Selection of BW 5147 subclones devoid of non-specific suppressive activity for use in cell hybridization.

Culture supernatants of BW 5147 cells widely used for T-cell hybridization often manifest non-MHC-restricted, non-antigen-specific regulatory activities on the mixed lymphocyte reaction (MLR) of mouse cells. This report demonstrates that, whereas supernatants of BW 5147 cells grown at low concentrations (2 X 10(5)/ml) enhanced MLR, high cell concentration (2 X 10(6)/ml) supernatants markedly inhibited this reaction. BW 5147 cell-free extracts significantly inhibited MLR and in vitro antibody production (PFC), as well as the mitogenic response to lipopolysaccharide E. coli (LPS) of mouse spleen cells, but did not affect the response to an optimal dose of phytohaemagglutinin (PHA). Both supernatant and cell-free extract inhibitory activities were located in 60,000 MW fraction. Inhibitory material of low MW (less than 12,000) was also found in high cell concentration supernatants. A similar suppressive activity was exerted by cell-free extracts of P3 X 63 NS cells used for B-cell hybridization. The suppressive activity seemed to stem from some kind of interaction between BW 5147 cells and the fetal calf serum (FCS) of the culture medium. Supernatants from subclones of BW 5147 cells obtained in selected batches of FCS and maintained in the same serum, even at high cell concentrations, did not affect MLR, whereas the supernatants from the same subclones maintained in other batches definitely suppressed this reaction. Thus, provided that culture conditions are chosen carefully, subclones of BW 5147 devoid of effect on in vitro immune reactions can be obtained.