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

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. Optimun colony formation with about two colonies per 10(2) peripheral blood lymphocytes was obtained by adding, in addition to phytohemagglutinin, autologous plasma, autoologous 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 10(5) 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 1-1-1-49) has shown that phytohemagglutinin-induced colonies can be derived from single cells and are, therefore, clones. No colonies were formed by lethally irradiated cells. Incubation with pokeweed mitogen also induced the formation of colonies. With autologous plasma and autologous red blood cells, pokeweek mitogen induced about one colony per 5 X 10(3) cells seeded and no colonies at seeding levels below 10(5) cells per petri dish. The minimum number of cells needed for colony formation by pokeweed mitogen was not decreased by fresh L-glutamine or L-cystine. The results indicate that normal human lymphocytes can be cloned in vitro and that induction of these lymphocyte colonies can be regulated by lectins and other specific factors.