Abstract
In normal mice, more than 10% of thymocytes in the CD4^+^CD8^–^ and CD4^–^CD8^+^ single‐positive (SP) subsets express a medium level of CD3 on the cell surface. However, the fate of CD3^medium^ cells is unclear. The CD3^medium^ SP subpopulations might contain (i) cells in an immature stage of the pathways leading to CD3^high^ cells, (ii) cells in developmental pathways that do not lead to CD3^high^ cells, or (iii) cells that have been negatively selected. We found that sorted CD3^medium^ CD4^+^CD8^–^ thymocytes from adult mice up‐regulated CD3 to high levels in reaggregation thymus organ culture. Unlike their CD3^high^ counterparts, CD3^medium^ CD4^+^CD8^–^ thymocytes wereunable to undergo chemotaxis towards the chemokines CCL19 and CCL21. CD3^medium^ thymocytes of both CD4^+^CD8^–^ and CD4^–^CD8^+^ subsets were also considerably more responsive than CD3^high^ SP cells to apoptotic signals induced in vitro by ligation of CD95 (Fas/APO‐1) or by dexamethasone. In both SP subsets, a higher frequency of thymocytes expressing forbidden Vβ^+^ T cell receptors reactive with endogenous mammary tumor virus superantigens was found in CD3^medium^ subpopulations than in CD3^high^ subpopulations.These findings argue that the CD3^medium^ SP thymocyte subpopulations contain apoptosis‐susceptible precursor cells of CD3^high^ SP cells and are subject to negatively selecting pressures.