Analysis of γ4 germline transcription in human B cells

Background: Allergic reactions occur rarely in patients with chronic helminth infections, although FcεRI-bearing cells are sensitized with anti-parasite IgE and are exposed to antigen. The inhibition of allergic reactivity is due to 'blocking antibodies', predominantly IgG4. IgG4 antibodies represent 50-95% of anti-filarial IgG, and have blocking activity because they compete with cell-bound IgE for allergen binding. Blocking IgG4 antibodies have also been detected in patients treated with immunotherapy. However, the molecular mechanisms of IgG4 regulation have remained unexplored. We address this issue starting with the IL-4-dependent induction of γ4 germline transcription, an essential step in the commitment to isotype switching. Results: γ4 germline transcripts were cloned and shown to contain Iγ4 spliced to the 5' portion of Cγ4 using the splice donor site shared by γ1 and γ3 germline transcripts. Sequence analysis located the human Iγ4 exon ≃ 0.6 kb upstream of the Sγ4 region. Four major transcription start sites located 547-583 bp upstream of the Iγ4 splice donor site were identified by primer extension analysis. A Hind/III/Nael construct (-413/+466) had the highest activity in reporter assays. Transcription was induced 4.6-fold by IL-4, 2.1-fold by CD40 engagement, and 14.5-fold by a combination of the two signals. Thus CD40 crosslinking enhanced IL-4-dependent transcription by 3.1-fold, showing that the two stimuli act synergistically. Conclusion: As we understand more about IgG4 regulation, our hope is to apply to allergy the lesson we learnt from helminth infections.