The role of Toll-like receptors and 14-3-3 proteins in the induction and targeting of immunoglobulin class switch DNA recombination.

摘要

We investigated the roles that Toll-like receptors (TLRs) play in B cell activation, immunoglobulin (Ig) class switch DNA recombination (CSR) and plasma cell formation, in the context of BCR, CD40 and cytokine signals. While one TLR signal alone directly activated B cells, the response was limited in terms of the antibody isotypes produced. Combinations of two or more signals together (e.g. surface TLR and cytokine) broadened the scope of the class switched antibody response as driven by the TLR and guided by the cytokines. Combinations of signals from multiple activated TLRs usually led to increased CSR; however, certain combinations resulted in suppressed CSR possibly due to paralysis or signaling antagonism.;We found that TLR9 stimulation in the presence of BCR crosslinking, induced CSR from IgM to all isotypes (IgG1, IgG2a, IgG2b, IgG3, IgE, IgA), as directed by germline transcription in response to appropriate cytokines, and measured by experiments employing flow cytometry to analyze the surface expression of Igs, PCR to measure germline and switch transcripts, and ELISA to measure secreted Ig levels. However, we also found that TLR9 stimulation without BCR crosslinking led to suppression of LPS- or CD154-induced CSR to most isotypes, regardless of their Th1 or Th2 affiliation.;A variety of molecular and cell biology techniques were used to analyze the recruitment of 14-3-3 proteins and the concomitant targeting of activation-induced cytidine deaminase (AID) to Ig heavy chain (IgH) Switch (S) DNA regions to initiate CSR. 14-3-3 proteins were specifically recruited to '5-AGCT-3'-rich S regions upstream of CH genes. In addition, 14-3-3 proteins were found to interact with proteins that are known to play key roles in CSR, such as activation-induced cytidine deaminase (AID). Several 14-3-3 isoforms were greatly upregulated upon B cell activation. Studies on mice genetically deficient in 14-3-3 isoforms, as well as studies using the 14-3-3 inhibitor difopein, showed profound impairment of CSR. Taken together, these findings implicate 14-3-3 proteins as key mediators of targeting of CSR machinery to IgH S regions.