Expression of RAGs in Peripheral B Cells outside Germinal Centers Is Associated with the Expression of CD5 This information is current as Sophie Hillion, Alain Saraux, Pierre Youinou and Christophe of September 24, 2021. Jamin J Immunol 2005; 174:5553-5561; ; doi: 10.4049/jimmunol.174.9.5553 http://www.jimmunol.org/content/174/9/5553 Downloaded from References This article cites 58 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/174/9/5553.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Expression of RAGs in Peripheral B Cells outside Germinal Centers Is Associated with the Expression of CD51 Sophie Hillion, Alain Saraux, Pierre Youinou,2 and Christophe Jamin 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. The Journal of Immunology, 2005, 174: 5553–5561. Downloaded from econdary V(D)J rearrangement of Ig genes has been ex- process may generate cells bearing BCRs with various affinities. tensively described in immature bone marrow B cells. This The mutant cells will be selected during their differentiation into S process is referred to as receptor editing, contributes to the centrocytes Bm4 (CD38ϩϩIgDϪCD77Ϫ), depending on their BCR maintenance of immunological tolerance (1), and could rescue po- affinity for the Ag that is trapped as an immune complex on the tentially autoreactive B cells from apoptosis (2). Receptor editing surface of follicular dendritic cells. High-affinity Bm4 may interact http://www.jimmunol.org/ is initiated upon BCR engagement by Ag, resulting in the up- strongly with the Ag, process, and present it to GC T cells. These regulation of RAG gene expression, which then participates in ed- T cells will be induced to express CD40L, to secrete cytokines, and iting the autoreactive BCR-encoding Ig V(D)J rearrangements. to promote survival, proliferation, and isotype switching of the B Secondary V(D)J recombination has been shown to also occur in cells. These activated B cells will thus terminally differentiate ei- murine spleen and lymph node B cells in response to immunization ther into early memory (CD38ϩIgDϪ) and memory Bm5 (3–5). This peripheral V(D)J rearrangement, termed receptor revi- (CD38ϪIgDϪ) cells or into high-affinity Ab-forming cells. In con- sion, might contribute to the generation of high-affinity Abs in trast, autoreactive Bm4 cells would be deleted, because they do not germinal centers (GCs)3 following the process of somatic hyper- receive survival signal from T cells (11). Mutant Bm4 cells with mutation in response to stimulation by T-dependent Ags (6, 7). low affinity for Ag may have the potential to revise their receptor due by guest on September 24, 2021 There is evidence that human B cells also undergo secondary Ig to an increased RAG gene transcription (12). This could raise the recombination in the periphery (8, 9). Seven peripheral subpopu- affinity of the BCR for the Ag (8, 9). In this setting, strong BCR lations of B cells have been identified in human tonsils based on engagement would switch off RAG gene expression (13), suggesting the surface expression of CD38 and IgD in conjunction with other that receptor revision, which might be involved in affinity maturation markers (reviewed in Ref. 10), which have led to the proposition of Abs, will be terminated when BCR is strongly cross-linked. This of a model of T cell-dependent mature B cell differentiation. The process will permit the positive selection of high-affinity B cells to Ϫ ϩ Ϫ naive mature B (Bm) cells Bm1 (CD38 IgD CD23 ) and Bm2 terminally differentiate. ϩ ϩ ϩ (CD38 IgD CD23 ) would be activated in extrafollicular areas Recently, RAG expression in peripheral B cells has been ob- through interaction with interdigitating cells and Ag-specific T served outside the GC in a number of mouse models (14, 15) and cells. Activated blasts may either terminally differentiate into low- in humans (16). In the human model, RAG-positive B cells were affinity Ab-forming cells or become GC founder cells Bm2Ј found in the follicular mantle zone (FMZ) of the GCs where naive (CD38ϩϩIgDϩ). In GCs, Bm2Ј cells would differentiate into cen- ϩϩ Ϫ ϩ Bm1 and Bm2 cells are positioned (10). The T cell marker CD5 is troblast Bm3 (CD38 IgD CD77 ), in which somatic hypermu- expressed by Ͻ10% of the B cells in the adult human spleen and tation in V gene region might take place during proliferation. This Ͻ30% in lymph nodes (17). It is interesting that the CD5ϩ B cell population is also enriched in the FMZ (18, 19). Interestingly, in hen egg lysosyme (HEL)/anti-HEL transgenic Laboratory of Immunology, Brest University Medical School Hospital, Brest, France mice, CD5 expression is observed on mature anergic B cells (20). Received for publication August 25, 2004. Accepted for publication February Moreover, immature anergic B cells may activate the machinery 16, 2005. for V(D)J rearrangement to maintain B cell tolerance (21). Al- The costs of publication of this article were defrayed in part by the payment of page though CD5ϩ B cells have been shown (22) to express RAG in the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. peritoneal cavity, it is unknown whether CD5 is involved in in- 1 This work was supported by grants from Ministe`re de l’Enseignement Supe´rieur et ducing the recombination process. Mature anergic B cells that fail de la Recherche and from Acade´mie Nationale Franc¸aise de Me´decine. to rearrange may express CD5 to negatively regulate the BCR and 2 Address correspondence and reprint requests to Prof. Pierre Youinou, Laboratory of avoid elimination, yet maintaining tolerance to self-Ag. However, Immunology, Brest University Medical School Hospital, BP 824, F 29609 Brest, by raising the threshold required for activation (23), CD5 may also France. E-mail address: [email protected] contribute to the triggering of the BCR revision. This view implies 3 Abbreviations used in this paper: GC, germinal center; Bm, mature B; FMZ, fol- licular mantle zone; HEL, hen egg lysosyme; biot, biotinylated; LM-PCR, ligation- that CD5 expression in the FMZ could be induced on mature B mediated PCR; ECD, energy-coupled dye. cells with BCR affinity insufficient to enter GC. RAG expression Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 5554 ASSOCIATION OF CD5 WITH BCR REVISION would, therefore, be found in activated cells expressing CD5. The 30 s at 94°C, 1 min at 56°C, and 1 min at 72°C with a final 10-min present study was designed to test this hypothesis. extension at 72°C. The second PCR round was for 35 cycles of 30 s at 94°C, 1 min at 56°C, and 1 min at 72°C with a final extension step at 72°C for 10 min. For the GAPDH RT-PCR amplification, only one round of PCR Materials and Methods was conducted for 40 cycles. Because the GAPDH primers spanned a short Preparation of B cells intron of 100 bp, genomic DNA could be amplified, but easily distinguish- able from the specific cDNA product. In such cases, samples were ex- Tonsils were obtained from 5- to 18-year-old children undergoing routine cluded from the RAG studies. Moreover, RAG1 and RAG2 first-round tonsillectomy. Tissues were minced up, diluted in PBS, and filtered to PCR primers spanned an intron at 5168 and 1174 bp, respectively, which deplete larger cells and clumps. Filtered cells were layered onto Ficoll- thus could not amplify contaminating genomic DNA. Taken together, these ϫ Hypaque density medium and centrifuged for 30 min at 450 g. Cells precautions certify that the PCR products originate specifically from were then incubated with neuraminidase-treated SRBC for1hat4°C. mRNA. RT-PCR products were analyzed on 2% agarose gels stained with Depletion of T cells was achieved by a second round of centrifugation on ethidium bromide.