Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.

TLDR

A cytokine that synergizes with IL‑2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally named cytotoxic lymphocyte maturation factor (CLMF), was isolated from a phorbol ester– and calcium ionophore–stimulated human B‑lymphoblastoid cell line and purified by classical and high‑performance liquid chromatography to a specific activity of 8.5 × 10⁷ units mg⁻¹ in a T‑cell growth factor assay. CLMF is a 75‑kDa heterodimer of disulfide‑linked 40‑ and 35‑kDa subunits that is unrelated to known cytokines, stimulates proliferation of PHA‑activated lymphoblasts alone and synergistically with suboptimal IL‑2, and enhances IL‑2–induced lymphokine‑activated killer cell formation.

Abstract

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human B-lymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.5 x 10(7) units/mg in a T-cell growth factor assay. Analysis of the purified protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated that CLMF is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. Determination of the N-terminal amino acid sequences of the two subunits revealed that both subunits are not related to any previously identified cytokine. Purified CLMF stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. Furthermore, the purified protein was shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells.