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

Abstract

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 Ca(2+) 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.

Figure 1. BCR crosslinking enables TLRs to induce efficient CSR.

(a) Proportions of surface IgG1⁺ (sIgG1⁺) B cells after stimulation of CFSE-labelled sIgδ⁺ B cells with TLR1/2 ligand Pam₃CSK₄, TLR4 ligand lipid A, TLR7 ligand R-848 or TLR9 ligand CpG at indicated doses, in the absence or presence of anti-δ mAb/dex at indicated doses, plus IL-4 (3 ng ml⁻¹). (b) Proportions of sIgG1⁺ B cells at each cell division after stimulation of sIgδ⁺ B cells with LPS or mCD154 at indicated doses, in the absence or presence of anti-δ mAb/dex (100 ng ml⁻¹), plus IL-4 (3 ng ml⁻¹). (c) Proportions of sIgG1⁺ B cells within each B cell division after stimulation of CFSE-labelled sIgδ⁺ B cells with Pam₃CSK₄ (100 ng ml⁻¹), lipid A (1 μg ml⁻¹), R-848 (30 ng ml⁻¹), CpG (1 μM), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹), in the absence (teal) or presence (plum) of anti-δ mAb/dex (100 ng ml⁻¹), plus IL-4 (3 ng ml⁻¹). Data are representative of four independent experiments.

Figure 2. BCR crosslinking potentiates TLR-dependent CSR in a proliferation-independent fashion.

(a,b) Proportions of sIgG1⁺ B cells (a) and average B-cell divisions (b) after stimulation of CFSE-labelled sIgδ⁺ B cells with CpG, LPS or mCD154, at indicated doses, plus IL-4 (3 ng ml⁻¹) in the absence (teal) or presence (plum) of anti-δ mAb/dex (100 ng ml⁻¹) (mean and s.e.m. of data from four independent experiments). Also depicted (blue brackets) are higher CSR rates in B cells stimulated with low dosages of CpG or LPS in the presence of anti-δ mAb/dex than in B cells stimulated with high dosages of CpG or LPS in the absence of anti-δ mAb/dex (despite comparable proliferation). P values: t-test. (c) Levels of germline Iγ1-Cγ1 transcripts in B cells stimulated for 48 h with TLR ligand Pam₃CSK₄ (100 ng ml⁻¹), lipid A (1 μg ml⁻¹), R-848 (30 ng ml⁻¹), CpG (1 μM), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹), plus IL-4 (3 ng ml⁻¹) in the absence (teal) or presence (plum) of anti-δ mAb/dex. Data were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in B cells stimulated in the absence of anti-δ mAb/dex (mean and s.e.m. of data from three independent experiments; P values: t-test). (d) Levels of circle Iγ1-Cμ transcripts and post-recombination Iμ-Cγ1 transcripts in B cells stimulated with CpG (1 μM), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) plus IL-4 (3 ng ml⁻¹) in the absence (teal) or presence (plum) of anti-δ mAb/dex for 48 h. Data were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in B cells stimulated in the absence of anti-δ mAb/dex (mean and s.e.m. of data from three independent experiments; P values: t-test).

Figure 3. LPS induces BCR-signalling, and blocking of CD79a inhibits CSR induced by dual BCR/TLR engagement or LPS.

