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.

I nnate immune receptors recognize conserved microbe-associated molecular patterns and have important roles in the early stages of the immune response 1 . Toll-like receptors (TLRs) are a family of conserved innate immune receptors highly expressed in macrophages, dendritic cells, epithelial cells and B cells [2][3][4] . Naïve B cells express a surface receptor for antigen (BCR) consisting of immunoglobulin (Ig)µ-chain (Igµ) and Igδ and react to antigens by making IgM and IgD antibodies (Abs). These are later diversified by somatic hypermutation (SHM) and class switch DNA recombination (CSR) 5 . SHM provides the structural substrate for antigen-mediated selection of higher affinity antibody mutants by introducing point-mutations with rare deletions or insertions into rearranged Ig variable regions. CSR replaces the Ig heavy chain (IgH) constant (C H ) region, for example, Cµ, with a downstream Cγ, Cα or Cε region, resulting in IgG, IgA or IgE antibodies with diverse biological effector functions 6 . Both SHM and CSR require activation-induced cytidine deaminase 6 (AID), which deaminates deoxycytidine, leading to the emergence of deoxyuracil 6 . This is not germane to DNA and is dealt with by elements of the DNA repair machinery, eventually leading to insertion of point-mutations in variable Ig regions for SHM and double-strand DNA breaks (DSBs) in switch (S) regions 6,7 , which lie 5′ of each of C H regions (except for Cδ), for CSR.
CSR entails IgH locus germline I H -C H transcription, which is initiated by the I H promoter (Iµ, Iγ, Iα or Iε) and proceeds through the upstream and downstream S and C H regions engaged in the CSR process, giving rise to germline Iµ-Cµ, Iγ-Cγ, Iα-Cα or Iε-Cε transcripts 6 . S regions contain a high density of 5′-AGCT-3′ repeats in their 'core' sequences. 5′-AGCT-3′ repeats recruit 14-3-3 adaptor proteins, which, in turn, target AID to S region DNA to generate DSBs 8 . CSR then proceeds through deletion of the intervening DNA and re-ligation of DSB free ends to form S-S junctions. The post-recombination sequences are transcribed, giving rise to post-recombination Iµ-Cγ, Iµ-Cα or Iµ-Cε transcripts and 'mature' V H DJ H -Cγ, V H DJ H -Cα or V H DJ H -Cε transcripts for the IgG, IgA or IgE H chain 6 . The efficiency of CSR critically depends on the level of AID 6,9 , which is under tight transcriptional regulation in a B-cell differentiation stage-specific fashion 10 , depending on HoxC4 (ref. 11), NF-κB (refs 11,12) and other transcription factors [12][13][14] . As shown by us 11 and others 12 , the NF-κB p52 subunit (a hallmark of the non-canonical NF-κB pathway 15 ) and p65 subunit (a hallmark of the canonical NF-κB pathway) bind to the AID gene (AICDA/Aicda) promoter and upstream enhancers, respectively.
AID and CSR are efficiently induced in T-dependent antibody responses by engagement of CD40 on B cells by surface CD154 (CD40 ligand) expressed on CD4 + T cells 15 and in the presence of appropriate cytokines (for example, IL-4, TGF-β or IFN-γ). However, IgG and IgA Abs can arise early in viral and bacterial infections before T-cell help becomes available 16 , indicating that CSR can also unfold in a T-cell-independent fashion. Accordingly, mice deficient in T cells or CD154/CD40 can generate class-switched specific Abs that are protective against some bacterial or viral infections 16,17 . In addition, class-switched Abs arise against bacterial or viral antigens that elicit weak or no T-cell responses (T-independent antigens), such as bacterial polysaccharides and lipolysaccharides (LPS) 18,19 . Further, polysaccharide-specific class-switched IgG Abs, which are critical for protection against infections by capsular bacteria 20 , are decreased in about half of patients deficient in the TLR-signalling molecule MyD88 or IRAK4 despite normal levels of total IgG/IgA Abs 21 . Finally, TLR ligands, for example, TLR4 ligand lipid A and TLR9 ligand CpG, can significantly boost specific antibody responses to proteinic (T-dependent) antigens 3,4,22 and, as such, are widely used as vaccine adjuvants.
