Selection and characterization of monoclonal antibodies to the idiotype-like structure of an interleukin-2-producing human leukemia T-cell line.
A variant of the Jurkat leukemia cell line termed JA3 (surface phenotype: T11+., T3+, T4-, T8-, T6-, T1+, 3A1+, HLA-DR-, Tac-, 4F2+) has been used for mouse immunization and production of monoclonal antibodies (MAbs) to molecules carrying clonotypic determinants that are thought to serve as receptor for antigen on human T cells. JA3 had been selected because of its ability to release large amounts of IL-2 following stimulation with phytohemagglutinin (PHA) or anti-T3 antibody in the presence of phorbolmyristate acetate (PMA). This functional property has been exploited for screening of MAbs potentially directed to idiotype-like structures of JA3 cells. Thus supernatants of hybridomas (obtained by fusing mouse splenocytes with P3-UI myeloma cells) were analyzed for their ability to induce JA3 cells to release IL-2 in the presence of PMA. Four stimulatory antibodies reacted with JA3 but not with polyclonal T-cell populations, T-cell clones, or T and B tumor cell lines as assessed by indirect immunofluorescence and fluorescence-activated cell sorter (FACS) analysis. All 4 antibodies immunoprecipitated the same disulphide-linked heterodimeric molecules having a molecular mass (Mr) of approximately 85,000 under non-reducing conditions that was resolved in 2 major peptides of 40,000 and 45,000 under reducing conditions. These data indicate that these antibodies (termed anti-JTi) were directed to the clonotypic-restricted structures of the JA3 T-cell-receptor molecules. Unlike the anticlonotypic antibodies described so far, anti-JTi MAbs were capable of triggering IL-2 production even in unbound, soluble form, in the absence of adherent cells or PMA. Competitive inhibition experiments, in which 35S-labelled anti-JTi MAbs have been used, provided evidence that they may be directed against different epitopes on the same clonotypic structure.
A
A
Moretta
G
G
Pantaleo
M
M
Lopez-Botet
L
L
Moretta
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology; D002460:Cell Line; D011232:Chemical Precipitation; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007120:Immunochemistry; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D007376:Interleukin-2 / Q000096:biosynthesis; D007945:Leukemia, Lymphoid / Q000097:blood*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D000939:Epitopes; D007130:Immunoglobulin Idiotypes; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell