Lymphocytes isolated from normal human peripheral blood can be induced to form colonies in vitro by incubation with the appropriate inducer. Phytohemagglutinin can induce colonies with T (thymus-derived) lymphocytes. Optimum colony formation with about two colonies per 102 peripheral blood lymphocytes was obtained by adding, in addition to phytohemagglutinin, autologous plasma, autologous red blood cells, and fresh L-glutamine or L-cystine. In the absence of these fresh amino acids, no colonies were formed at seeding levels below 105 cells per 35 mm petri dish. The addition of either of these amino acids gave a 10-fold decrease in the minimum number of cells that had to be seeded for colony formation. Lipopolysaccharide did not induce the formation of colonies, but enhanced the formation of T cell colonies by phytohemagglutinin. The mixing of lymphocytes from persons with and deficient in glucose-6-phosphate-dehydrogenase (EC 1. The development of a technique for the in vitro cloning in semi-solid medium of normal macrophages and granulocytes (1-4) has made it possible to elucidate the control of growth and differentiation of these two types of white blood cells and the changes in this control in myeloid leukemia and nonmalignant granulocyte diseases (5-8). The formation of macrophage and granulocyte colonies requires a specific protein inducer which we now call MGI (macrophage and granulocyte inducer) (6) but does not seem to require the addition of a foreign antigen. However, normal lymphocytes require antigenic stimulation for differentiation in mass culture in liquid medium (9, 10), and this suggested that normal lymphocytes could be cloned in semi-solid medium by adding the appropriate antigenic inducer.We have previously shown (11) that normal lymphocytes isolated from human peripheral blood and incubated with the appropriate inducer can form colonies in semi-solid medium.The inducers used were the T (thymus-derived) cell lectin phytohemagglutinin (PHA) and the T and B (bone-marrowderived) cell lectin pokeweed mitogen (PWM indicated that colony formation requires a critical concentration of some factors produced by the seeded cells in addition to the presence of the appropriate lectin (11). The present experiments were undertaken to clarify the specific factors required for optimum colony formation, to determine the clonal origin of the colonies, and to study the inducing effect of lipopolysaccharide (LPS) and its interaction with PHA and PWM. 2-Mercaptoethanol, which has been reported to induce the formation of B cell colonies in cultures of unfractionated cells from mouse hemapoietic organs (12), did not induce the formation of colonies with isolated human peripheral blood lymphocytes (11). MATERIALS AND METHODSCollection and Isolation of Peripheral Blood Lymphocytes. Peripheral blood from healthy adult volunteers was collected in citrate/phosphate/dextrose anticoagulant solution, and the plasma and buffy coat were separated. The cells were separated from the plasma by centrifugation and resuspe...
Unfractionated human peripheral blood lymphocytes seeded at 5 × 105 cells per 35-mm Petri dish with phytohemagglutinin (PHA) can be induced to form T cell colonies with a cloning efficiency of 1%. Fractionated cells with T cell markers did not form any colonies at this seeding level. T cells were induced to form colonies either by increasing the number of cells seeded, or by adding conditioned medium (CM) from syngeneic or allogeneic T cells cultured with PHA. This CM induced a cloning efficiency of 10% at a seeding level of 5 × 103 cells per Petri dish. The induction of T cell colonies by CM did not require the presence of phagocytic or B cells. The T cell colony-inducing activity in the CM appears to be a trypsin-sensitive protein(s) and was not substituted by thymopoietin. Incubation of T lymphocytes with this inducing activity for 30 min was sufficient to induce colony formation. Colony-inducing activity for human T cells was also found in CM from normal human unfractionated lymphocytes incubated with concanavalin A, pokeweed mitogen, or lipopolysaccharide, human peripheral blood cells adherent to a Petri dish, Krebs mouse ascites tumor cells, and MGI+D+ mouse myeloid leukemia cells cultured with PHA. The results indicate that the colony-inducing activity for normal human T cells (TCI) can be found in CM from normal human T lymphocytes, human adherent cells, and some malignant mouse cells.
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