ISOLATION OF HUMAN LYMPHOCYTE MITOGENIC FACTOR

Abstract

Stimulation of sensitized human peripheral T lymphocytes with tetanus toxoid release lymphocyte mitoqenic factor (LMF). LMP induces DNA and antibody synthesis in allogenic and autologous B lymphocytes. Initial studies showed that the biologically active fraction was precipitated by 40-60% ammonium sulfate and 33-50% ethanol. It sedimented in cesium chloride with a density of 1.250. To purify the activity, 1000ml of T cell supernate obtained from cells of a single donor were concentrated twenty fold by Amicon ultrafiltration on a xm-50 membrane. Concentrated supernate was chromatographed on Sephadex G-200. The biological activity eluted post albumin. It was electrophoresed on an LKB iso-electrofocusing column. A major peak of proliferative activity was found at pH=6.7±0.1. A second minor peak of activity was found at pH=7.2±0.1. In one instance, when the major peak was assayed for its ability to initiate de novo synthesis of IgG by tonsillar B lymphocytes, it was found to cause a twofold increase of precipitable 14C activity over control. The data suggest that stimulation of human peripheral blood T lymphocytes by antigen results in production of two factors that can induce DNA synthesis in autologous or allogenic B lymphocytes. It appears that both of these factors are of molecular weight greater than 50,000. At least one of the factors increases IgG synthesis in B cells derived from human tonsils, while the other was present in concentrations too low to study.