Abstract
A variant of the Jurkat leukemia cell line termed JA3 (surface phenotype: T11 +, T3 +, T4‐, T8‐, T6‐, T1 +, 3A1 +, HLA‐DR‐, Tac‐, 4FZ+) 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‐Z 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 (M′) of approximately 85,000 under non‐reducing conditions that was resolved in Z 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‐Z production even in unbound, soluble form, in the absence of adherent cells or PMA. Competitive inhibition experiments, in which ^35^S‐la‐belled anti‐JTi MAbs have been used, provided evidence that they may be directed against different epitopes on the same clonotypic structure.