ANALYSIS OF CLONES DERIVED FROM HUMAN CD7+CD4-CD8-CD3- THYMOCYTES

The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR-gamma-delta-cells, 12% of clones were TCR-alpha-beta-cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR-alpha,beta, TCR-gamma-delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR-gamma, delta, and beta-gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR-delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR-alpha-beta and TCR-gamma-delta-lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR-alpha-beta and TCR-gamma-delta-lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR-gamma-delta and NK cells.