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 which the L4 lymphoid clone was cocultured with the St3 stromal line derived from the same murine thymic tumor, 15#4T.L4 cells in L4-St3 cocultures sequentially express Thy-1 and CD4 in a manner typical of normal thymocytes. In contrast, L4 cells grown in medium alone retain their Thy-1-CD4- phenotype. We also isolated L4 subclones from the coculture with increasingly differentiated phenotypes with respect to Thy-1 and CD4. We now report induction of an additional thymocyte differentiation marker, terminal deoxynucleotidyl transferase (TdT) in 15#4T cells (and to a lesser extent subcloned L4 cells) upon coculture with St3 stroma. Coculture of 15#4T cells with St3 stroma resulted in expression of TdT as measured by ribonuclease protection for TdT RNA and Western 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 the 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 undetectable by Western immunoblotting and immunofluorescent staining of the L4E subclone on or off stroma. Recombination-activating gene-1 (RAG-1), which is expressed in immature thymocytes during T-cell receptor rearrangement, but suppressed in mature thymocytes, was also examined using the ribonuclease protection assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Brightman
B K
BK
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717, USA.
Belli
B
B
Simon
M
M
Fan
H
H
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003239:Connective Tissue Cells; D004253:DNA Nucleotidylexotransferase / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins; D051379:Mice; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000201:enzymology*; D013950:Thymus Gland / Q000166:cytology*
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D011506:Proteins; D012333:RNA, Messenger; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004253:DNA Nucleotidylexotransferase