Observations on the Proliferation of Human Leucocytes Cultured with Phytohaemagglutinin

Abstract

ANDA REMARKABLE finding reported recently by Nowell (1960) is the mitogenic effect of a bean extract in short-term cultures of normal human peripheral blood leucocytes. The bean material, named phytohaemagglutinin (PHA), was originally introduced by Li and Osgood (1949) for the separation of leucocytes from erythrocytes for in vitro culture. Nowell's observation is the basis of a culture method (Moorhead, Nowell, Mellman, Battips and Hungerford, 1960) that is now widely used for the study of human chromosomes. However, the nature of the PHA effect and the morphological and biochemical characteristics of the cell population on which it acts appear to be largely unexplored.Since deoxyribonucleic acid (DNA) replication is the essential biochemical basis of cellular proliferation, we directed particular attention to this aspect of cell metabolism in the culture system. During the course of these studies observations were also made on the morphological changes occurring in the cultured cells. In addition, information was sought about their oxygen requirements and about the presence of certain intracellular enzymes. MATERIALS AND METHODS Culture TechniquesPeripheral blood cultures were set UP as described by Moorhead and associates (1960); in some instances antibiotics were omitted. For the earlier experiments PHA was obtained from Difco Laboratories, Detroit, U.S.A. (Batch 0528-3 3). The later experiments were done with PHA prepared by the method of Rigas and Osgood (1955). Both preparations were dissolved in 0.89 per cent NaCl for use (10 mg. PHA per ml.). Leucocyte suspensions were also prepared using 6 per cent dextran in 0.89 NaCl (Benger) and 6 per cent human fibrinogen (Lister Institute) in 0.89 per cent NaCl to remove the erythrocytes (I part dextran or fibrinogen to 9 parts of blood).Human thoracic duct lymph lymphocytes, obtained during thoracotomy, were set up in culture by dduting the lymph to give 1.0-1.5 x 106 lymphocytes per ml. and 0.2 ml. of PHA was added to a 10 ml. culture. Isotopes and Autoradiography3H-Thymidme (1.9 c. per mmole) was used to study the metabolism of DNA. Smears were fixed either with Carnoy's reagent or in formaldehyde vapour followed by Feulgen or Giemsa staining. Kodak AR 10 stripping film was used for the autoradiography.

Kinetics and chromosome analyses of tissue culture lines derived from Burkitt lymphomata.

“…This method of analysis has been used previously for the study of euploid cells (Cooper, Barkhan and Hale, 1963;Balfour, Cooper and Meek. 1965).…”

Glucose transporter 1 expression identifies a population of cycling CD4+CD8+human thymocytes with high CXCR4-induced chemotaxis

“…Interestingly, however, GLUT1 does not appear to be expressed on either quiescent human T cells (2) or their murine counterparts (3,4). Rather, GLUT1 expression is induced upon T cell receptor (TCR) activation, a process long recognized as being associated with increased glucose metabolism (5)(6)(7)(8). We recently determined that GLUT1 is, indeed, an early marker of human T cell activation, with surface expression detected as early as 4 h after TCR stimulation (9).…”

Phosphatidylinositol 3-Kinase-dependent Modulation of Carnitine Palmitoyltransferase 1A Expression Regulates Lipid Metabolism during Hematopoietic Cell Growth

“…Together, these signal-induced changes function to direct uptake and utilization of nutrients, channeling metabolites into biosynthetic pathways (2)(3)(4). One important example of this phenomenon occurs during lymphocyte stimulation, where receptor-induced signaling directly increases glucose transport, glycolysis, lactate production, and synthesis of lipids (5)(6)(7)(8)(9).…”