Souto-Carneiro M Margarida, Fritsch Ruth, Sepúlveda Nuno, Lagareiro M João, Morgado Nuno, Longo Nancy S, Lipsky Peter E
Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892-1560, USA.
J Immunol. 2008 Jan 15;180(2):1040-9. doi: 10.4049/jimmunol.180.2.1040.
V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-kappaB-binding sites are present in the human and mouse IgH, Igkappa, and Iglambda enhancer modules and RAG expression is controlled by NF-kappaB, it is not known whether NF-kappaB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-kappaB 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-kappaB essential modulator (NEMO/Ikkgamma). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkgamma-mediated canonical NF-kappaB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3(H) 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-kappaB 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-kappaB 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.
V(D)J重组对于产生具有广泛抗原特异性的免疫球蛋白库至关重要。尽管在人类和小鼠的免疫球蛋白重链（IgH）、免疫球蛋白κ链（Igκ）和免疫球蛋白λ链（Igλ）增强子模块中存在核因子κB（NF-κB）结合位点，且重组激活基因（RAG）的表达受NF-κB调控，但尚不清楚NF-κB在RAG介导的双链DNA断裂后是否调节V(D)J重组机制。为了阐明NF-κB在人类V(D)J重组中的作用，我们从患有X连锁无汗性外胚层发育不良伴高IgM综合征（HED-ID）的患者的单个外周B细胞中扩增免疫球蛋白基因重排，这些患者的NF-κB必需调节因子（NEMO/Iκγ）表达缺陷。从HED-ID B细胞中扩增出的无功能免疫球蛋白基因重排反映了Iκγ介导的经典NF-κB途径对V(D)J重组中特定分子机制的影响。我们发现，HED-ID B细胞的互补决定区3（CDR3）（重链）异常长，这是由于V、D和J基因座编码末端的核酸外切酶活性显著降低所致，而随机N核苷酸添加和回文突出端（P核苷酸）与对照组相当。这表明完整的经典NF-κB途径对于人类V(D)J重组过程中的正常核酸外切酶活性至关重要，而末端脱氧核苷酸转移酶、Artemis和DNA依赖性蛋白激酶催化亚基的活性不受影响。记忆B细胞的产生和体细胞高频突变明显缺陷，这证实了NF-κB在这些B细胞成熟事件中的作用。然而，初始B细胞库的选择似乎是完整的，并且部分能够纠正由异常V(D)J重组产生的缺陷。
