Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 is online at: average * The Journal of Immunology , 22 of which you can access for free at: 2013; 191:1210-1219; Prepublished online 3 July from submission to initial decision 4 weeks from acceptance to publication 2013; doi: 10.4049/jimmunol.1203462 http://www.jimmunol.org/content/191/3/1210 Increased ID2 Levels in Adult Precursor B Cells as Compared with Children Is Associated with Impaired Ig Locus Contraction and Decreased Bone Marrow Output Kristin Jensen, Magdalena B. Rother, Berit Sletbakk Brusletto, Ole K. Olstad, Hans Christian Dalsbotten Aass, Menno C. van Zelm, Peter Kierulf and Kaare M. Gautvik J Immunol cites 51 articles Submit online. 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The Journal of Immunology Increased ID2 Levels in Adult Precursor B Cells as Compared with Children Is Associated with Impaired Ig Locus Contraction and Decreased Bone Marrow Output Kristin Jensen,*,† Magdalena B. Rother,‡,1 Berit Sletbakk Brusletto,*,1 Ole K. Olstad,* Hans Christian Dalsbotten Aass,* Menno C. van Zelm,‡ Peter Kierulf,* and Kaare M. Gautvik*,x Precursor B cell production from bone marrow in mice and humans declines with age. Because the mechanisms behind are still unknown, we studied five precursor B cell subsets (ProB, PreBI, PreBII large, PreBII small, immature B) and their differentiation- stage characteristic gene expression profiles in healthy individual toddlers and middle-aged adults. Notably, the composition of the precursor B cell compartment did not change with age. The expression levels of several transcripts encoding V(D)J recombination Downloaded from factors were decreased in adults as compared with children: RAG1 expression was significantly reduced in ProB cells, and DNA- PKcs, Ku80, and XRCC4 were decreased in PreBI cells. In contrast, TdT was 3-fold upregulated in immature B cells of adults. Still, N-nucleotides, P-nucleotides, and deletions were similar for IGH and IGK junctions between children and adults. PreBII large cells in adults, but not in children, showed highly upregulated expression of the differentiation inhibitor, inhibitor of DNA binding 2 (ID2), in absence of changes in expression of the ID2-binding partner E2A. Further, we identified impaired Ig locus contraction in adult precursor B cells as a likely mechanism by which ID2-mediated blocking of E2A function results in reduced bone marrow http://www.jimmunol.org/ B cell output in adults. The reduced B cell production was not compensated by increased proliferation in adult immature B cells, despite increased Ki67 expression. These findings demonstrate distinct regulatory mechanisms in B cell differentiation between adults and children with a central role for transcriptional regulation of ID2. The Journal of Immunology, 2013, 191: 1210–1219. ommitment and differentiation of early precursor B cells whereas the production of other hematopoietic lineages seems to to Ig-producing B lymphocytes is a key requirement for continue unchanged (9). With advancing age, the capacity to induce C a competent adaptive immune system. Each differentia- protective Ab responses decreases, leading to reduced ability to deal tion step is tightly controlled by integrated activities of multiple with new and previously encountered pathogens (10). Some studies by guest on September 27, 2021 transcription factors in a complex gene regulatory network (1). In in mice suggest that the impact of aging on lymphoid progenitors is fact, a handful of key transcription factors is used in multiple con- first manifested in hematopoietic stem cells (11, 12). Other studies texts and distinct combinations to initiate and maintain the com- link the waning B cell production in mice to the inability of im- mitment process throughout the lifespan of the B cell (2). mature B cells to replenish the peripheral compartments (6), to Production of B lymphocytes from bone marrow (BM) continues decreased transition of ProB cells into PreB cells (6, 13), and/or to throughout life. Similar to T lymphocytes (3), the precursor B cell microenvironmental changes causing altered Ig gene rearrange- pool is decreasing with age in both humans (4, 5) and mice (6–8), ments (14, 15). The question has also been raised whether specific transcriptionally regulated mechanisms obstruct B cell generation *Department of Medical Biochemistry, Oslo University Hospital, 0407 Oslo, Nor- with age, such as changes in expression of inhibitor of DNA binding † way; Department of Pediatrics, Oslo University Hospital, 0407 Oslo, Norway; 2 (ID2) (16–20), a physiological regulator of the essential transcrip- ‡Department of Immunology, Erasmus MC, University Medical Center Rotterdam, 3015GE Rotterdam, The Netherlands; and xInstitute of Basic Medical Sciences, tion factor E2A. E2A protein levels in mice have been reported to University of Oslo, 0407 Oslo, Norway decrease with age (17), but, to date, no age-related changes were 1M.B.R. and B.S.B. contributed equally to this study. found for ID2 protein using in vitro expanded ProB/early PreB cells Received for publication December 31, 2012. Accepted for publication May 29, from aged and young mice (16). 