Accurate and rapid assessment of MHC antigen upregulation following cytokine stimulation.
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.
Gillott
D J
DJ
Department of Medical Oncology, Royal London Hospital, UK.
Nouri
A M
AM
Compton
S J
SJ
Oliver
R T
RT
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
D000951:Antigens, Neoplasm; D016207:Cytokines; D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; D009363:Neoplasm Proteins; D007371:Interferon-gamma