CD8 CD4 LINEAGE COMMITMENT OCCURS BY AN INSTRUCTIONAL/DEFAULT PROCESSFOLLOWED BY POSITIVE SELECTION/

In the present study. we investigated the developmental potential of p urified populations of transitional CD4(in) CD8(hi) and CD4(hi)CD8(in) thymocytes that were further defined according to their differentiati on 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 suspens ion culture assay that showed that CD-4(in) CD8(hi) TCR(hi) are precur sors of CDS single-positive cells. whereas CD4(hi) CD8(in) TCR(inhi) a re precursors of both CD4 and CD8 single-positive thymocytes. The anal ysis of transitional subsets in mutant mice for either beta 2-microglo bulin or major histocompatibility complex (MHC) class II further revea led that lineage commitment to the CDS lineage requires a TCR-MHC clas s I engagement. presumably at the immature double-positive stage of th ymic development. while CD4 commitment does not require an MHC class I I-mediated signal. but rather occurs by default. Using the addition of MHC class I- or class II-expressing cells or the addition of total th ymocytes to purified sorted transitional precursors for the duration o f the cultures in vitro, we identified an additional stage of differen tiation for both CD-4 and CDS lineages that requires a positive select ion signal. Examination of protein tyrosine phosphorylation of transit ional precursors revealed that CD4(in) CD8(hi) transitional cells cont ain a high level of a 70-kDa phosphorylated Protein consistent with a role for ZAP70 in the signal transduction during the positive selectio n of CD8(-) cells.