Single or multicellular origin of human T lymphocyte colonies in vitro: modification by 12-o-tetradecanoylphorbol 13-acetate (TPA).

Abstract

Abstract The assumption that human T lymphocyte colonies have a unicellular origin has been directly tested with peripheral blood mononuclear cells from 2 women heterozygous for the common X-linked glucose-6-phosphate dehydrogenase (G-6-PD) gene (GdB) and the variant GdA. T cells were cultured in semisolid medium in the presence of phytohemagglutinin (PHA) and T lymphocyte growth factor with or without preincubation in suspension culture with PHA (2-stage and 1-stage assays, respectively). The enzyme type of individual T cell colonies was then determined electrophoretically at the lowest colony density with adequate growth (usually less than 100 colonies/dish). In the 2-stage system, 90 of 97 tested colonies had equal amounts of A and B enzyme activities suggesting multicellular origin of the colonies. Similarly, in the single-stage system, 21 of 31 colonies had both A and B enzymes. Increasing the density of the soft agar did not influence the frequency of A/B colonies. However, when 12-O-tetradecanoylphorbol 13-acetate (TPA), a promoter of T cell colony growth shown in other systems to inhibit metabolic cooperation, was added, a striking decrease in frequency of colonies with both G-6-PD types was found. In the 2-stage culture, 0 of 9 colonies had a double-enzyme type and in the single-stage system, the frequency of A/B colonies declined to 9 of 34 (p less than 0.025). The data suggest that despite the apparent multicellular origin of T cell colonies in cultures with TPA, most colonies do originate from single cells when cultured with TPA at low colony densities. Stimulation of cell growth or inhibition of metabolic cooperation between cells by TPA are possible explanations for these differences.