Abstract
The correlation between surface phenotype and function in subpopulations of murine thymocytes was investigated using flow microfluorometry (FMF). C57BL/6 thymocytes stained with monoclonal antibodies directed against Lyt-2, H-2Kb, and Thy-1.2 and passed on a fluorescence-activated cell sorter (FACS II flow cytometer) could be resolved into at least 4 distinct subpopulations on the basis of fluorescence and forward light scatter measurements: (a) medium-sized Lyt-2+ cells that stained strongly with H-2Kb and weakly with Thy-1.2 (5% of total cells); (b) medium-sized Lyt-2- cells with other properties as in (a) (10%); (c) small Lyt-2+ cells that stained weakly with H-2Kb and strongly with Thy-1.2 (60%); and (d) large Lyt-2+ cells that stained weakly with H-2Kb and very strongly with Thy-1.2 (23%). Cortisone-resistant (CR) thymocytes were found to correspond phenotypically to populations (a) and (b). The distribution of cytolytic T lymphocyte precursors (CTL-P) directed against H-2d alloantigens in subpopulations of C57BL/6 thymocytes that were sorted according to the phenotypic criteria described above was then investigated. CTL-P in sorted and control populations were quantitated by limiting dilution analysis of mixed leukocyte microcultures established in an excess of interleukin 2 (IL-2). All thymus CTL-P apparently could be quantitatively recovered in a subpopulation of cells that was CR, medium-sized, Lyt-2+, H-2Kb+ and weakly stained with Thy-1.2. In parallel studies, the production of IL-2 by subpopulations of C57BL/6 thymocytes was quantitatively assessed using a sensitive microassay system. Graded numbers of sorted or control thymocytes were stimulated with irradiated T cell-depleted allogeneic cells and assayed for their ability to support the growth of an IL-2-dependent cytolytic T lymphocyte clone. IL-2 production was found to reside entirely in a subpopulation of CR, medium-sized, Lyt-2- thymocytes. Further phenotypic analysis of this subpopulation of cells indicated that it was homogeneously H-2Kb+ and weakly staining with Thy-1.2. Thus, subpopulation of thymocytes with a mature phenotype (i.e., Cr, medium-sized, H-2Kb+ and weakly staining with Thy-1.2) accounts for all the functional activity in the thymus. Reasons for the apparent discrepancy between these results and other recent studies are discussed.