IL-4 plus CD40 monoclonal antibody induces human B cells γ subclass-specific isotype switch: switching to γ 1, γ 3, and γ 4, but not γ 2

Abstract

Stimulation with IL-4 plus CD40 mAb is known to induce production of IgE and IgG4. In this study, we determined the IgG subclass specificity of IL-4 plus CD40 mAb stimulation for human purified B cells. We determined true in vitro switching by the generation of switch circular DNA (S γ/S mu) representing primary S mu/S γ events and production of γ subclass-specific germ-line transcripts by a combination of reverse transcription-PCR and restriction endonuclease digestion. We simultaneously measured changes in the levels of IgG subclass proteins produced. Forty-two clones of circular switch DNA were identified and sequenced. The IgG subclass of S γ fragment in the S γ/S mu chimeric PCR products was determined by analyzing key S γ nucleotides. The switch-deleted clones were found to consist of S γ 1/S mu, S γ 3/S mu, and S γ 4/S mu chimeric switch sequences, showing that such switching had occurred. No S γ 2/S mu chimeric switch sequences were found. While a consensus sequence was not identified at the S γ/S mu breakpoints, four contiguous guanines (GGGG) were noticeably present in the S γ region near the breakpoint. The induction of γ 1, γ 3, and γ 4 switch circles in human purified B cells was accompanied by enhanced production of IgG1, IgG3, and IgG4 but not IgG2. Similarly, stimulation with IL-4 alone induced γ 1, γ 3, and γ 4 but not γ 2 germ-line transcripts. These results demonstrate that IL-4 plus CD40 mAb induces Ig isotype switch from mu to γ 1, γ 3, and γ 4 but not to γ 2.