CD8/CD4 lineage commitment occurs by an instructional/default process followed by positive selection

In the present study. we investigated the developmental potential of purified populations of transitional CD4(in) CD8(hi) and CD4(hi)CD8(in) thymocytes that were further defined according to their differentiation stage by their levels of T cell receptor (TCR) expression into TCR(lo). TCR(in) and TCR(hi) subpopulations. The differentiation potential of each of these subsets was tested in vitro in a single-cell suspension culture assay that showed that CD-4(in) CD8(hi) TCR(hi) are precursors of CDS single-positive cells. whereas CD4(hi) CD8(in) TCR(inhi) are precursors of both CD4 and CD8 single-positive thymocytes. The analysis of transitional subsets in mutant mice for either beta 2-microglobulin or major histocompatibility complex (MHC) class II further revealed that lineage commitment to the CDS lineage requires a TCR-MHC class I engagement. presumably at the immature double-positive stage of thymic development. while CD4 commitment does not require an MHC class II-mediated signal. but rather occurs by default. Using the addition of MHC class I- or class II-expressing cells or the addition of total thymocytes to purified sorted transitional precursors for the duration of the cultures in vitro, we identified an additional stage of differentiation for both CD-4 and CDS lineages that requires a positive selection signal. Examination of protein tyrosine phosphorylation of transitional precursors revealed that CD4(in) CD8(hi) transitional cells contain a high level of a 70-kDa phosphorylated Protein consistent with a role for ZAP70 in the signal transduction during the positive selection of CD8(-) cells.