2013. In contrast to mouse studies, the effects of aging on human This work was supported by grants from Torsteds legat, Rakel og Otto Kr. Bruuns precursor B cell development have been scarcely studied to date. legat, and Olav Raagholt og Gerd Meidel Raagholts stiftelse for forskning. This work was also supported by ZonMW/NWO Veni Grant 916.110.90 (to M.C.v.Z.). Global gene expression has only been studied in successive mat- The microarray data cited in this article were deposited in the ArrayExpress database uration stages of precursor B cells in children (21) and adults (22), (http://www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-1422. separately. We have previously reported that the total pool of Address correspondence and reprint requests to Dr. Kristin Jensen or Dr. Menno C. van precursor B cells in human BM decreased rapidly during the first Zelm, Departments of Medical Biochemistry and Pediatrics, Oslo University Hospital, 2 y of life concomitantly with reduced expression of RAG1 (5). HF Ulleva˚l, 0407 Oslo, Norway (K.J.) or Department of Immunology, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. (M.C.v.Z.). E-mail addresses: Still, these studies were unable to address the issue of decreased [email protected] (K.J.) or [email protected] (M.C.v.Z.) BM output of B cells. Therefore, we analyzed precursor B cell The online version of this article contains supplemental material. subsets and their gene expression profiles in BM from healthy Abbreviations used in this article: BM, bone marrow; CDK, cyclin-dependent kinase; young children and adults. Through differentiation stage-dependent ID2, inhibitor of DNA binding 2; IRF, IFN regulatory factor; KREC, recombination analysis of five precursor B cell subsets and pairwise comparisons excision circles. between children and adults, we identified several mechanisms that Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 suggest tighter checkpoint control in adult precursor B cells. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203462 The Journal of Immunology 1211 Materials and Methods DNA probe preparation and three-dimensional BM samples immunofluorescence in situ hybridization We obtained BM samples from healthy children aged 18 6 2 mo (mean 6 The three-dimensional DNA immunofluorescence in situ hybridization was range) and healthy adults aged 50 6 5 y (mean 6 range). The children were performed, as described previously (23, 24), with fosmid clones 761A10 and eligible for minor surgery, and the adults for elective orthopedic surgery. 3777B2 (BACPAC Resources, Oakland, CA), BAC clones 47P23, 101G24 Both groups were hematologically healthy, and none of the middle-aged (BACPAC Resources), and 3087C18 (Open Biosystems, Huntsville, AL) adults had active inflammatory disease requiring regular anti-inflammatory recognizing regions within the human IGH locus. Probes were either di- medication. Written informed consent was obtained using protocols ap- rectly labeled with Chromatide Alexa Fluor 488-5 dUTP or Chromatide proved by the Regional Medical Research Ethics Committee of Eastern Alexa Fluor 568-5 dUTP (Invitrogen) using Nick Translation Mix (Roche Norway (REK Øst, accession number 473-02132) (https://helseforskning. Diagnostics); or they were indirectly labeled using DIG-Nick Translation etikkom.no/). The study was performed according to the Norwegian Health Mix (Roche Diagnostics). Just prior to use, the probes were precipitated and Regulations. a hybridization mixture was prepared containing 600 ng each labeled probe, 4 mg human Cot-1 DNA (Invitrogen), 5 mg salmon sperm DNA dissolved in Isolation of CD10-positive cells 23 SSC, 50% formamide, and 10% dextran sulfate. The probes were BM aspirates (∼20 ml from children and 120 ml from adults) were subjected denatured at 75˚C for 5 min prior to hybridization. m 3 6 to Ficoll density gradient centrifugation (Ficoll-Paque PLUS). CD10+ pre- Approximately 100 l1 10 cells/ml suspension of freshly sorted cursor B cells were positively selected using streptavidin-coated Dynabeads cells was directly attached to poly(L-lysine)–coated coverslips.