Reexpression of RAG-1 and RAG-2 Genes in Activated Mature Mouse B cells

Abstract

Recombination activating genes (RAG-1 and RAG-2), involved in V(D)J rearrangement of immunoglobulin genes, have been thought to be expressed only in immature stages of B-cell development. However, RAG-1 and RAG-2 transcripts were found to be reexpressed in mature mouse B cells after culture with interleukin-4 in association with several different co-stimuli. Reexpression was also detected in draining lymph nodes from immunized mice. RAG-1 and RAG-2 proteins could be detected by immunofluorescence microscopy in the nuclei of B cells cultured in vitro and in the germinal centers of draining lymph nodes. These findings suggest that RAG gene products play a heretofore unsuspected role in mature B cells.The rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes is a crucial step in the maturation process of B cells and T cells. Recombination of Ig and TCR genes is catalyzed by the products of the two recombination activating genes, RAG-1 and RAG-2 (1-3). A defect in either RAG-1 or RAG-2 results in the retention of Ig and TCR loci in germline configuration and blocks the development of mature lymphocytes (4). RAG-1 and RAG-2 have been considered to be expressed exclusively in immature lymphocytes in bone marrow and thymus, being readily down-regulated in mature lymphocyte populations, such as IgD ϩ B cells and TCR ϩ T cells (2,5,6). RAG expression in early B cells occurs in two waves, the first being responsible for V(D)J (V, variable; D, diversity; J, joining) rearrangement of Ig heavy-chain genes in proB and preB-I cells and the second for VJ recombination of Ig light-chain genes in small preB-II cells (5). We describe a third wave of RAG-1 and RAG-2 expression, induced in activated mature B cells in vitro and in vivo.Spleen B cells from C3H/HeN mice cultured for 2 days with lipopolysaccharide (LPS) plus interleukin-4 (IL-4) expressed RAG-1 and RAG-2, as assessed by the combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern (DNA) blotting (Fig. 1A). The presence of bands of correct size was strictly dependent on RT, indicating that PCR-amplified materials were not derived from contaminated genomic DNA. RAG-1 and RAG-2 expression was not detected in the cells before the culture, in unstimulated B cells, and in cells stimulated with either LPS or IL-4 alone. LPS combined with other cytokines-including IL-2, IL-3, or IL-5-was ineffective (7). To confirm that surface IgD ϩ (sIgD ϩ ) mature B cells respond to LPS plus IL-4, we undertook similar experiments in B cells purified by panning on plates coated with monoclonal antibody (mAb) to IgD (Fig. 1B). These enriched B-cell preparations also expressed RAG genes in response to LPS plus IL-4.Various stimuli other than LPS were examined for their capacity to elicit the expression of RAG genes in the presence of IL-4 (Fig. 1C). Monoclonal antibody to CD40 was as effective as LPS, and antibody to heavy chain plus 8-mercaptoguanosine, a potent B-cell activator (8), was also effective; both these stimuli were ineffective in the absen...

Recent Progress in the Understanding of B‐Cell Functions in Autoimmunity

“…These secondary rearrangements have indeed been described in a number of systems [43]. For example, it has been reported that RAG1 and RAG2 may be re-expressed in a subset of activated mature murine B cells population the GCs of draining lymph nodes [45], spleen and Peyer's patches [46]. A similar phenomenon has recently been reported to occur in human tonsillar B lymphocytes [47].…”

Antigen Receptor Editing in Anti-DNA Transitional B Cells Deficient for Surface IgM

“…RAG expression and L chain editing in the SPL have been reported earlier in immunized mice (67)(68)(69)(70)(71) and in LPS/IL-4-stimulated splenic B cells (72). However, such RAG expression and L chain editing may not occur, as initially thought, in mature splenic (germinal center) B cells.…”

Localization of recombination activating gene 1/green fluorescent protein (RAG1/GFP) expression in secondary lymphoid organs after immunization with T-dependent antigens in rag1/gfpknockin mice

“…[15][16][17][18][19][20] Recent studies proposed an interesting model in which mature B cells also undergo a similar process, termed receptor editing, in GCs of secondary lymphoid follicles after stimulation by Tdependent (TD) Ags. [21][22][23][24][25] This notion has been supported by the detection of rag1/rag2 transcripts and by the evidence of immunoglobulin gene rearrangements in situ, suggesting that receptor editing occurs in the GC light zone. 21 In addition, stimulation with anti-CD40 monoclonal antibody (mAb) plus interleukin-4 (IL-4), or lipopolysaccharide (LPS) plus IL-4, induced the up-regulation of rag1/rag2 transcripts and immunoglobulin gene rearrangements in mature surface immunoglobulin M (sIgM) ϩ B cells in vitro.…”

“…Line 5 shows intermediate GFP signal, and line 7 shows a mixture of bright positive cells and cells with various levels of GFP signal during culture in the presence of IL-7. We also examined whether the same stimuli, reported previously by others, 22,[24][25][26] …”