Separation of a serum-derived tumoricidal factor from a helper factor for plaque-forming cells

C3H/HeN mice administered BCG followed by lipopolysaccharide 14 days later released into their serum a cytotoxic factor for tumor cells and a factor that restored the anti-SRBC plaque-forming cell response of nude mouse spleen cells (helper activity). Gel filtration of serum containing the cytotoxic and the helper activities indicated that both factors exhibited an apparent m.w. of 125,000 to 150,000. The helper activity was also found at lower m.w. (60,000 and 13,000) suggesting the possibility that this factor existed in aggregated forms. Gel filtration of ammonium sulfate (40 to 60% saturation) precipitated serum in a high ionic strength buffer (1.6 M NaCl) resulted in shifts in the apparent m.w. of both factors. The cytotoxic factor now exhibited a m.w. of 55,000. The helper activity eluted with an apparent m.w. of 13,000, and thus was clearly separated from the cytotoxic factor. The helper activity was further shown to co-elute with macrophage-derived lymphocyte activating factor (LAF). This as well as other data represent the first demonstration of in vivo produced LAF.