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

Abstract

Allergic asthma is the most frequent chronic disorder in childhood. Although IgE is a central effector molecule in allergic diseases, the nature of the IgE response is still under debate. The objective of our study was to clarify whether the IgE repertoire in the circulation of allergic children represents a classical Ag-driven and oligoclonal B cell response, a superantigen-like activation of a subset of B cells, or a polyclonal B-1 cell expansion. Using a highly sensitive RT-PCR method, we amplified, cloned, and sequenced IgE H chain transcripts from 13 children with allergic asthma. We gained 1366 functional IgE sequences, which currently represent the most extensive collection of human IgE transcripts. Compared to IgM transcripts from the same children, the somatic mutation rate was significantly enhanced in IgE transcripts (21‰ versus 72‰; p < 0.001), which renders a polyclonal B-1 response unlikely. Moreover, IgE sequences displayed significantly enhanced Ag selection and hence were indicative of a classical Ag-driven immune response with affinity maturation (p < 0.001). In contrast to several recent studies, the usage pattern of variable gene segment of the H Ig chain in IgE transcripts followed the germline complexity, arguing against a superantigen-like interaction. We conclude that IgE transcripts in the circulation of children with allergic asthma reflect a classical adaptive B-2 cell response. This study provides reference data for a better characterization of the IgE response under immunomodulating therapies, such as anti-IgE therapy or allergen-specific immunotherapy.

IgG4 and IgE Transcripts in Childhood Allergic Asthma Reflect Divergent Antigen-Driven Selection

“…Moreover, IgG4 can also mediate autoimmunity (e.g., pemphigus vulgaris) and might play a pathogenic role in newly identified "IgG4-related disease" (34). We have shown that, in contrast to earlier assumptions, the IgE transcripts in PBMCs of allergic children reflect a classical Ag-driven B2 cell response and not a B1 cell response or a superantigen-like activation (35). Even in the absence of Ag, B1 cells spontaneously secrete "natural" Abs that are more "germline-like" because of minimal N-region addition and low somatic mutation frequency compared with B2 cell-derived Igs (36).…”

“…As a reference, we used 341 previously published unique V H DJ H C ε sequences (IgE transcripts) from the same blood samples (GenBank accession nos. FJ840062-FJ840439) (35).…”

“…35, who were diagnosed with allergic asthma according to standardized criteria of the PRACTALL consensus report (40) and the GINA (Global Initiative against Asthma), were accessible for IgG4 analysis. After informed consent, 1.2 ml blood was collected subsequent to a routinely performed blood withdrawal.…”

“…The following program was performed on a thermocycler (Sensoquest, Göttingen, Germany): 5 min at 94˚C; 40 cycles with 20 s at 94˚C, 30 s at 60˚C, and 90 s at 72˚C; 10 min at 72˚C. As a control for RNA quality, GAPDH transcripts were amplified from each sample, using human GAPDH-1 and human GAPDH-2 primers (35). PCR products were gel purified, and DNA was extracted with QIAquick Gel Extraction Kit (QIAGEN).…”

“…RNA was reverse transcribed into cDNA according to standard protocols with SuperScript III (Invitrogen, Karlsruhe, Germany). A combination of framework region (FR) 1-based forward primers for each VH gene family was used (35) . The reverse primer is specific for the hinge region of the Cg4 C region (59-GCA TGA TGG GCA TGG GGG ACC ATA TTT GGA-39).…”