Despite the putative role of TLRs in class-switched antibody responses, only few studies exist on CSR induction by TLR ligands, including TLR3 ligand poly(I:C) 23 , TLR7 ligand 8-mercaptoguanosine or loxoribine 24 or CpG 25 . In these studies, TLR ligands alone triggered only marginal CSR, if at all 16,26 , suggesting that another B-cell-activating stimulus is required for TLR-dependent CSR induction. Indeed, engagement of selected surface B-cell molecules, such as CD38 (ref. 24), CD40 (ref. 25), CD180 (ref. 27), TACI (refs 23,25,28) and IL-10R (ref. 25), has been suggested to enhance TLRdependent CSR. Likewise, TLR7-and TLR9-dependent CSR would be enhanced by BCR engagement 24,25,29 , albeit only marginally (1-4% B-cell switching) 24,25 . The mechanisms underlying a putative BCR enhancement of TLR-triggered CSR are unknown, in spite of findings suggesting an integration of BCR-and TLR-signalling for activation of NF-κB 30 and/or other transcription factors [31][32][33][34] . In contrast to other TLR ligands, LPS consists of a proper TLR-binding moiety (TLR4 ligand lipid A) and a repetitive polysaccharide moiety (O-antigen). This can elicit specific anti-polysaccharide antibody responses 19 through mobilization of O-antigen-binding B cells 35 . However, in spite of wide and empirical usage of LPS in mouse B cells, the mechanism underlying LPS-dependent CSR and its enhancement by BCR crosslinking 36 remain to be determined.
Here we have tested the hypothesis that BCR-signalling synergizes with TLR-signalling for AID and CSR induction. We used a well-defined in vitro B-cell system to address the synergistic induction of CSR by BCR crosslinking 18 and the agonistic ligand of each one of the five TLRs highly expressed in B cells, that is, surface TLR1 and TLR2 (which form TLR1/2 heterodimer) and TLR4, and endosomal TLR7 and TLR9 (ref. 37). We further addressed the molecular mechanisms, underlying the synergy of BCR and TLR in inducing AID and CSR, by measuring activation of central components of the non-canonical and canonical NF-κB pathways and levels of AID expression, and by using B cells deficient in p85α (p85α -/-), the predominant class IA regulatory subunit of the PI(3)K lipid kinases that critically transduce BCR signals 38 . Finally, we analysed the unique properties of LPS in triggering dual BCR/ TLR-signalling and, therefore, activating both the non-canonical and canonical NF-κB pathways for induction of AID and CSR.

BCR synergizes with TLRs to induce CSR.
To test our hypothesis that BCR crosslinking provides a B-cell-specific signal enabling TLRs to induce CSR, we analysed CSR in B cells stimulated with agonistic ligands of surface TLR1/2 and TLR4 and endosomal TLR7 and TLR9. TLR1/2 ligand Pam 3 CSK 4 , TLR4 ligand lipid A, TLR7 ligand R-848 or TLR9 ligand CpG showed various B-cell mitogenic activities and, in the presence of appropriate cytokines, all induced only marginal CSR to IgG1, IgG2a, IgG3, IgA and IgE, even at a tenfold higher dose than that typically used ( Fig. 1; Supplementary  Fig. S1). An agonistic anti-δ chain monoclonal Ab (mAb) conjugated to dextran (anti-δ mAb/dex), which crosslinks Igδ to trigger BCRsignalling and induces limited B-cell proliferation but no CSR 18 , synergized with these TLR ligands to induce CSR in a potent and dose-dependent fashion, as shown by analysis of surface (s)Ig ( Fig. 1; Supplementary Fig. S1), germline I H -C H , circle I H -Cµ, postrecombination Iµ-C H and mature V H DJ H -C H transcripts ( Fig. 2; Supplementary Fig. S2), and secreted Ig ( Supplementary Fig. S3). Anti-δ mAb/dex synergized with TLR ligands to induce CSR but not by enhancing B-cell proliferation, as it increased switched B cells at each cell division and over a tenfold range of CpG or LPS concentrations ( Fig. 1 and Fig. 2a,b), and increased circle Iγ1-Cµ transcripts, an accurate parameter of ongoing CSR (Fig. 2d). Anti-δ mAb/dex did not further enhance CSR induced by membrane CD154 (mCD154) (Fig. 1b). Unlike anti-δ mAb/dex, BAFF or APRIL only moderately enhanced TLR-dependent CSR to IgG1, despite synergizing with anti-δ mAb/dex to induce CSR to IgA and, to a lesser extent, IgG1 ( Supplementary Fig. S4). BCR and TLR synergy in CSR induction was further emphasized by a decrease in anti-δ mAb/dex and CpG-induced CSR by chloroquine, an inhibitor of endosomal TLR maturation, and the negligible CSR induction by GpC, a weak TLR9 agonist 39 , and anti-δ mAb/dex ( Supplementary  Fig. S5a). Thus, BCR synergizes with TLR1/2, TLR4, TLR7 or TLR9 to induce efficient CSR; in the absence of BCR crosslinking, engagement of these TLRs alone results in only marginal CSR.
