Putative early thymocytes, the Ly-2-L3T4-(CD8-CD4-) cells representing 3 to 4% of adult CBA mouse thymic lymphocytes, were isolated in high purity (99.5%). They were then stained by using mAb and analyzed by flow cytometry for the expression of six additional surface antigenic markers. Cross-correlation of the data obtained from a complete series of successive two-parameter analyses revealed the existence of about 11 discrete subsets, falling into four-main groups, within the Ly-2-L3T4- population. All subsets consisted of relatively large lymphoid cells. The most numerous group of Ly-2-L3T4- cells was Ly-1 low B2A2-M1/69 high Thy-1 high Pgp-1 low and by these markers resembled Ly-2+L3T4+ cortical blasts. Many of the cells in this group were positive for the IL-2R and/or for MEL-14. A second major group of Ly-2-L3T4- cells was Ly-1 high B2A2-M1/69 low Pgp-1 high, and resembled in some respects activated mature T cells. This group had previously been shown to be absent from the embryonic thymus. The group could be divided into Thy-1 high and Thy-1 low subsets. None of the cells in this group were positive for the IL-2R and very few expressed MEL-14. A third group, 13% of the Ly-2-L3T4- population, was Ly-1 low B2A2-M1/69 low Pgp-1 high, and could also be divided into Thy-1 high and Thy-1 low subsets. A final minor group, 9% of the Ly-2-L3T4- population, was Ly-1 high B2A2-M1/69 high Pgp-1 low Thy-1 high. The particular pattern of markers on these subsets, combined with subsequent information on their properties, makes it unlikely that they all represent sequential steps in one continuous developmental stream, and indicates that complex developmental steps have occurred, even at this supposedly early stage of T cell differentiation.

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