Identification and purification of human T-lymphocyte colony-enhancing factor, TLCEF: increased production by phorbol myristate acetate

• Published in: Immunology Vol. 56; no. 2; pp. 285 - 294
• Main Authors: Werber, M M, Goldman, J, Radnay, J, Klein, S, Rozenszajn, L A
• Format: Journal Article
• Language: English
• Published: England 01.10.1985
• Subjects: Chromatography, Gel; Colony-Stimulating Factors - biosynthesis; Colony-Stimulating Factors - isolation & purification; Culture Media; Humans; In Vitro Techniques; Interleukin-2 - analysis; Molecular Weight; Phytohemagglutinins - pharmacology; Proteins - analysis; T-Lymphocytes - immunology; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0019-2805; 1365-2567

T-lymphocyte colony-enhancing factor (TLCEF), a growth factor for a minute subpopulation of T lymphocytes, is produced, along with other factors, by conditioned media (CM) of mononuclear cells following stimulation with T mitogens, such as phytohaemagglutinin (PHA). A combination of PHA and a co-mitogen, phorbol 12-myristate 13-acetate (PMA), has been shown to have a synergistic effect on the production of TLCEF, yielding levels of activity eight to fifteen times higher than those obtained with either PHA or PMA alone. TLCEF was purified by ammonium sulphate, fractionation hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration. The last step of this purification procedure yielded two peaks of activity purified 13,000- and 29,000-fold, respectively. The first peak eluted from the column with an average molecular weight of 125,000, whereas the second peak was retained on the gel, probably due to non-specific interactions with it. The purified fractions contained none of the following activities: T-cell growth factor (TCGF), colony-stimulating factor-1 (CSF-1), interleukin-3 (IL-3) and interferon. TCGF activity, which is known to be unstable at high degrees of purification, was already lost after the ammonium sulphate fractionation step, most probably because of the low protein and albumin concentrations (0.33 and 0.11 mg/ml, respectively). TLCEF is thus a more stable immunoregulatory factor than TCGF and has a much higher apparent molecular weight.