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 γ-globulin (NP-CGG) and the adjuvant aluminum-hydroxide (alum) have been proposed to be mature B cells that reexpress RAG after an antigen encounter in the germinal center (GC), a notion supported by findings of RAG expression in peripheral B lymphocyte populations activated in vitro. However, recent 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 cells do appear in the spleen after the administration of alum alone, and that their appearance is independent of T cell interactions via the CD40 pathway. Moreover, splenic RAG⁺ B lineage cells were detectable in immunized RAG2-deficient mice adoptively transferred with bone marrow (BM) cells, but not with spleen cells from RAG⁺ mice. Although splenic RAG⁺ B cells express 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 (interleukin [IL]-4 and IL-7). Together, our studies indicate that RAG⁺ B lineage cells from BM accumulate in the spleen after immunization, and that this accumulation is not the result of an antigen-specific response.