LPS induces BCR-signalling for CSR. LPS, unlike its lipid A moiety, induced AID and CSR to all Ig isotypes ( Fig. 1b; Supplementary  Fig. S1 and ref. 6), suggesting LPS fulfills the requirement of dual BCR/TLR-signalling by engaging TLR4 through the lipid A moiety and BCR through the polysaccharidic moiety, respectively. In the normal B-cell repertoire, many B cells express BCRs that bind with variable affinities to repetitive polysaccharides 35 , which include the polysaccharidic moiety of LPS. Indeed, CD79a Tyr182, one of the two ITAM Tyr residues, which, on phosphorylation, transduce BCR-signalling 29 , was phosphorylated in B cells stimulated with anti-δ mAb/dex or anti-µ F(ab′) 2 or LPS (within 3 h, and more within 24 h), but not lipid A (Fig. 3a). In addition, unlike anti-µ F(ab′) 2 , which induced a rapid increase of free cytosolic Ca 2 + ions ([Ca 2 + ] cytosol ), anti-δ mAb/dex induced a slower, but continuous increase in [Ca 2 + ] cytosol (Fig. 3b). As sustained extracellular Ca 2 + entry is required upon BCR engagement for activation of Ca 2 +dependent signalling pathways, we pretreated B cells with LPS, antiδ mAb/dex, lipid A or mCD154, followed by labelling with Ca 2 + indicator dyes and then probing for Ca 2 + mobilization in response to anti-µ F(ab′) 2 . B cells, pretreated for 16 h with LPS or anti-δ mAb/dex, showed only a transient Ca 2 + flux on further stimulation with anti-µ F(ab′) 2 , likely resulting from Ca 2 + release from ER stores only and without the sustained entry of Ca 2 + from the plasma membrane 40 , as it occurred in B cells that were pretreated with lipid A or mCD154 (Fig. 3c). This suggests that LPS or anti-δ mAb/dex likely triggered sustained Ca 2 + elevation before the B cells were rendered refractory to extra Ca 2 + entry. Finally, LPS-induced CSR could be further increased by anti-δ mAb/dex ( Fig. 1b; Supplementary Fig. S1), which inhibited LPS-induced plasmacytoid differentiation ( Supplementary Fig. S5b), resulting in virtually no increase in secreted IgG1 or IgE ( Supplementary Fig. S3). These experiments show that LPS triggers dual BCR/TLR-signalling by engaging TLR4 through lipid A and by inducing CD79a Tyr phosphorylation and Ca 2 + mobilization (hallmark BCR-signalling events).

BCR-signalling increases TLR-dependent Aicda expression.
