Colony growth of human T lymphocytes in agar: effect of a soluble factor from adherent cells

Abstract

Abstract When mononuclear cells from peripheral human blood were plated directly in agar medium containing phytohemagglutinin (PHA) and sheep red blood cells and cultured for 6–7 days, discrete colonies of 4–50 lymphocytes were formed. The plating efficiency was one colony per 50–100 cells plated, but this was reduced by approximately 85 % when adherent cells were removed from the mononuclear cell suspension prior to culture. PHA‐induced colony formation by autologous or homologous nonadherent cells could be completely restored by culturing nonadherent cells over a feeder layer of adherent cells or addition of the medium in which adherent cells had been cultured. Thus, adherent cells were presumed to secrete a stable soluble factor which supported PHA‐induced colony formation. This active factor in conditioned medium from adherent cells could be stored for several months at –10°C, and was relatively stable when heated to 56°C for 30 min. It did not bind to concanavalin A‐Sepharose, had a molecular weight greater than 10 000 daltons, was inactivated by treatment with pronase but not by DNAse, RNAse or lipase, and was active at a concentration of 1 %. Adherent cells and culture media from human peritoneal macrophages and fibroblasts did not stimulate colony formation in nonadherent cells, and likewise culture medium from nonadherent cells or purified T lymphocytes was inactive. When concentrates of conditioned medium from cultures of adherent cells were fractionated on a Sephadex G‐100 column two peaks of activity were obtained: a sharp peak with a molecular weight of approximately 100 000 daltons and a smaller peak with a molecular weight between 12 000 and 15 000 daltons. Our data indicate that a factor or factors of high molecular weight are produced by cultures of adherent mononuclear cells; these factors support the formation of PHA‐induced colonies by human T lymphocytes.