Clayton A. White

BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-κB pathway

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.

14-3-3 adaptor proteins recruit AID to 5′-AGCT-3′-rich switch regions for class switch recombination

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

Scaffold Functions of 14-3-3 Adaptors in B Cell Immunoglobulin Class Switch DNA Recombination

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5′-AGCT-3′ repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S–S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180–198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

Histone Deacetylase Inhibitors Upregulate B Cell microRNAs That Silence AID and Blimp-1 Expression for Epigenetic Modulation of Antibody and Autoantibody Responses

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require activation-induced cytidine deaminase (AID), and plasma cell differentiation, which requires B lymphocyte–induced maturation protein-1 (Blimp-1), are critical for the generation of class-switched and hypermutated (mature) Ab and autoantibody responses. We show that histone deacetylase inhibitors valproic acid and butyrate dampened AICDA/Aicda (AID) and PRDM1/Prdm1 (Blimp-1) mRNAs by upregulating miR-155, miR-181b, and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a, and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1, and X-box binding protein 1, thereby inhibiting CSR, SHM, and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of histone deacetylase inhibitor–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, valproic acid blunted class-switched and hypermutated T-dependent and T-independent Ab 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 X-box binding protein 1) that are critical to the B cell differentiation processes that underpin Ab and autoantibody responses. They also provide therapeutic proof-of-principle in autoantibody-mediated autoimmunity.

B cell TLRs and induction of immunoglobulin class-switch DNA recombination.

Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors (PRRs). Engagement of B cell TLRs 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. 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 inducing immunoglobulin (Ig) class switch DNA recombination (CSR), which crucially diversifies the antibody biological effector functions. Dual BCR/TLR engagement induces CSR to all Ig isotypes, as directed by cytokines, while TLR engagement alone induces 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 IgH switch (S) regions. A critical role of B cell TLRs in CSR and the antibody response is emphasized by the emergence of several TLR ligands as integral components of vaccines that greatly boost humoral immunity in a B cell-intrinsic fashion.

AID dysregulation in lupus-prone MRL/Fas lpr/lpr mice increases class switch DNA recombination and promotes interchromosomal c-Myc/IgH loci translocations: Modulation by HoxC4

Immunoglobulin gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) play important roles in the generation of autoantibodies in systemic lupus erythematosus. Systemic lupus is characterized by the production of an array of pathogenic high-affinity mutated and class-switched, mainly IgG, antibodies to a variety of self-antigens, including nuclear components, such as dsDNA, histones, and chromatin. We previously found that MRL/Faslpr/lpr mice, which develop a systemic autoimmune syndrome sharing many features with human lupus, display greatly upregulated CSR, particularly to IgG2a, in B cells of the spleen, lymph nodes, and Peyers patches. In MRL/Faslpr/lpr mice, the significant upregulation of CSR is associated with increased expression of activation-induced cytidine deaminase (AID), which is critical for CSR and SHM. We also found that HoxC4 directly activates the promoter of the AID gene to induce AID expression, CSR and SHM. Here, we show that in both lupus patients and lupus-prone MRL/Faslpr/lpr mice, the expression of HoxC4 and AID is significantly upregulated. To further analyze the role of HoxC4 in lupus, we generated HoxC4− / − MRL/Faslpr/lpr mice. In these mice, HoxC4-deficiency resulted in reduced AID expression, impaired CSR, and decreased serum anti-dsDNA IgG, particularly IgG2a, autoantibodies, which were associated with a reduction in IgG deposition in kidney glomeruli. In addition, consistent with our previous findings in MRL/Faslpr/lpr mice that upregulated AID expression is associated with extensive DNA lesions, comprising deletions and insertions in the IgH locus, we found that c-Myc to IgH (c-Myc/IgH) translocations occur frequently in B cells of MRL/Faslpr/lpr mice. The frequency of such translocations was significantly reduced in HoxC4− / − MRL/Faslpr/lpr mice. These findings suggest that in lupus B cells, upregulation of HoxC4 plays a major role in dysregulation of AID expression, thereby increasing CSR and autoantibody production and promoting c-Myc/IgH translocations.

Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions

Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs), particularly staggered DSBs, in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID), subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However, consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung, we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases, such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single and double-strand DNA, primarily at dG/dC residues, the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA, as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG(-/-) mice and EndoG(-/-) B cells, we found that EndoG deficiency resulted in a two-fold reduction in CSR in vivo and in vitro, as demonstrated by reduced cell surface IgG1, IgG2a, IgG3 and IgA, reduced secreted IgG1, reduced circle Iγ1-Cμ, Iγ3-Cμ, Iɛ-Cμ, Iα-Cμ transcripts, post-recombination Iμ-Cγ1, Iμ-Cγ3, Iμ-Cɛ and Iμ-Cα transcripts. In addition to reduced CSR, EndoG(-/-) mice showed a significantly altered spectrum of mutations in IgH J(H)-iEμ DNA. Impaired CSR in EndoG(-/-) B cells did not stem from altered B cell proliferation or apoptosis. Rather, it was associated with significantly reduced frequency of DSBs. Thus, our findings determine a role for EndoG in the generation of S region DSBs and CSR.

TSPAN33 is a novel marker of activated and malignant B cells.

We have identified Tspan33 as a gene encoding a transmembrane protein exhibiting a restricted expression pattern including expression in activated B cells. TSPAN33 is a member of the tetraspanin family. TSPAN33 is not expressed in resting B cells, but is strongly induced in primary human B cells following activation. Human 2E2 cells, a Burkitts lymphoma-derived B cell model of activation and differentiation, also upregulate TSPAN33 upon activation. TSPAN33 is expressed in several lymphomas including Hodgkins and Diffuse large B cell lymphoma. TSPAN33 is also expressed in some autoimmune diseases where B cells participate in the pathology, including rheumatoid arthritis patients, systemic lupus erythematosus (SLE), and in spleen B cells from MRL/Fas(lpr/lpr) mice (a mouse model of SLE). We conclude that TSPAN33 may be used as a diagnostic biomarker or as a target for therapeutic antibodies for treatment of certain B cell lymphomas or autoimmune diseases.