Consistent with its potentiation of TLR-dependent CSR, anti-δ mAb/dex, which alone did not induce AID expression, significantly increased Aicda transcripts induced by TLR ligand Pam 3 CSK 4 , lipid A, R-848 or CpG (in the presence of IL-4), resulting in Aicda levels (peaking at 60 h) comparable to those induced by LPS (Fig. 4a,b). Anti-δ mAb/dex decreased mCD154 and IL-4-induced AID expression, consistent with its reduction of CD40-dependent CSR to IgG1 (Fig. 1b). Anti-δ mAb/dex enhanced, to variable extents, germline Iγ1-Cγ1 transcription induced by IL-4 or IL-4 and TLR ligands (peaking as early as at 12 h) or mCD154. It also upregulated B-cell expression of PNA-binding glycoproteins, which are markers of germinal centre B cells, as induced by CpG, but not CD40-high levels of PNA-binding glycoproteins induced by LPS were not further increased by anti-δ mAb/dex ( Fig. 4c; Supplementary Fig. S5c). Anti-δ mAb/dex downregulated Blimp1 and CD138 expression (Fig. 4a,d), consistent with inhibition of plasmacytoid differentiation by BCR-signalling 41 . To further confirm that BCR-signalling increases TLR-dependent Aicda expression and CSR, we blocked BCR-signalling with an anti-CD79a mAb, which inhibited LPSinduced CD79a phosphorylation (Fig. 3a). This blocking anti-CD79a mAb significantly reduced the levels of Aicda transcripts and inhibited CSR to IgG1, resulting in decreased levels of sIgG1 and circle Iγ1-Cµ transcripts, as induced by the BCR/TLR dual engagement or LPS (Fig. 3d,e). Finally, that BCR signalling enabled TLR-dependent CSR by upregulating AID expression was demonstrated by the ability of exogenous AID to substitute anti-δ mAb/dex in TLR9-dependent CSR (Fig. 4e,f). Thus, BCR-signalling enables TLR-dependent induction of efficient CSR by enhancing TLR-dependent AID induction. It also enhances TLR-dependent expression of PNA-binding glycoproteins and suppresses plasmacytoid differentiation.

BCR and TLR activate both NF-κB pathways for AID induction.
We next addressed the mechanisms underlying the synergy of BCR and TLRs in inducing AID expression, which is regulated by the HoxC4 and NF-κB transcription factors 11,12 . Anti-δ mAb/dex did not enhance TLR-induced expression of HoxC4, prompting us to hypothesize that BCR and TLRs synergize to induce AID by activating both the canonical NF-κB (p65) and non-canonical NF-κB (p52) pathways, as CD40 does 15 . To test this hypothesis, we tracked the kinetics of IκBα phosphorylation at Ser32 and p65 phosphorylation at Ser536 (canonical) and expression of p100, the precursor of p52, and processing of p100 to p52 (non-canonical) in B cells stimulated with TLR ligands, anti-δ mAb/dex, or both (all in the presence of IL-4). Consistent with the findings that TLRs activate the canonical NF-κB pathway 2 , CpG or lipid A induced IκBα and p65 phosphorylation that did not peak until 40 m after stimulation. These phosphorylation events were accelerated and enhanced by anti-δ mAb/dex, as were IκBα and p65 phosphorylation induced by LPS, but not CD40 ( Fig. 5; Supplementary Fig. S6). Anti-δ mAb/dex alone, however, did not activate appreciable p65 phosphorylation ( Supplementary Fig. S6a). Rather, it increased the level of both p52 and p100 within 24-48 h, the time frame of AID induction, as mCD154 did ( Fig. 6a; Supplementary Fig. S7a). Like anti-δ mAb/ dex, LPS increased the levels of p100 and p52. Lipid A, or CpG, mainly increased p100, thereby providing more substrate for anti-δ mAb/dex-processing to p52 (Fig. 6a). Thus, BCR and TLRs each activates only one NF-κB pathway (non-canonical and canonical, respectively), but reciprocally enhance the main pathway of activation by the other receptor, thereby synergizing in inducing AID expression, as mediated by both NF-κB pathways.

BCR/TLR-engaging NP-LPS induces switched NP-specific IgGs.
