Structural valency of APRIL identifies and controls B cell intrinsic class switch recombination 2259580

Abstract

Abstract Introduction CSR is an intrachromosomal DNA rearrangement of the IgH locus in which constant regions are replaced by another isotype, diversifying antibody effector function but not antigen specificity. Conventionally, CSR is triggered and directed by T cell help but can also be initiated by T independent cytokines, including APRIL. APRIL is a homotrimer produced primarily in myeloid cells that initiates T cell independent CSR by engaging its receptor TACI on B cells. In this project I define how the valency of APRIL controls the T independent CSR. Methods Recombinant APRIL (Ala96-Leu 240) was expressed in Expi293 cells and purified using size exclusion chromatography where trimeric and monomeric fractions were collected separately. Naïve splenic B cells from C57BL/6 mice (1 × 10^6 cells/mL) were stimulated with APRIL trimer or monomer (2ug/mL)±IL-4 (20ng/mL); media served as control. CSR was assessed by sterile transcripts at 48 hours (RT-qPCR) and surface IgG and IgM at 96 hours (flow cytometry). Results After 48-hour culture, sterile transcripts, formed by the opening of a transcription bubble at the switch region, were detected using qPCR, and after a 96-hour culture surface Ig isotype was detected using flow cytometry. At the protein level, APRIL trimer induced significantly more CSR to IgG than APRIL monomer. Monomer CSR activity to IgG can be rescued with the addition of IL4 to the level of trimer. At the transcript level, trimer did not induce significantly more sterile transcripts to any IgG subtype than monomer, pointing to trimeric ligation of TACI by APRIL as a requirement for generating productive spliced DNA at the IgM to IgG switch region. In the monomeric state, CSR can be induced as evidenced by sterile transcripts, but intrachromosomal DNA rearrangements to form productive IgG transcripts cannot. Conclusion We present a model for T independent CSR, where trimeric structure of APRIL is needed to push CSR beyond initiation steps to a fully spliced DNA and productive mRNA IgG transcript. Funding Source NIH Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)