Colony formation by subpopulations of human T lymphocytes. III. Antigenic phenotype and function of colony-forming cells, colony cells, and their expanded progeny

The antigenic phenotype of individual PHA-induced T lymphocyte colonies was studied with a direct immunofluorescence technique using fluorescein-labeled anti-Leu-2a and anti-Leu-3a antibodies. Of the colonies grown from mononuclear peripheral blood cells 85% were Leu-3a+ (inducer/helper phenotype), 12% were Leu-2a+ (suppressor/cytotoxic phenotype), and 3% contained equal numbers of Leu-2a+ and Leu-3a+ cells. Fluorescence-activated cell sorter (FACS) separated T-cell subsets showed that Leu-2a+ cells and Leu-3a+ cells form exclusively Leu-2a+ and Leu-3a+ colonies, respectively. Leu-3a+ cells formed colonies in both the absence and presence of conditioned medium (PHA-CM), whereas colony formation by Leu-2a+ cells was absolutely dependent on PHA-CM. Mixing experiments with FACS-separated T-cell subsets showed that Leu-2a+ cells inhibit colony formation by Leu-3a+ cells in a cell dose-dependent manner both in the presence and absence of PHA-CM. Phenotype analysis of individual colonies from mixing experiments strongly suggested monoclonal proliferation in the present colony assay system. The majority of expanded T-cell colonies showed helper activity in a reverse hemolytic plaque-forming B-cell assay, although to a lesser degree as compared to that of freshly isolated T lymphocytes.