We next extended our in vitro findings to show that dual BCR/TLR engagement induces efficient CSR in vivo by injecting normal C57BL/6 mice with NP-LPS conjugate. This avoids proteinic linkages (such as in streptavidin used for conjugation to biotinylated moieties) that would introduce a T-cell-dependent component in the antibody response, thereby complicating the interpretation of results. The NP-LPS conjugate would also maximize the number of B cells hit with concomitant (NP-specific) BCR-crosslinking and TLR4-engagement, as the frequency of B cells expressing NP-specific BCR in the normal mouse B cell repertoire is only ~10 -7 (in our in vitro cultures, virtually all B cells were BCR-crosslinked by anti-δ mAb/dex). The presence of a large proportion of B cells expressing BCRs binding to the LPS polysaccharidic moiety was irrelevant, because such BCRs do not cross-react with NP. As predicted, NP-LPS induced a strong classswitched (IgG3 and IgG2b, and to a lesser extent IgG1 and IgG2a) NP-specific antibody response, which was readily detectable 6 days after injection and sustained through day 12 ( Supplementary Fig. S9). By contrast, a comparable class-switched NP-specific antibody response was not elicited by NP 26 (which efficiently crosslinks NPspecific BCRs but does not engage TLRs), or LPS alone. NP-binding to IgMs were detectable not only in mice injected with NP-LPS, but also and, to a lesser extent, in mice injected with NP 26 , likely owing to the decavalency and high avidity of this Ig class. Thus, these experiments show that, as predicted by our in vitro findings, dual BCR/TLR engagement in vivo induces class-switched and specific antibodies. The IgG subclasses (IgG3 and IgG2b) elicited by NP-LPS reflect those characteristically induced by LPS 6 .

Discussion
Here we have shown that BCR-signalling synergized with TLR1/2-, TLR4-, TLR7-or TLR9-signalling to induce AID and enable CSR. Either signalling alone resulted in marginal (TLRs) or no (BCR) CSR. Importantly, BCR crosslinking synergized with all TLR ligands analysed for CSR induction, thereby emphasizing the critical role of dual BCR/TLR-signalling in inducing specific and isotypediverse antibodies at early stages of the response to bacteria or  viruses, before T-cell help becomes available 3,4 . In addition, as emphasized by our in vivo demonstration that NP-LPS conjugate elicited a strong NP-specific IgG3 and IgG2b response, CSR induced by dual BCR/TLR engagement underpins the generation of classswitched Abs throughout T-independent responses, such as those involving bacterial or viral BCR-crosslinking antigens, together with TLR-engaging microbe-associated molecular patterns, such as LPS, Pam 3 CSK 4 , single-stranded RNA, polymerized flagellin, double-stranded RNA or unmethylated microbial DNA. The important role of B-cell TLRs in antibody responses to microbial pathogens is emphasized by reduced IgGs, particularly against pyogenic bacteria, in patients with defective TLR-signalling pathways 21 and decreased IgGs to protein antigens admixed with TLR ligands in TLR-, MyD88-and/or TRIF-deficient mice 3,4 . BCR and TLR synergy in inducing AID and CSR reflects the activation by these two receptors of the non-canonical and canonical NF-κB pathways. As we showed here, TLR ligands activated NF-κB in B cells only through the canonical pathway 2 . This was potentiated by BCR crosslinking 30 (particularly by anti-δ mAb/dex, which initiates sustained BCR-signalling 18,42 ), which also activated the noncanonical NF-κB pathway. BCR and TLRs reciprocally enhanced the main NF-κB pathway activated by the other receptor; integration of the two signalling pathways would occur at the level of p100 induction (non-canonical) and TRAF6 activation (canonical), leading to induction of p52 and p65, respectively (Fig. 8). NF-κB p52 and p65 subunits, which are also induced by CD154 and, as we showed here, LPS, bind to the AICDA/Aicda promoter and enhancer elements, respectively, to upregulate AID gene expression. Impairment of BCR-signalling, as in p85α -/-B cells, virtually abolished the generation of p52 following stimulation with anti-δ mAb/dex or LPS, thereby decreasing AID expression and CSR. This, together with the largely normal phosphorylation of p65 (despite a slight decrease in IκBα phosphorylation) in p85α -/-B cells and virtually no difference in proliferation and (limited) CSR between p85α -/and p85α + / + B cells on stimulation by CpG plus IL-4, strongly suggests that PI(3)K-signalling is more important for transducing BCR-signalling, than TLR-signalling, in B cells. The same time-dependence of p52 and AID induction, both of which progressively increased within 48 h and were perhaps linked to cell divisions, further emphasizes the critical role of the non-canonical NF-κB pathway in AID and CSR induction. Accordingly, unlike anti-δ mAb/dex, soluble/monomeric anti-Ig Abs cannot extensively crosslink BCR to initiate sustained signalling for activation of the non-canonical NF-κB pathway 43 or enhancement of AID and CSR 44,45 . Finally, BCR signalling, while upregulating AID and CSR and PNA-binding glycoproteins, downregulated Blimp1 and CD138, suggesting that antigen engagement of BCR delays plasmacytoid differentiation of TLR-stimulated B cells until CSR completion.
