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B Canonical Pathway Is Involved in the Control of the Exonucleolytic Processing of Coding Ends during V(D)J Recombination1
,
* Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892;
Instituto Gulbenkian de Ciência, Oeiras, Portugal;
Medical University of Vienna, Vienna, Austria; and
Center of Statistics and Applications, University of Lisbon, Lisbon, Portugal
V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-
B-binding sites are present in the human and mouse IgH, Ig, and Ig
enhancer modules and RAG expression is controlled by NF-B, it is not known whether NF-B regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-B in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-B essential modulator (NEMO/Ikk
). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikk-mediated canonical NF-B pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3H from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-B pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-B in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Intramural Research Program. M.M.S.-C. was partially supported by the Marie Curie Intra-European Fellowship LIF-025885 and Fundação para a Ciência e Tecnologia Fellowship SFRH/BPD/20418/2004. N.S. was supported by Fundação para a Ciência e Tecnologia Fellowship SFRH/BD/19810. M.J.L. was supported by Instituto do Emprego e Formação Profissional and Fundação Calouste Gulbenkian.
2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Room 6D47C, 9000 Rockville Pike, Bethesda, MD 20892-1560. E-mail address: lipskyp{at}mail.nih.gov
3 Abbreviations used in this paper: FR, framework region; TdT, terminal deoxynucleotide transferase; HED-ID, anhidrotic ectodermal dysplasia with hyper-IgM syndrome; NEMO, NF-B essential modulator; DNA-PKcs, DNA-dependent protein kinase catalytic subunit.
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