By diversifying antibody biological effector functions, class switch DNA recombination has a central role in the maturation of the antibody response. Here we show that BCR-signalling synergizes with Toll-like receptor (TLR) signalling to induce class switch DNA recombination. BCR-signalling activates the non-canonical NF-κB pathway and enhances the TLR-dependent canonical NF-κB pathway, thereby inducing activation-induced cytidine deaminase (AID), which is critical for class switch DNA recombination. Escherichia coli lipopolysaccharide (LPS) triggers dual TLR4/BCR-signalling and induces hallmarks of BCR-signalling, including CD79a phosphorylation and Ca2+ mobilization, and activates both the NF-κB pathways to induce AID and class switch DNA recombination in a PI(3)K p85α-dependent fashion. CD40-signalling activates the two NF-κB pathways to induce AID and class switch DNA recombination independent of BCR-signalling. Finally, dual BCR/TLR-engaging NP–lipopolysaccharide effectively elicits class-switched NP-specific IgG3 and IgG2b in mice. Thus, by integrating signals of the non-canonical and canonical NF-κB pathways, BCR and TLRs synergize to induce AID and T-cell-independent class switch DNA recombination.

Class switch DNA recombination (CSR) is the mechanism that diversifies the biological effector functions of antibodies. Activation-induced cytidine deaminase (AID), a key CSR player, targets IgH switch (S) regions, which contain 5′-AGCT-3′ repeats in their core. How AID is recruited to S regions remains unclear. Here we show that 14-3-3 adaptor proteins play an important role in CSR. 14-3-3 proteins specifically bind 5′-AGCT-3′ repeats, are upregulated in B cells undergoing CSR and are recruited together with AID to the S regions involved in CSR events (Sμ→Sγ1, Sμ→Sγ3 or Sμ→Sα). Moreover, blocking 14-3-3 by difopein, deficiency in 14-3-3γ or expression of a dominant negative 14-3-3σ mutant impaired recruitment of AID to S regions and decreased CSR. Finally, 14-3-3 proteins interact directly with AID and enhance AID-mediated in vitro DNA deamination, further emphasizing the important role of these adaptors in CSR.

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require AID, and plasma cell differentiation, which requires Blimp-1, are critical for the generation of class-switched and hypermutated (mature) antibody and autoantibody responses. We showed here that the histone deacetylase (HDAC) inhibitors (HDI) valproic acid (VPA) and butyrate upregulated miR-155, miR-181b and miR-361, which silenced AICDA/Aicda (AID) mRNA, and miR-23b, miR-30a and miR-125b, which silenced PRDM1/Prdm1 (Blimp-1) mRNA, in human and mouse B cells. This led to downregulation of AID, Blimp-1 and Xbp-1 expression, thereby dampening CSR, SHM and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of HDI-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2 or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell intrinsic epigenetic mechanisms, VPA blunted class-switched and hypermutated T-dependent and T-independent antibody responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease and extended survival in lupus MRL/Faslpr/lpr mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and Xbp-1) that critical to the B cell differentiation processes that underpin antibody and autoantibody responses. They also provide therapeutics proof-of-principle in autoantibody-mediated autoimmunity.

Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors (PRRs). Engagement of TLRs in B cells by microbe-associated molecular patterns (MAMPs) induces T-independent (TI) antibody responses and plays an important role in the early stages of T-dependent (TD) antibody responses before specific T cell help becomes available, in part by facilitating B cell entry into the germinal center reaction. The role of B cell TLRs in the antibody response is magnified by the synergy of B cell receptor (BCR) crosslinking and TLR engagement in promoting B cell proliferation and efficiently inducing immunoglobulin (Ig) class switch DNA recombination (CSR), which crucially diversifies the antibody biological effector functions. Dual engagement of TLRs and BCR can be mediated by complex MAMPs such as lipopolysaccharides (LPS), which engages TLR4 through its lipid A moiety and crosslinks the BCR through its polysaccharidic moiety (O-antigen). Dual BCR/TLR engagement induces CSR to all Ig isotypes, as directed by different cytokines, while engagement of any TLR alone induces only marginal CSR. Integration of BCR and TLR signaling results in activation of the canonical and non-canonical NF-κB pathways, induction of activation-induced cytidine deaminase (AID) and germline transcription of switch (S) regions in the IgH locus. The last two are essential events for CSR to unfold. A critical role of dual BCR/TLR engagement in induction of CSR and generation of neutralizing antibodies is emphasized by the emergence of TLR ligands as integral components of vaccines that greatly boost humoral immunity in a B cell-intrinsic fashion. Further, dual BCR/TLR engagement by complex self-antigens will result in dysregulation of AID expression and CSR in autoreactive B cells, leading to generation of isotype-switched pathogenic autoantibodies. Finally, an important aspect of dual BCR/TLR engagement is the boosting of specific antibody response to tumor antigens, as suggested by high titers of anti-tumor antibodies in response to tumor vaccines that contain TLR agonists.