We showed that BCR and TLRs synergized to induce CSR by enhancing AID expression rather than by merely promoting B-cell proliferation or survival. We also showed that BCR-enhanced and the IκBα degradation and subsequent re-synthesis led to more than one peak of IκBα phosphorylation). Levels of phosphorylated IκBα and p65 were normalized to the level of total IκBα and p65, respectively, and data are depicted as the ratio to the expression levels in p85α -/-B cells before stimulation (0 m, set as 1; right panels). In p85α -/-B cells, p65 phosphorylation induced by CpG or mCD154 was not impaired despite changes in IκBα phosphorylation, that is, decrease following stimulation with CpG and increase following stimulation with mCD154. Data are representative of three independent experiments.
IL-4-dependent germline Iγ1-Cγ1 transcription peaked earlier than Aicda expression, thereby suggesting a synergy of the (BCRactivated) non-canonical NF-κB pathway, with the (IL-4Ractivated) JAK-Stat6 pathway, to induce rapid gene expression. BCR enhancement of cytokine (IL-4, IFNγ and/or TGFβ)-promoted germline I H -C H transcription and TLR-dependent AID induction would underpin T-cell-independent CSR induction. Also, anti-δ mAb/dex, together with LPS, which induced B cells to undergo CSR to IgG3, induced high levels of post-translational modifications of histones in the Sγ3 DNA region 46 ; modified histones would stabilize 14-3-3 binding to S regions to unfold CSR 8 . The requirement for dual BCR/TLR engagement to induce efficient CSR can be fully satisfied by LPS alone. As we have shown, LPS, but not lipid A, induced CD79a phosphorylation (a unique hallmark of signalling emanating from BCR, but not from other receptors) and Ca 2 + mobilization, and activated the non-canonical NF-κB pathway, indicating that the LPS polysaccharidic moiety triggers BCR-signalling, which synergizes with lipid A moietyengaged TLR4-signalling to induce AID expression and CSR. Indeed, blocking CD79a or deficiency in PI(3)K p85α hampered induction of CSR by LPS in the same fashion as it hampered induction of CSR by TLR ligands plus anti-δ mAb/dex. Findings that mice deficient in PI(3)K display negligible serum IgG3 and that B cells deficient in BCR-signalling elements, such as CD19, Btk, Blnk, Lyn or Vav, show greatly reduced responses to LPS 47-51 further support our conclusion that LPS activates BCR-signalling in addition to TLR4-signalling for CSR. The ability of O-antigen to bind (specific) BCRs to induce antibody responses to LPS in vivo 19 also provides strong support for the argument that the LPS polysaccharidic moiety engages the BCR and synergizes with the lipid A-engaged TLR4 to induce efficient CSR; polysaccharides have not been reported to interact with receptors on B cells other than the BCR 35 .
