Identification and partial purification of human T lymphocyte colony-enhancing factor (LCEF), distinct from T cell-growth factor

Abstract

Colony growth of T lymphocytes in a soft agar culture system, following stimulation with phytohaemagglutinin (PHA), is enhanced by T cell-derived conditioned medium (CM). The putative enhancing factor present in this CM has been termed lymphocyte colony-enhancing factor (LCEF). The purification of LCEF from CM has been complicated by the fact that active CM could be obtained only in the presence of 7-8 mg/ml of serum proteins. In order to circumvent this problem, CM which was shown to be active in the colony formation assay, was produced in the presence of a serum fraction precipitated by 1.6 M ammonium sulphate (AS). The first step of the purification of this CM was fractionation by 1.6 M AS, whereby the active material was fully recovered in the supernatant. The 20-fold purified material was applied on a DEAE-cellulose column. Two peaks of activity were obtained using the colony formation assay. The purification achieved by this second step was 5-10 fold for the first peak, eluting around 0.06 M NaCl, and two- to three-fold for the second peak, eluting around 0.19 M NaCl. Two peaks of activity were also obtained upon hydrophobic chromatography on a phenyl-Sepharose column, with enhancement factors of 5-8 for the first peak and 25-60 for the second peak. These results imply the presence of more than one LCEF molecule in the original CM. None of the LCEF-active fractions contained any T cell-growth factor (TCGF) activity; LCEF seems therefore to be a molecular entity distinct from TCGF.