Low molecular weight factors displaying augmenting activity for human antibody production in vitro

Dialyzable low molecular weight antibody-augmenting factors (LMAAF) were found in the culture supernatant of human tonsillar lymphocytes which were not stimulated by antigen and/or mitogen in vitro. Phagocyte-depleted nylon wool-adherent lymphocytes (M-Ny+ cells) were responsible for the release of the LMAAF. Marbrook's culture system was adopted to assay for the LMAAF. The M-Ny+ cells, which were cultured without antigen and/or without mitogen in the reservoir of Marbrook's diffusion culture vessel, released the LMAAF, which diffused across a dialysis membrane and significantly augmented the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) response of phagocyte-depleted lymphocytes (M-cells) cultured in the inner vessel. Phagocyte-depleted nylon wool-passed lymphocytes (M-Ny- cells) cultured in the reservoir could not augment the PWM-induced PFC response of the M- cells cultured in the inner vessel. The exuded fluid, which was the dialysate of the culture supernatant of the M-Ny+ cells ultrafiltrated with dialysis tubing, also enhanced the PFC response of M- cells cultured in 24-well multi plates. The exuded fluid also augmented the total IgM and IgG production of human tonsillar and peripheral blood lymphocytes measured by enzyme-linked immunosorbent assay (ELISA) systems. Gel filtration chromatography on Sephadex G-25 Superfine column showed that the LMAAF activity was demonstrated in the fractions corresponding to a molecular weight (m.w.) of 362 to 1,355 and a m.w. of 3,560 to 5,700, with a peak activity at about 4,500 dalton. The LMAAF were inactivated by treatment with proteinase K, but not by trypsin, alpha-chymotrypsin, RNase, and DNase, and were stable when treated at 56 C for 60 min. The dialysates of culture supernatants from two out of seven Epstein-Barr virus (EBV)-transformed M-Ny+ cell lines showed LMAAF-like activity. These results indicate that phagocyte-depleted nylon wool-adherent lymphocytes, possibly B cells, release low molecular weight factors displaying augmenting activity for human antibody production in vitro.