No abstract

SummaryAntibody diversity is created by imprecise joining of the variability (V), diversity (D) and joining (J) gene segments of the heavy and light chain loci. Analysis of rearrangements is complicated by somatic hypermutations and uncertainty concerning the sources of gene segments and the precise way in which they recombine. It has been suggested that D genes with irregular recombination signal sequences (DIR) and chromosome 15 open reading frames (OR15) can replace conventional D genes, that two D genes or inverted D genes may be used and that the repertoire can be further diversified by heavy chain V gene (V H ) replacement. Safe conclusions require large, well-defined sequence samples and algorithms minimizing stochastic assignment of segments. Two computer programs were developed for analysis of heavy chain joints. JOINTHMM is a profile hidden Markow model, while JOINTML is a maximum-likelihood-based method taking the lengths of the joint and the mutational status of the V H gene into account. The programs were applied to a set of 6329 clonally unrelated rearrangements. A conventional D gene was found in 80% of unmutated sequences and 64% of mutated sequences, while D-gene assignment was kept below 5% in artificial (randomly permutated) rearrangements. No evidence for the use of DIR, OR15, multiple D genes or V H replacements was found, while inverted D genes were used in less than 1& of the sequences. JOINTML was shown to have a higher predictive performance for D-gene assignment in mutated and unmutated sequences than four other publicly available programs. An online version 1Á0 of JOINTML is available at www.cbs.dtu.dk/services/VDJsolver.

Background

BackgroundNaturally occurring IgE-specific IgG autoantibodies have been identified in patients with asthma and other diseases, but their spectrum of functions is poorly understood.ObjectiveAddress the hypothesis that: (i) IgG anti-IgE autoantibodies are detectable in the serum of all subjects but elevated in asthmatic patients regardless of atopic status as compared with controls; (ii) some activate IgE-sensitized basophils; and (iii) some inhibit allergen-induced basophil activation.MethodsIgE-specific IgG autoantibodies were detected and quantified in sera using ELISA. Sera were examined for their ability to activate IgE-sensitized human blood basophils in the presence and absence of allergen using a basophil activation test, and to inhibit allergen binding to specific IgE on a rat basophilic cell line stably expressing human FcεRI.ResultsIgG autoantibodies binding to both free and FcεRI-bound IgE were detected in patients with atopic and non-atopic asthma, as well as controls. While some were able to activate IgE-sensitised basophils, others inhibited allergen-induced basophil activation, at least partly by inhibiting binding of IgE to specific allergen.ConclusionNaturally occurring IgG anti-IgE autoantibodies may inhibit, as well as induce, basophil activation. They act in a manner distinct from therapeutic IgG anti-IgE antibodies such as omalizumab. They may at least partly explain why atopic subjects who make allergen-specific IgE never develop clinical symptoms, and why omalizumab therapy is of variable clinical benefit in severe atopic asthma.

The initial event of somatic hypermutation (SHM) is the deamination of cytidine residues by activation-induced cytidine deaminase (AID). Deamination is followed by the replication over uracil and/or different error-prone repair events. We sequenced 659 nonproductive human IgH rearrangements (IGHV3-23*01) from blood B lymphocytes enriched for CD27-positive memory cells. Analyses of 6,912 unique, unselected substitutions showed that in vivo hot and cold spots for the SHM of C and G residues corresponded closely to the target preferences reported for AID in vitro. A detailed analysis of all possible four-nucleotide motifs present on both strands of the VH gene showed significant correlations between the substitution frequencies in reverse complementary motifs, suggesting that the SHM machinery targets both strands equally well. An analysis of individual JH and D gene segments showed that the substitution frequencies in the individual motifs were comparable to the frequencies found in the VH gene. Interestingly, JH6-carrying sequences were less likely to undergo SHM (average 15.2 substitutions per VH region) than sequences using JH4 (18.1 substitutions, p = 0.03). We also found that the substitution rates in G and T residues correlated inversely with the distance to the nearest 3′ WRC AID hot spot motif on both the nontranscribed and transcribed strands. This suggests that phase II SHM takes place 5′ of the initial AID deamination target and primarily targets T and G residues or, alternatively, the corresponding A and C residues on the opposite strand.