THE SELECTIVE GROWTH OF HUMAN LYMPHOCYTE-T COLONIES FROM WHOLE-BLOOD IN A SEMISOLID CULTURE SYSTEM

  • 1 January 1981
    • journal article
    • research article
    • Vol. 9  (9) , 926-937
Abstract
Human T lymphocyte colonies may be selectively grown from whole blood in a single phase semisolid culture system following stimulation with PHA-P [phytohemagglutin], Con-A [concanavalin A] or PPD. This technique eliminates the requirement for gradient-enriched lymphocyte fractions and provides a sensitive system for the study of T lymphocyte progenitors that more closely approximates the in vivo milieu. Whole blood colonies were composed of lymphoblasts and mature lymphocytes. Individual colony cells, identified as T lymphocytes, lacked lipase and specific esterase activity, formed E rosettes, did not phagocytize latex beads and were largely ANAE [.alpha.-naphthyl acetate esterase] positive. Whole blood was plated at a final concentration of 3%. Optimal mitogen/antigen concentrations were 125 .mu.g Con-A, 80 .mu.g PHA-P and 50 .mu.g PPD/ml culture media. Peak colony growth occurred between days 7 and 8. Colony formation increased as a power function over a wide range of cell concentrations (5 .times. 103-5 .times. 104 lymphocytes plated). Maximal whole blood colony formation occurred when 5 .times. 104-105 lymphocytes were plated. There was a significant increase in the cloning efficiency using whole blood as compared to gradient-separated cells. This method has wide application for the study of radiation effects, lymphocyte alterations in various disease states, antigen recognition and the induction and amplification of T cell function.