Characterization of triple negative clones isolated from post-natal human thymus

Human triple negative (CD3- CD4- CD8-) thymocytes and double negative (CD4- CD8-) thymocytes purified from post-natal thymus were cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells and PHA and expanded with IL-2. The cloning efficiency of triple negative thymocytes was less than 1% and the majority of the clones were triple negative. One out of 11 clones was TCR alpha beta+ CD4+. No TCR gamma delta+ clones were isolated. On the other hand, the cloning efficiency of double negative thymocytes was about 10% and most of the clones isolated were TCR gamma delta+. We could not find any evidence of in vitro differentiation of triple negative thymocytes into TCR gamma delta+ cells. Some of the triple negative clones expressed CD16 brightly and were apparently NK cells. All CD16- clones isolated from triple negative thymocytes, however, expressed NKH1, which is also an NK cell marker. Cytoplasmic CD3-delta and CD3-epsilon Ag which have been reported to be expressed in the most immature thymocytes were not detected in any of these clones. Furthermore, the CD16- triple negative clones exhibited significant cytolytic activity against K562. Phenotype of the clones seems to be stable under various conditions in vitro including coculture with human thymic epithelial cells. These data indicate that the CD16- triple negative clones isolated from triple negative thymocytes are similar to a minor subset of NK cells which is CD16- NKH-1+. It is not clear whether they originated from a distinct subset of mature or immature NK cells resident in the thymus tissue or from common precursors of both T and NK lineage.