Expression of RAGs in Peripheral B Cells outside Germinal Centers Is Associated with the Expression of CD5

Abstract

Previous studies have indicated that mature B cells reactivate secondary V(D)J recombination inside and outside the germinal center (GC) of peripheral lymphoid organs. The nature of the B cells undergoing Ig rearrangement before they enter GC is unknown. In this study, we present evidence that activated mature CD5-positive human tonsil B cells coexpress both RAG1 and RAG2 mRNA and protein, and display DNA cleavage resulting from their recombinase activity. Furthermore, in vitro activation of CD5-negative naive mature B cells by IgR and CD40 cross-linking induces expression of CD5 on a subset of cells, and leads to the up-regulation of RAG1 and RAG2 only in cells turned positive for CD5. Thus, RAG gene expression is closely related to CD5 expression outside GCs. These data suggest that CD5 is associated with receptor revision in activated mature B cells and likely to promote expression of suitable IgR capable of initiating the GC reaction.

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin‐6 to induce the expression of recombination‐activating gene in B lymphocytes

“…Once prompted by FLS-mediated signals, synovial B cells required a second signal, which was generated by IL-6. Similar combinations of factors have been reported to induce RAG expression in immature (22) and mature (23) B cells, most notably in systemic lupus erythematosus (SLE) B cells (24). The current data indicate that B cells require 2 separate signals to induce RAG gene expression, the first from transmembrane BAFF on RA FLS and the second from one or more cytokines, including IL-6.…”

“…The RNAble method (Eurobio, Paris, France) was used to extract mRNA, which was then reverse-transcribed in 20 l of SuperScript II RNase H reverse transcriptase (Invitrogen, Cergy-Pontoise, France). RAG-1 and RAG-2 mRNA were amplified by nested RT-PCR using 1 l of complementary DNA (cDNA), with the primer pairs described elsewhere (22) and Taq DNA polymerase (Invitrogen). In the first round of PCR, cDNA was amplified for 25 cycles of 30 seconds at 94°C, 1 minute at 56°C, and 1 minute at 72°C, with a final 10-minute extension at 72°C.…”

“…Individual B cells were sorted using an Epics Elite FACS instrument outfitted with an Autoclone single-cell deposition unit (Beckman Coulter). RT and nested PCR for mRNA for the RAG-1, RAG-2, and GAPDH genes were performed as described previously (22)(23)(24). Briefly, after mRNA conversion for 2 hours at 42°C with SuperScript II RNase H reverse transcriptase, a first round of PCR for 25 cycles was performed as described above, which was followed by a second round for 40 cycles.…”

“…B cells were collected from the cultures and prepared as described elsewhere (22). Cells were stained first with rabbit anti-RAG-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by FITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch) and stained second with goat anti-RAG-2 antibody (Santa Cruz Biotechnology) followed by biotinylated anti-goat IgG (Jackson ImmunoResearch) plus tetramethylrhodamine isothiocyanate (TRITC)-streptavidin.…”

“…In previous studies, we established that BCR ligation requires CD40 engagement to promote RAG gene expression in B cells (22,23). Therefore, it is possible that the cell-cell signal delivered by the RA FLS to normal B cells is mediated by CD40L.…”