ACCURATE AND RAPID ASSESSMENT OF MHC ANTIGEN UP-REGULATION FOLLOWING CYTOKINE STIMULATION

Three human tumour cell lines, A431 (cervical), Scaber (bladder) and F en (bladder), were studied using immunohistochemical staining (IC), ra diobinding (RB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA) and dot-blot (DB) techniques in order to assess major h istocompatability antigen (MHC) induction in response to interferon-ga mma (IFN-gamma). Induction of class II antigens by IFN-gamma was obser ved on all three cell lines using all techniques. Monoclonal antibody (Mab) staining showed that both Scaber and A431 lines were positive fo r intact class I (Mab W6/32), class I free heavy chain (Mab HC10) and beta2 microglobulin (beta2-M) (Mab BBM.1), while Fen cells were positi ve 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 po sitive staining which was unregulated by IFN-gamma. Transfection of th e Fen cells with the beta2-M gene resulted in the surface expression o f fully assembled class I molecules. The DB technique showed upregulat ion of class I antigens following IFN-gamma stimulation, while RB dete cted no significant increase in cell surface expression (t test; p = 0 .104). The binding values for transfected Fen cells before and after I FN-gamma stimulation were 2000 +/- 48 and 2161 +/- 156 cpm respectivel y. These results demonstrate that the DB technique facilitates an accu rate assessment of cytokine induced antigens, corrected against a back ground of total cellular protein synthesis. The case of execution, sim plicity, non-radioactive nature and economy make it the method of choi ce for routine screening prior to the selection of suitable patients f or cytokine therapy.