Lymphocytes responsible for cell-mediated immunity. Two types have been identified - cytotoxic (T-LYMPHOCYTES, CYTOTOXIC) and helper T-lymphocytes (T-LYMPHOCYTES, HELPER-INDUCER). They are formed when lymphocytes circulate through the THYMUS GLAND and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T Cell,T Lymphocyte,T-Cells,Thymus-Dependent Lymphocytes,Cell, T,Cells, T,Lymphocyte, T,Lymphocyte, Thymus-Dependent,Lymphocytes, T,Lymphocytes, Thymus-Dependent,T Cells,T Lymphocytes,T-Cell,T-Lymphocyte,Thymus Dependent Lymphocytes,Thymus-Dependent Lymphocyte
D014162 Transfection The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES. Transfections
D014407 Tumor Cells, Cultured Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely. Cultured Tumor Cells,Neoplastic Cells, Cultured,Cultured Neoplastic Cells,Cell, Cultured Neoplastic,Cell, Cultured Tumor,Cells, Cultured Neoplastic,Cells, Cultured Tumor,Cultured Neoplastic Cell,Cultured Tumor Cell,Neoplastic Cell, Cultured,Tumor Cell, Cultured

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