Like dual BCR/TLR-signalling, including that triggered by LPS, CD40-signalling activates the two (non-canonical and canonical) NF-κB pathways for AID induction, demonstrating that T-cellindependent and T-cell-dependent CSR, despite their significant difference in the nature of triggering stimuli, entail the same set of transcription factors critical for AID induction (Fig. 8). CD40, however, employs different signal transducers, utilizing TRAFs (TRAF2/TRAF3, in particular) to activate both NF-κB pathways and induce AID and CSR 52 -TRAF2 and TRAF3 have no role in the BCR-signalling pathway, thereby explaining the lack of synergy of BCR and CD40 in CSR. That CD40 activation of NF-κB and induction of AID and CSR were not impaired in p85α -/-B cells is also consistent with findings that BCR/PI(3)K-signalling has no significant role, or possibly even a negative role through Aktmediated phosphorylation and, therefore, nuclear exclusion of Foxo1 (a transcription factor shown to enhance Aicda expression in CD40-dependent CSR 53 ); BCR-signalling, however, is required for CD40-dependent SHM 54 . BCR-triggered and time-dependent activation of the non-canonical NF-κB pathway versus nuclear exclusion of Foxo1 might influence the outcome of CD40-dependent CSR. Also, consistent with the dependence of IgA switching on TACI, which activates the canonical NF-κB pathway 55 , anti-δ mAb/ dex synergized with TACI ligand BAFF or APRIL to induce CSR to IgA (our findings and ref. 28).
Our findings that dual BCR/TLR engagement induced CSR in, up to, 40% of B cells indicate that T-cell-independent CSR can be as efficient as CD40-dependent CSR, as induced by specific CD4 + T-helper cells. They also provide much needed evidence for the argument that dual BCR/TLR engagement is highly relevant to T-independent antibody responses, a notion further supported by our in vivo experiments showing a strong NP-specific IgG response induced by NP-LPS, which is devoid of any proteinic component and, therefore, would not elicit T-cell help. In spite of (conflicting) suggestions on the ability of NP 26 (NP-Ficoll) to induce modest levels of class-switched antibodies (for example, refs 56,57), our experiments have clearly shown that NP 26 could not induce an in vivo class-switched NP-specific antibody response comparable to that induced by NP-LPS, further extending our in vitro findings that BCR crosslinking alone did not efficiently induce AID or CSR. Collectively, our in vitro and in vivo findings indicate that among the pool of B cells that can be activated by TLR ligands, only those that can also bind to antigen can undergo TLR-dependent differentiation, thereby focusing the early adaptive (antibody) response when specific T-cell help is not yet available or effectively mediating antibody response to some T-independent antigens. They outline a major T-independent B-cell differentiation pathway, as underpinned by dual BCR/TLR induction of AID and CSR, to diversify antibody responses. Such a pathway would be evolutionarily conserved in jawed vertebrates, which express germline encoded and nonclonally distributed TLRs and clonally distributed BCRs. In organisms with a primitive adaptive immune system, for example, jawless vertebrates, cooperation of TLRs or perhaps other innate immune receptors with antigen receptor VLRB 58 might have an important role in fighting invading pathogens.

Methods B cells.
Single B-cell suspensions were prepared from murine axillary, inguinal and cervical lymph nodes using a 70-µm cell strainer. Spleen B cells were resuspended in ACK Lysis Buffer (Lonza) to lyse red blood cells, quenched with RPMI containing 10% FBS (RPMI-FBS) and then resuspended in same medium before further B-cell purification. Lymph node and spleen B cells were purified by negative depletion of cells expressing CD43, CD4 or Ter-119 using the B cell Isolation kit (Miltenyi), following manufacturer's protocol, resulting in a preparation of more than 99% B220 + sIgδ + B cells (Supplementary Fig. S10). In most experiments, we supplemented the kit with biotinylated mAb to CD11c (clone N418, eBioscience) and mAb to IgG1 (clone A85-1, BD Biosciences), thereby achieving an even greater degree of sIgδ + B cell purity.
B-cell-surface analysis and proliferation. sIgδ + B cells were stimulated for 4 days and collected for flow cytometry analysis of surface expression of Ig, B220, CD138    or PNA-binding glycoproteins (Supplementary Table S1), as we had described 8,11 . For proliferation studies, B cells were resuspended in PBS at 10 7 cells per ml and incubated with 4 µM of carboxyfluorescein diacetate succinimidyl ester (Invitrogen) at 37°C for 5 minutes and immediately quenched with 10 vol of RPMI-FBS. Cells were analysed using a BD FACSCalibur (BD Biosciences) and data analysis was performed using the FlowJo software (Tree Star). Dead cells (7-AAD + ) were excluded from analysis. Cell divisions were determined using the proliferation platform of FlowJo.
