We investigated responsiveness to cytokines and differentiating potential of early human T cell precursors in vitro. Human CD3- CD4- CD8- (triple negative) thymocytes were highly purified by using magnetic bead columns and cell sorting. These cells proliferated for the first 3 to 4 days and then remained viable for up to 14 days in the presence of IL-7, IL-2 or IL-4 had only limited growth-promoting activity on these cells and could not maintain the cell viability. We followed the phenotypic change of triple negative thymocytes during culture with IL-7. After 7 to 14 days of culture with IL-7, a considerable proportion became CD4+ CD8+ (double positive). These cells were found to be CD3- CD4+ CD8 alpha+ beta- in contrast to common double positive thymocytes, which express low levels of CD3 and both alpha- and beta-chains of CD8. By using four-color immunofluorescence and multi-parameter cytofluorometric analysis, we could identify this novel subset in fresh thymocytes. These results suggest that the CD3- CD4+ CD8 alpha+ beta- subset exists physiologically in the human thymus and may represent an intermediate stage between triple negative and common double positive thymocytes.

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