CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway

Thymocytes with a low expression of CD4 and an intermediate density of the TCR (CD4low TCRint) were analyzed for phenotype, MHC dependence, production kinetics, and TCR repertoire to investigate their position in the intrathymic T cell maturation process. Comparison of normal and MHC-deficient mice showed that the CD4low TCRint cell subset was MHC class II dependent, as this subpopulation could not be defined in MHC class II- or double (class I and II)-deficient mice. These thymocytes were heat-stable Aghigh and CD69+, thus immature and recently engaged in a TCR interaction, probably with MHC class II molecules. Their generation kinetics were studied in two systems: development of exogenous bone marrow cells transferred into RAG-2-/- mice, and pulse labeling with bromodeoxyuridine. In both systems, CD4low TCRint cells were produced well before CD4low TCRhigh cells, the direct precursors of CD8 single-positive cells. Their production paralleled that of CD4high TCRint cells, but they were different than these thymocytes in their smaller cell size. Moreover, they had the same V beta 6 frequency in Mls-1a and Mls-1b mice, suggesting that these cells could be undergoing a negative selection process. The data here clearly demonstrate that CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway, and suggest that these cells could represent a MHC class II-restricted dead-end subset.