Analysis of IgE Antibodies from a Patient with Atopic Dermatitis: Biased V Gene Usage and Evidence for Polyreactive IgE Heavy Chain Complementarity-Determining Region 3

Abstract

To better understand V gene usage, specificity, and clonal origins of IgE Abs in allergic reactions, we have constructed a combinatorial Ab library from the mRNA of an adult patient with atopic dermatitis. Sequence analysis of random clones revealed that 33% of clones used the IGHV6-1 H chain V gene segment, the only member of the VH6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the VH6 rearrangements included short complementarity-determining region 3, frequent use of IGHD7-27 D gene, and little nucleotide addition at the D-J junction. There was also a low level of mutation compared with VH1, VH3, and VH4 rearrangements. The library was expressed as phage-Fab fusions, and specific phage selected by panning on the egg allergen ovomucoid. Upon expression as soluble IgE Fabs, 12 clones demonstrated binding to ovomucoid, skim milk, and BSA by ELISA. Nucleotide sequencing demonstrated that the IGHV6-1 V gene segment encoded each of the 12 multiply reactive IgE Fabs. A cyclic peptide was designed from the complementarity-determining region 3 of several of these clones. The cyclic peptide bound both self and nonself Ags, including ovomucoid, human IgG, tetanus toxoid, and human and bovine von Willebrand factor. These results suggest that some IgE Abs may bind more than one Ag, which would have important implications for understanding the multiple sensitivities seen in conditions such as atopic dermatitis.

IgE Transcripts in the Circulation of Allergic Children Reflect a Classical Antigen-Driven B Cell Response and Not a Superantigen-Like Activation

“…First, evidence of a heavily biased use of specific V H gene families in IgE transcripts from allergic patients (5-9) has been interpreted as indicative of a superantigen-driven B cell proliferation. Second, IgE sequences were found to contain a strikingly low level of somatic hypermutation and include significantly shorter CDR-3 (8). These characteristics have also been reported for transcripts from CD5 + B cells (10), which share similarities with murine B-1a cells (11) and arise from polyclonal activation (12).…”

“…These characteristics have also been reported for transcripts from CD5 + B cells (10), which share similarities with murine B-1a cells (11) and arise from polyclonal activation (12). Therefore, it was hypothesized that B-1 cell activation might also play a role during allergic immune responses (8,13).…”

“…B-1 cells are normally produced during fetal and neonatal development (34) and represent a major B cell subset in peripheral blood throughout childhood (35). The first study to imply a potential role for B-1 cells in the allergic response came from Edwards et al (8), who analyzed 31 unique IgE sequences from one 40-y-old patient with atopic dermatitis. In uncommonly overrepresented sequences containing the otherwise scarce IGHV6-1 gene segment, they found a low level of somatic hypermutation in the V H region and very short CDR-H3 regions due to reduced N nucleotide addition, which both have also been reported for transcripts from B-1 cells (10,11).…”

Plasma Cells and Nonplasma B Cells Express Differing IgE Repertoires in Allergic Sensitization

“…It is subject to ongoing discussion whether allergic sensitization represents a misled but classical Ag-driven and oligoclonal B cell response (43,44). However, recent observations of a heavily biased use of specific V H gene families in IgE transcripts from allergic patients (45)(46)(47)(48)(49) have led to the hypothesis that some allergens might also act in a superantigen-like fashion (superallergen hypothesis, reviewed in Refs. 50,51).…”

Isoallergen Variations Contribute to the Overall Complexity of Effector Cell Degranulation: Effect Mediated through Differentiated IgE Affinity

“…Still, the very narrow epitope specificity of the isolated IgE clones underscores that we have not successfully been able to isolate all mite-specific IgE Abs from the HDM-allergic individual, as productive allergen-mediated IgE cross-linking triggering an effector cell response requires an absolute minimum of at least two different IgE clones binding two nonoverlapping allergen epitopes. In the fairly limited number of papers (19)(20)(21)(22)(23), including this one, in which human allergenspecific IgE Ab fragments have been isolated, combinatorial libraries have always been cloned from peripheral blood samples. However, an essential aspect of the evolving IgE response is the accumulating evidence that both class-switch recombination (24)(25)(26)(27) and somatic hypermutation (25) followed by clonal expansion and IgE production (28,29) occur in local airway mucosa under the influence of the inflamed tissue (reviewed in Ref.…”