Real-time qRT-PCR transcript analysis. RNA was extracted from 5×10 6 B cells stimulated for 48 h (or as indicated) using the RNeasy Mini Kit (Qiagen). First-strand complementary DNA was synthesized from equal amounts of total RNA (4 µg) using the SuperScript III System (Invitrogen) and analysed by the SYBR Green dye (Bio-Rad) incorporation in PCR reactions involving specific primers (Supplementary  Table S2) and performed in an iCycler and MyIQ real-time qPCR system (Bio-Rad). The levels of transcripts were quantified by the ∆∆CT (cycle threshold) method.

Ca 2 + mobilization assay.
For analysis of the elevation of [Ca 2 + ] cytosol induced by transient BCR-signalling, B cells were loaded with Fluo-3 AM (5 µg ml − 1 , Invitrogen) and Fura-Red AM (10 µg ml − 1 ) in 1 ml of RPMI containing 2% (v/v) of FBS and 0.04% (v/v) of pluronic F-127 at 37°C for 45 m. After washing with HBSS containing 1% (g/v) of BSA, 1 mM of CaCl 2 and 1 mM of MgCl 2 (HBSS-Ca), cells were pelleted and resuspended in 500 µl of HBSS-Ca and stained with APCconjugated anti-B220 mAb (eBiosciences). After washing, cells were resuspended in HBSS-Ca, stimulated with F(ab′)2 of goat anti-mouse µ chain (anti-µ F(ab′)2, Southern Biotech) or anti-δ mAb/dex at indicated concentrations and immediately analysed by flow cytometry. The level of free intracellular Ca 2 + was determined by the ratio of the Fluo-3 intensity to the Fura-Red intensity using the 'kinetics' tool of the FlowJo software. For analysis of Ca 2 + mobilization following a long period of stimulation, resting B cells were stimulated for 16 h with anti-δ mAb/dex or LPS, or nil, lipid A or mCD154 and labelled with Fluo-3 AM and Fura-Red as above, and then stimulated with anti-µ F(ab′) 2 (5 µg ml − 1 ) and analysed.
CD79a phosphorylation assay. Spleen B cells (10 7 ) were stimulated as indicated, collected and resuspended in lysis buffer (20 mM of Tris-Cl, pH 7.5, 150 mM of NaCl, 0.5 mM of EDTA, 1% (v/v) of NP-40) supplemented with phosphatase inhibitors sodium pyrophosphate (1 mM), NaF (10 mM) and NaVO 3 (1 mM) and a cocktail of protease inhibitors (Sigma). After sonication and centrifugation, protein lysates were pre-cleared with protein A-agarose (Pierce) for 2 h at 4°C and then incubated with Armenian hamster anti-mouse CD79a (2 µg ml − 1 , BioLegend) or control IgGs with irrelevant specificities overnight at 4°C before immunocomplexes a  (a) BCR crosslinking activates the non-canonical nF-κB pathway by inducing expression of p100 and its processing to p52 in a PI(3)K-dependent fashion. TLR engagement (as exemplified by TLR4 engagement by lipid A) activates the canonical nF-κB pathway, resulting in limited Aicda expression and marginal CsR. BCR enhances the TLR-dependent canonical nF-κB pathway, likely through TRAF6 activation, and TLR enhances the BCR-activated non-canonical nF-κB pathway by inducing p100 expression, thereby synergizing in inducing AID and CsR. (b) unlike lipid A, LPs triggers both BCR-signalling through its polysaccharidic moiety and TLR4-signalling through its lipid A moiety, thereby activating both the non-canonical and canonical nF-κB pathways to induce AID and CsR in a PI(3)K-dependent fashion. (c) CD40 activation of the non-canonical and canonical nF-κB pathways and induction of CsR depend on TRAF2/TRAF3, which, however, play no role in the BCR-signalling pathway; TRAF6 mediates activation of the canonical nF-κB pathway only and is dispensable for CsR induced by CD40-signalling.