Regulation of the induction of colonies in vitro by normal human lymphocytes.

Abstract

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...

Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

“…Another possibility is that lipopolysaccharide (endotoxin) may be present in CM and cause formation of colonies. This seems unlikely since the mitogenic effect apears to be exerted predominantly on B cells (15), and recent experiments have shown that although lipopolysaccharide potentiated the colony-inducing activity of mitogenic factors such as PHA, it did not induce colony formation de novo (16). Furthermore the cell lines used in this study were free from bacterial contamination, and the CM did not contain endotoxin as judged by the limulus assay (17).…”

Growth of Human T Lymphocyte Colonies From Whole Blood: Culture Requirements and Applications

“…The presence of erythrocytes in the culture system may contribute to the enhanced growth of colonies from WB. Addition of RBC [9,13,14] or RBC membrane preparations [I51 to lymphocyte cultures has been previously reported to potentiate T lymphocyte colony formation. It is possible that soluble mediators or membrane interactions may be responsible for this enhancement.…”

Ph¹-Negative T Lymphocytic Colonies in Agar Cultures of Peripheral Blood in Chronic Myeloid Leukemia

“…It has been shown that human lympho cytes can be induced to form colonies in semisolid culture media [8,11,20]. T cell colony formation in agar culture has been thus evaluated under various experimental conditions [2,12,19,21], and is currently investigated in several hematological diseas es [3,5,6,9,10,16].…”