Induction of recombination activating gene expression in a human lymphoid progenitor cell line: requirement of two separate signals from stromal cells and cytokines.

The activation and expression of recombination activating genes (RAGs) plays critical roles in V(D)J gene recombination machinery and lymphocyte development. We showed that RAG gene expression was induced in freshly isolated human bone marrow cells and a human lymphoid progenitor cell line, FL8.2.4.4, by coculture on a monolayer of a murine bone marrow-derived stromal cell line, PA6, in the presence of a mixture of recombinant cytokines. The RAG transcripts were detected 12 hours after initiation of culture, and the increased level was sustained at 24 hours. Among recombinant cytokines, interleukin-3 (IL-3), IL-6, and IL-7, but not IL-2, IL-4, stem cell factor (SCF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) could induce RAG-1 activation in FL8.2.4.4 cells, and a significant synergistic effect between IL-3, IL-6, and IL-7 was observed. Using a double chamber culture technique, it was shown that a cognate interaction between FL8.2.4.4 cells and PA6 stromal cells was a prerequisite for RAG-1 activation. Furthermore, RAG-1 transcripts were induced in FL8.2.4.4 cells when they were cocultured on paraformaldehyde-fixed PA6 stromal cells in the presence of cytokines. These results suggest that two separate signals are both required for induction of RAG-1 activation in lymphoid progenitors; one from the cell surface molecule(s) on stromal cells, and the other from the recombinant cytokine(s). Finally, we showed that expression of RAG mRNA in FL8.2.4.4 cells was concomitant with induction of recombinase activity. This system may provide useful means for further understanding the mechanisms controlling RAG activation and lymphocyte development in the human system.