The majority of postselection CD4+ single-positive thymocytes requires the thymus to produce long-lived, functional T cells.

We have previously isolated, and characterized in vitro, two subsets of CD4hi T cell receptor (TCR)hi single positive (SP) thymocytes: CD8- and CD8lo. In this report, we have analyzed phenotypic, functional, and developmental characteristics of these "late" CD4hi SP thymocyte subsets. The TCRhi phenotype and the elimination of T cells expressing TCR V beta segments reactive with endogenous mouse mammary tumor virus (MMTV) products suggested that both subsets had undergone positive and negative selection. CD8-4hi thymocytes were functional, as judged by their ability to: (a) induce lethal graft versus host disease (GVHD); (b) survive and expand in peripheral lymphoid organs; and (c) proliferate, rather than undergo apoptosis, in response to in vitro TCR cross-linking. By contrast, CD8lo4hi cells could not induce GVHD, were unable to expand (and perhaps even survive) in peripheral organs and underwent apoptosis upon TCR cross-linking. However, when reintroduced into the thymus, these cells matured into functional, long-lived CD8-4hi lymphocytes. These results document an obligatory requirement for the thymic microenvironment in the final maturation of the majority of CD4hi SP postselection thymocytes, and demonstrate the existence of a previously unrecognized control point in T cell development.