STAGE-SPECIFIC INDUCTION OF TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE IN A T-LYMPHOID LINE UPON COCULTURE WITH A THYMIC STROMAL LINE

We previously reported an in vitro T-cell differentiation system in wh ich the L4 lymphoid clone was cocultured with the St3 stromal line der ived from the same murine thymic tumor, 15#4T. L4 cells in L4-St3 cocu ltures sequentially express Thy-1 and CD4 in a manner typical of norma l thymocytes. In contrast, L4 cells grown in medium alone retain their Thy-1(-)CD4(-) phenotype. We also isolated L4 subclones from the cocu lture with increasingly differentiated phenotypes with respect to Thy- 1 and CD4. We now report induction of an additional thymocyte differen tiation marker, terminal deoxynucleotidyl transferase (TdT) in 15#4T c ells (and to a lesser extent subcloned L4 cells) upon coculture with S t3 stroma. Coculture of 15#4T cells with St3 stroma resulted in expres sion of TdT as measured by ribonuclease protection for TdT RNA and Wes tern immunoblotting for TdT protein. Cocultured L4 cells were induced for TdT expression to a lesser degree and for a shorter period of time . The magnitude of TdT RNA induction was maximal for cell lines with t he least mature differentiation phenotype (15#4T and L4: Thy-1(-)CD4(- )) and decreased proportionally for subclones with increasingly mature phenotype, e.g., L4E cells (Thy-1(+)CD4(+)). TdT protein was undetect able by Western immunoblotting and immunofluorescent staining of the L 4E subclone on or off stroma. Recombination-activating gene-1 (RAG-1), which is expressed in immature thymocytes during T-cell receptor rear rangement, but suppressed in mature thymocytes, was also examined usin g the ribonuclease protection assay. In contrast to TdT, RAG-1 express ion was suppressed by coculture with St3 cells for 15#4T and also more mature subclones, indicating regulation by a mechanism independent fr om TdT. The ordered induction of TdT, Thy-1, and CD4, as well as regul ation of RAG-1 in the 15#4T-St3 system, supports the conclusion that t his in vitro system is a valuable model for characterizing regulation of these markers in normal thymocytes. (C) 1995 Academic Press, Inc.