title: Accurate and rapid assessment of MHC antigen upregulation following cytokine stimulation.
abstract: Three human tumour cell lines, A431 (cervical), Scaber (bladder) and Fen (bladder), were studied using immunohistochemical staining (IC), radiobinding (RB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA) and dot-blot (DB) techniques in order to assess major histocompatibility antigen (MHC) induction in response to interferon-gamma (IFN-gamma). Induction of class II antigens by IFN-gamma was observed on all three cell lines using all techniques. Monoclonal antibody (Mab) staining showed that both Scaber and A431 lines were positive for intact class I (Mab W6/32), class I free heavy chain (Mab HC10) and beta 2 microglobulin (beta 2-m) (Mab BBM.1), while Fen cells were positive only with HC10. The IP technique demonstrated the presence of a 45 kDa band on precipitation of the Fen lysate with HC10 Mab, whereas no such band was observed when W6/32 was used. The DB technique confirmed the negative reaction with W6/32 and BBM.1 Mabs, while HC10 showed positive staining which was upregulated by IFN-gamma. Transfection of the Fen cells with the beta 2-m gene resulted in the surface expression of fully assembled class I molecules. The DB technique showed upregulation of class I antigens following IFN-gamma stimulation, while RB detected no significant increase in cell surface expression (t test; p = 0.104). The binding values for transfected Fen cells before and after IFN-gamma stimulation were 2000 +/- 48 and 2161 +/- 156 cpm respectively. These results demonstrate that the DB technique facilitates an accurate assessment of cytokine induced antigens, corrected against a background of total cellular protein synthesis. The ease of execution, simplicity, non-radioactive nature and economy make it the method of choice for routine screening prior to the selection of suitable patients for cytokine therapy.
authors.affiliation: Department of Medical Oncology, Royal London Hospital, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Gillott
authors.forename: A M
authors.initials: AM
authors.lastname: Nouri
authors.forename: S J
authors.initials: SJ
authors.lastname: Compton
authors.forename: R T
authors.initials: RT
authors.lastname: Oliver
mesh_terms: D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016207:Cytokines / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D008214:Lymphocytes / Q000276:immunology; D009363:Neoplasm Proteins / Q000502:physiology; D011233:Precipitin Tests; D013268:Stimulation, Chemical; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects*; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000276:immunology; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D016207:Cytokines; D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; D009363:Neoplasm Proteins; D007371:Interferon-gamma