Secondary V(D)J Rearrangements and B Cell Receptor-Mediated Down-Regulation of Recombination Activating Gene-2 Expression in a Murine B Cell Line

Abstract

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD− murine B cell line, 38C-13, which has previously been found to undergo κ light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vκ-Jκ and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD− B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.

The MAPK/ERK and PI(3)K Pathways Additively Coordinate the Transcription of Recombination-Activating Genes in B Lineage Cells

“…In the case of the 38c13 cells, it is unclear why Rag is continuously expressed. However, as has been shown earlier, the response of the cells to BCR ligation by suppressing Rag expression resembles that of peripheral B cells undergoing L chain gene rearrangement, rather than that of immature B cells in the bone marrow (34). We show in this study that this response is mediated through the MEK/ERK pathway.…”

“…Indeed, elevation of these signals, imposed by BCR ligation, suppresses Rag expression (this study and Ref. 34), whereas inhibition of these signals enhances the expression of Rag (this study). Overall, these findings indicate that BCR-mediated regulation of Rag gene expression in 38c13 cells is intact.…”

“…In 38c13 B lymphoma cells Rag genes are constitutively expressed, but they are rapidly suppressed upon cross-linking of the BCR (34). To study the intracellular mechanisms regulating Rag gene expression in this cell line, we first conducted kinetic experiments to quantify the suppression effect.…”

“…To study the role of ERK kinase in regulating Rag gene expression we used the 38c13 murine B cell lymphoma cell line (32), which has been shown to express Rag and to undergo L chain gene replacement (33,34). This cell line is a carcinogen-induced lymphoid tumor (35) that effectively responds to BCR signals by activation of main signaling pathways (36).…”

“…This cell line is a carcinogen-induced lymphoid tumor (35) that effectively responds to BCR signals by activation of main signaling pathways (36). Ligation of the BCR in 38c13 cells effectively downregulates Rag expression, implicating that Rag genes in these cells are under stringent regulation of BCR signaling (34). In this study, we show in 38c13 cells that BCR-mediated regulation of the Rag gene expression is additively controlled by the MEK/ERK and PI(3)K pathways, in both ligand-dependent and ligand-independent manners.…”