Spontaneous generation of human CD8+ TCR alpha beta+ cells derived from precursors within the double negative...

Flow cytometric analysis demonstrated that fresh human thymocytes contain only a low level of mature CD8+ TCR alpha beta + or CD8+ TCR gamma delta + cells and they consist consist of approximately 70% double positive (DP) and approximately 10% double negative (DN) cells. These unfractionated thymocytes could be selectively expanded in vitro by stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) and PHA in the presence of IL-2. The majority of the cells expanded from unfractionated thymocytes expressed CD3, TCR alpha beta and CD8 molecules after long-term culture (18 days). When highly purified DN thymocytes were expanded over a period of 18 days in the presence of DP cells, they also co-expressed CD3, TCR alpha beta and CD8 molecules on their surface. However, when purified DN thymocytes were expanded alone, that is, in the absence of DP cells for 18 days, they expressed CD3-associated TCR gamma delta, but not CD8 or TCR alpha beta. Despite the expression of measurable levels of IL-2 alpha and beta receptors, as well as a significant level of TCR alpha beta, purified DP cells failed to proliferate. These findings provide the first evidence, in humans, that the progression of precursor cells in the DN compartment to a later stage of differentiation can be induced outside the thymus and that DP cells can affect the development of TCR expression in proliferating DN thymocytes.