(a) CD79a phosphorylation in B cells stimulated with anti-δ mAb/dex (100 ng ml⁻¹) for 5 m, anti-μ F(ab′)₂ (5 μg ml⁻¹) for 5 m, nil, lipid A (1 μg ml⁻¹) for 24 h, LPS (3 μg ml⁻¹) for 3 h or 24 h, or LPS (3 μg ml⁻¹) plus blocking anti-CD79a mAb for 24 h. (b) Ca²⁺ mobilization in B cells stimulated with anti-μ F(ab′)₂ or anti-δ mAb/dex at indicated doses for a short period (less than 30 m). (c) Anti-μ F(ab′)₂–triggered Ca²⁺ mobilization in B cells pretreated with nil, anti-δ mAb/dex (100 ng ml⁻¹), LPS (3 μg ml⁻¹), lipid A (1 μg ml⁻¹) or mCD154 (1 U ml⁻¹) for 16 h (elevated [Ca²⁺]_cytosol would reduce the amplitude of the Ca²⁺ flux and subsequent steady-state Ca²⁺ levels induced by newly applied anti-μ F(ab′)₂). Data are representative of three independent experiments. (d) Proportions of sIgG1⁺ B cells after stimulation of CFSE-labelled sIgδ⁺ B cells with lipid A in the absence (teal) or presence (plum) of anti-δ mAb/dex (100 ng ml⁻¹), LPS (3 μg ml⁻¹), or CpG (1 μM) in the presence of anti-δ mAb/dex, plus IL-4 (3 ng ml⁻¹) and treated with nil or blocking anti-CD79a mAb (1 μg ml⁻¹) (top panels). Also depicted are proportions of B cells at each cell division that were IgG1⁺ (bottom panels). (e) Levels of Aicda, germline Iγ1-Cγ1 and circle Iγ1-Cμ transcripts in B cells stimulated with CpG (1 μM) and anti-δ mAb/dex (100 ng ml⁻¹), or LPS (3 μg ml⁻¹), in the absence (teal) or presence (plum) of blocking anti-CD79a mAb (1 μg ml⁻¹) (all with 3 ng ml⁻¹ of IL-4). Data were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in B cells not treated with anti-CD79a mAb (mean and s.e.m. of data from three independent experiments; P values: t-test).

Figure 4. BCR crosslinking increases TLR-dependent AID expression.

(a) Aicda and Blimp1 transcripts in B cells stimulated for 48 h with Pam₃CSK₄ (100 ng ml⁻¹), lipid A (1 μg ml⁻¹), R-848 (30 ng ml⁻¹), CpG (1 μM), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) plus IL-4 (3 ng ml⁻¹) in the absence (teal) or presence (plum) of anti-δ mAb/dex (100 ng ml⁻¹). Data were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in B cells stimulated in the absence of anti-δ mAb/dex (mean and s.e.m. of data from three independent experiments; P values: t-test). (b) Kinetics of induction of AID expression and germline Iγ1-Cγ1 transcription by anti-δ mAb/dex alone (100 ng ml⁻¹, orange), IL-4 (3 ng ml⁻¹, purple triangles) or lipid A (1 μg ml⁻¹, green diamonds), CpG (1 μM, blue squares) or LPS (3 μg ml⁻¹, red circles) plus IL-4 in the absence (open symbols) or presence (filled symbols) of anti-δ mAb/dex. Levels of Aicda and germline Iγ1-Cγ1 transcripts were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in unstimulated sIgδ⁺ B cells (0 h, set as 1). Anti-δ mAb/dex alone induced only low levels of germline Iγ1-Cγ1 transcription. Data are representative of three independent experiments. (c) Proportions of PNA^hi (B220⁺) B cells after stimulation of sIgδ⁺ B cells with nil, anti-δ mAb/dex (100 ng ml⁻¹), or CpG (1 μM), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) in the absence (teal) or presence (plum) of anti-δ mAb/dex (all in the presence of 3 ng ml⁻¹ of IL-4, mean and s.e.m. of data from three independent experiments, P values: t-test). (d) Proportions of CD138⁺(Syndecan-1⁺) B220^lo plasmacytoid cells/plasmacytes after stimulation of sIgδ⁺ B cells with nil, anti-δ mAb/dex (100 ng ml⁻¹), or CpG (1 μM), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) in the absence (teal) or presence (plum) of anti-δ mAb/dex (all in the presence of 3 ng ml⁻¹ of IL-4, mean and s.e.m. of data from three independent experiments, P values: t-test). (e) Potentiation of TLR9-dependent CSR to IgG1 by exogenous AID. Proportions of sIgG1⁺ (B220⁺) B cells after sIgδ⁺ B cells were transduced with pTAC or pTAC–AID retrovirus (CD25⁺) and stimulated with CpG (1 μM), or CpG and anti-δ mAb/dex (100 ng ml⁻¹), plus IL-4 (3 ng ml⁻¹). (f) Levels of Aicda, germline Iγ1-Cγ1 and post-recombination Iμ-Cγ1 transcripts in B cells transduced with pTAC (teal) or pTAC–AID (plum) retrovirus and stimulated with CpG (1 μM) plus IL-4 (3 ng ml⁻¹). Data were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in the B cells transduced with pTAC virus (mean and s.d. of triplicate samples, P values: t-test).

