BACKGROUND: In contrast to evidences of Ig H chain receptor editing in transformed cell lines and transgenic mouse models, there has been no direct evidence that this phenomenon occurs in human developing B cells. METHODS: V(H)DJ(H) rearrangements were obtained from genomic DNA of individual IgM- B cells from liver and IgM+B cells from bone marrow of 18 wk of gestation human fetus by PCR amplification and direct sequencing. RESULTS: We found three examples of H chain receptor editing from IgM+ and IgM+human fetal B cells. Two types of V(H) replacements were identified. The first involved V(H) hybrid formation, in which part of a V(H) gene from the initial VDJ rearrangement is replaced by part of an upstream V(H) gene at the site of cryptic RSS. The second involved a gene conversion like replacement of CDR2, in which another V(H) gene donated a portion of its CDR2 sequence to the initial VDJ rearrangement. CONCLUSION: These data provide evidence of receptor editing at the H chain loci in developing human B cells, and also the first evidence of a gene conversion event in human Ig genes.
Animals ; B-Lymphocytes* ; Bone Marrow ; Cell Line, Transformed ; DNA ; Fetus ; Genes, Immunoglobulin ; Genes, vif ; Humans* ; Liver ; Mice ; Mice, Transgenic ; Polymerase Chain Reaction ; Pregnancy

OBJECTIVETo investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-kappaB (NF-kappaB) signaling pathway and whether CD40 signaling requires TRAF2.

METHODSHuman B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2, TRAF2-shRNA, or TRAF6-shRNA. The activation of NF-kappaB was detected by Western blot, kinase assay, transfactor enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-kappaB activity was examined following stimulation with recombinant CD154.

RESULTSTRAF2 induced activity of IkappaB-kinases (IKKalpha, IKKi/epsilon), phosphorylation of IkappaBalpha, as well as nuclear translocation and phosphorylation of p65/RelA. In contrast, TRAF6 strongly induced NF-kappaB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA, but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However, the two TRAFs competed for CD40 binding.

CONCLUSIONSThese results indicate that TRAF2 can signal in human B cells, but it is not essential for CD40-mediated NF-kappaB activation. Moreover, TRAF2 can compete with TRAF6 for CD40 binding, and thereby limit the capacity of CD40 engagement to induce NF-kappaB activation.

Animals ; B-Lymphocytes ; cytology ; physiology ; CD40 Antigens ; metabolism ; Cell Line ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fluorescence Resonance Energy Transfer ; Humans ; I-kappa B Kinase ; metabolism ; NF-kappa B ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Signal Transduction ; physiology ; TNF Receptor-Associated Factor 2 ; genetics ; metabolism ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Transcription Factor RelA ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism