Antigen-independent appearance of recombination activating gene (RAG)-positive bone marrow B cells in the spleens of immunized mice

Splenic B lineage cells expressing recombination activation genes (RAG(+)) in mice immunized with 4-hydroxy-3-nitrophenyl-acetyl coupled to chicken ga mma -globulin (NP-CGG) and the adjuvant aluminum-hydroxide (alum) have been proposed to be mature B cells that reexpress RAG after an antigen encounte r in the germinal center (GC), a notion supported by findings of RAG expres sion in peripheral B lymphocyte populations activated in vitro. However, re cent studies indicate that these cells might be immature B cells that have not yet extinguished RAG expression. Here, we employ RAG2-green fluorescent protein (GFP) fusion gene knock-in mice to show that RAG(+) B lineage cell s do appear in the spleen after the administration of alum alone, and that their appearance is independent of T cell interactions via the CD40 path-wa y. Moreover, splenic RAG(+) B lineage cells were detectable in immunized RA G2-deficient mice adoptively transferred with bone marrow (BM) cells, but n ot with spleen cells from RAG(+) mice. Although splenic RAG(+) B cells expr ess surface markers associated with GC B cells, we also find the same basic markers on progenitor/precursor BM B cells. Finally, we did not detect RAG gene expression after the in vitro stimulation of splenic RAG(-) mature B cells with mitogens (lipopolysaccharide and anti-CD40) and cytokines (inter leukin [IL]-4 and IL-7). Together, our studies indicate that RAG(+) B linea ge cells from BM accumulate in the spleen after immunization, and that this accumulation is not the result of an antigen-specific response.