Figure 5. BCR crosslinking enhances the TLR-dependent canonical NF-κB pathway in a p85α-dependent fashion.

(a–c) Levels of phosphorylated IκBα, total IκBα, phosphorylated p65, total p65 and β-actin in p85α^+/+ (plum) and p85α^–/– (teal) B cells stimulated with CpG (1 μM), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) plus IL-4 (3 ng ml⁻¹) in the absence (open symbols) or presence (filled symbols) of anti-δ mAb/dex (100 ng ml⁻¹) (left panels; 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.

Figure 6. BCR crosslinking activates the non-canonical NF-κB pathway in a p85α-dependent fashion.

(a) Induction of p52 and p100 in B cells stimulated with anti-δ mAb/dex (100 ng ml⁻¹), lipid A (1 μg ml⁻¹) or CpG (1 μM) in the absence or presence of anti-δ mAb/dex, LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) (all in the presence of 3 ng ml⁻¹ of IL-4) for 0, 3, 24 and 48 h. (b) Levels of p52, p100 and β-actin in p85α^+/+ and p85α^–/– B cells stimulated with anti-δ mAb/dex (100 ng ml⁻¹), lipid A (1 μg ml⁻¹) or CpG (1 μM) in the absence or presence of anti-δ mAb/dex, LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) (all in the presence of 3 ng ml⁻¹ of IL-4) for 48. Data are representative of three independent experiments.

Figure 7. BCR and TLR-induced CSR is impaired in p85α^–/– B cells.

(a) Proportions of sIgG1⁺ B cells after stimulation of CFSE-labelled sIgδ⁺ p85α^–/– B cells (teal) and their p85α^+/+ counterparts (plum) with Pam₃CSK₄ (100 ng ml⁻¹), lipid A (1 μg ml⁻¹), R-848 (30 ng ml⁻¹) or CpG (1 μM) in the absence or presence of anti-δ mAb/dex (100 ng ml⁻¹), LPS (3 μg ml⁻¹), all in the presence of IL-4 (3 ng ml⁻¹), (top panels), and proportions of B cells at each cell division that were sIgG1⁺ (bottom panels). (b) Proportions of sIgG1⁺ B cells after stimulation of CFSE-labelled sIgδ⁺ p85α^–/– B cells (teal) and their p85α^+/+ counterparts (plum) with mCD154 (1 U ml⁻¹) plus IL-4 (3 ng ml⁻¹), in the absence or presence of anti-δ mAb/dex (top panels), and proportions of B cells at each cell division that were IgG1⁺ (bottom panels). (c) Levels of Aicda, germline Iγ1-Cγ1, circle Iγ1-Cμ and post-recombination Iμ-Cγ1 transcripts in p85α^–/– B cells (teal) and their p85α^+/+ counterparts (plum) stimulated with CpG (1 μM) and anti-δ mAb/dex (100 ng ml⁻¹), LPS (3 μg ml⁻¹) or mCD154 (1 U ml⁻¹) in the absence of presence of anti-δ mAb/dex, plus IL-4 (3 ng ml⁻¹). Data were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in p85α^+/+ B cells (mean and s.e.m. of data from three independent experiments; P values: t-test).

Figure 8. BCR and TLR signalling are integrated to induce T-cell-independent CSR through both NF-κB pathways.

(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.