Growth at limiting dilution of human T cell colonies from T cell-depleted peripheral blood leukocytes

Using a new culture system, we found that up to one-fourth of nylon-wool nonadherent human peripheral blood lymphocytes (NAd) could give rise to a colony containing T cells. Even after NAd cells were depleted of T cells (rosette-forming and/or OKT3+ cells and/or OKT11+ cells), up to one-ninth of cells could still give rise to a colony containing T cells. Colonies were grown in microwell liquid cultures in a vol of 0.02 ml/culture seeded with from 2 to 40 cells. Each cell concentration was set up with 60 or more replicates, and the results were analyzed using limiting dilution theory. Growth had an absolute requirement both for lymphokine(s) present in the supernatant of PHA-stimulated peripheral blood leukocytes and for PHA. A fit to limiting dilution theory could be obtained only when heavily irradiated (3000 rad) peripheral blood leukocytes or NAd cells were also included, 300 cells/well being optimum. The system supported the development of OKT3+ and OKT11+ cells from OKT3-, OKT11-, OKM1-, and OKla1- precursors. All colonies contained some OKT3+ and OKT11+ cells, usually more than half the colony cells carrying the markers. Some colonies also contained OKTM1+ cells and OKla1+ cells at a frequency such that some colony cells must carry more than one marker.